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){T.07S

AGRICULTURAL
RESEARCH

Volume XXIl

OCTOBER I— DECEMBER 3, 192 1

PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE

WITH THE COOPERATION OF THE ASSOCIATION

OF LAND-GRANT COLLEGES

WASHINGTON, D. C.

CONTENTS

Page

Occurrence of Quercetin in Emerson's Brown- Husked Type of
Maize. Charles E. Sando and H. H. Bartleit i

Biological Analysis of the Seed of the Georgia Velvet Bean, Stilzo-
lobium deeringianum. BarnETT Sure and J. W. Read (15 text
figures) 5

Effect of Soil Temperature upon the Development of Nodules on
the Roots of Certain Legumes. FrEd reuel, Jones and W. B.
TiSDALE (4 text figures; 3 plates) 17

Influence of the Period of Transplanting Western White Pine Seed-
lings upon Their Behavior in Nursery and Plantation. E. C.
Rogers (7 text figures) 33

A Dryrot Canker of Sugar Beets. B. L. Richards (6 plates) .... 47

Comparative Vigor of Fj Wheat Crosses and Their Parents. Fred

Griffee 53

Temperature and Humidity Studies of Some Fusaria Rots of the
Irish Potato. R. W. Goss (2 plates) 65

Blackleg Potato Tuber-Rot under Irrigation. M. Shapovalov and

H. A. Edson (6 plates) 81

Microscopic Study of Bacteria in Cheese. G. J. HucKER (i plate) . 93

Further Studies on Relation of Sulphates to Plant Growth and

Composition. Harry G. Mii^l,er loi

Soybean Mosaic. Max W. Gardner and James B. Kendrick (2

plates) HI

Influence of the Plane of Nutrition on the Maintenance Require-
ment of Cattle. F. B. Mumford, A. G. Hogan, and W. D.
Salmon 115

Turnip Mosaic. Max W. Gardner and James B. Kendrick

(i plate) 123

Hydrocyanic Acid in Sudan Grass. C. O. Swanson 125

Nutrient Requirements of Growing Chicks: Nutritive Deficiencies
of Corn. F. E. Mussehl, J. W. Calvin, D. L. HalberslEben
and R. M. SandsuEdt (ii text figures) 139

Aecial Stage of the Orange Leafrust of Wheat, Puccinia triticina

Eriks. H. S. Jackson and E. B. Mains (i plate) 151

A Transmissible Mosaic Disease of Chinese Cabbage, Mustard,
and Turnip. E. S. SchulTz (4 plates) 173

Flora of Corn Meal. Charles Thom and Edwin LeFevre 179

, Hopkins Host-Selection Principle as Related to Certain Ceramby-

u. cid Beatles. F. C. Craighead 189

IV Journal of Agricultural Research voi. xxii

Page

Notes on the Organic Acids of Pyrus coronaria, Rhus glabra, and

Acer saccharum. Charles E. Sando and H. H. BartlETT. . . 221

FertiHty in Shropshire Sheep. Elmer Roberts 231

Relation of Soil Temperature and Other Factors to Onion Smut

Infection. J. C. Walker and L. R. Jones (2 text figures; 3

plates) 235

A Physiological Study of Grapefruit Ripening and Storage. Lon

A. Hawkins (i text figure) 263

Absorption of Copper from the Soil by Potato Plants. F. C.

Cook 281

Pale Western Cutworm (Porosagrotis orthogonia Morr.) J. R.

Parker, A. L. Strand, and H. L. Seamans (i text figure; 4

plates) 289

Biology of Embaphion muricatum. J. S. Wade and Adam H.

Boving (3 text figures; 2 plates) 323

Genetic Behavior of the Spelt Form in Crosses between Triticum

spelta and Triticum sativum. Clyde E- LeighTy and Sarkis

Boshnakian (3 text figm-es; i plate) 335

Plum Blotch, a Disease of the Japanese Plum Caused by Phyllo-

sticta congesta Heald and Wolf. John W. Roberts (2 text

figures; i plate) 365

A Comparison of the Pectinase Produced by Dififerent Species of

Rhizopus. L. L. Harter and J. L. Weimer (2 text figures) .... 371

Hemotoxins from Parasitic Worms. Benjamin Schwartz 379

Ash Content of the Awn, Rachis, Palea, and Kernel of Barley

during Growth and Maturation. Harry V. Harlan and

Merritt N, Pope (5 text figures) 433

Temperature Relations of Stone Fruit Fungi. Charles Brooks

and J. S. CoolEy (24 text figures) 45 1

Transportation Rots of Stone Fruits as Influenced by Orchard

Spraying. CharlES Brooks and D. F. Fisher (6 text figures) . 467
Storage of Coniferous Tree Seed. C. R. Tillotson (2 text figures) . 479
Susceptibility of the Different Varieties of Sweet Potatoes to

Decay by Rhizopus nigricans and Rhizopus tritici. L. L.

Harter and J. L. Weimer 511

Index 517

Vol. XXII OCXOBER 1, 1921 No. 1

JOURNAI. OF

AGRICULTURAL

RESEARCH

CONXENXS

PagQ

Occurrence of Quercetin in Emerson's Brown-Husked
Type of Maize -------- i

CHARLES E. SANDO and H. H. BARTLETT

(Contribution from Bureau of Plant Industry and University ot Michigan)

Biological Analysis of tlie Seed of the Georgia Velvet Bean,
Stilzolobium deeringianum - - - - - - 5

BARNETT SURE and J. W. READ

(Contribution from Arkansas Agricultural Experiment Station)

Effect of Soil Temperature upon the Development of
Nodules on the Roots of Certain Legumes - - - 17

FRED REUEL JONES and W. B. TISDALE

(Contribution from Bureau of Plant Industry and University of Wisconsin)

Influence of the Period of Transplanting Western White
Pine Seedlings upon Their Behavior in Nursery and
Plantation - -- - - - - - -33

E. C. ROGERS

(Contribution from Forest Service)

A Dryrot Canker of Sugar Beets ----- 47

B. L. RICHARDS

(Contribution from Utab Agricultural Experiment Station)

PUBUSHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE,

WITH THE COOPERATION OF THE ASSOCIATION OF

LAND-GRANT COLLEGES

WASHINOa^ON, D. C.

EDITORIAL COMMITTEE OF THE

UNITED STATES DEPARTMENT OF AGRICULTURE AND

THE ASSOCIATION OF LAND-GRANT COLLEGES

FOR THE DEPARTMENT
KARL F. KELLERMAN, Chairman

Physiologist and Associate Chief, Bureau
of Plant Industry

EDWIN W. ALLEN

Chief, Office of Experiment Stations

CHARLES L. MARLATT

Entomologist and Assistant Chief, Bureau
of Entomology

FOR THE ASSOCIATION
J. G. LIPMAN

Dean, Sfafe College of Agriculture, and
Director, New Jersey Agricultural Experi-
ment Station, Rutgers College

W. A. RILEY

Entomologist and Chief, Division of Ento-
mology and Economic Zoology, Agricuh
tural Experiment Station of the University
of Minnesota

R. L. WATTS,

Dean, School of Agriculture, and Director;
Agricultural Experiment Station, Tb4
Pennsylvania Staf* College

All correspondence regarding articles from the Department of AgricultiU'e should be
addressed to Karl F. Kellerman, Journal of Agricultural Research, Washington, D. C.

All correspondence regarding articles from State Experiment Stations should be
addressed to J. G. Lipnaan, New Jersey Agricultural Experiment Station, New
Bnxnswick, N. J.

J01M£ OF AGRICiriAL RESEARCH

Vol. XXII Washington, D. C, October i, 1921 No. i

OCCURRENCE OF QUERCETIN IN EMERSON'S BROWN-
HUSKED TYPE OF MAIZE

By Charles E. Sando, Junior Chemist, Office 0/ Physiological and Fermantation Investi-
gations, Bureau of Plant Industry, United States Department of Agriculture, and H. H.
BartlETT, Department of Botany, University of Michigan, Collaborator, Office of
Physiological and Fermentation Investigations, Bureau of Plant Industry, United
States Department of Agriculture '

In connection witli the genetical studies of pigmentation in maize
which have been carried on for several years at Cornell University by
Prof. R. A. Emerson and his students, a plan was made for the writers
to collaborate in parallel biochemical studies in the isolation and identifi-
cation of the pigments from material of known genetical constitution,
the latter to be furnished as a by-product of the Cornell experiments.

As a beginning in the work it seemed desirable to undertake a study
of the character pair purple versus brown. These are two of the general
plant colors recognized in the Cornell experiments, the series running as
follows: I, purple; II, sun-red; III, dilute purple; IV, dilute sun-red;
V, brown; and VI, green. Full details of the genetic relations of these
types are being published by Enierson,^ to whose memoir the reader
should refer for full details. Here it may suffice to say that purple is a
color type which is uncommon in cultivation and infrequent in experi-
mental cultm-es. It is distinguished from other types by the fact that
some purple color is developed even in seedlings grown in the dark. At
maturity nearly all parts are more or less purple, including the culm, the
brace roots, all leaf sheaths, the husks, even the inner ones, the cob, and
the staminate inflorescence. In intensity of coloration purple stands at
the head of the series of color types. Material of this type was furnished
by Prof. Emerson to Dr. John W. Calvin, of the University of Nebraska,
before we took over the general problem, and we have therefore confined
our attention for the present to the brown type, awaiting a report on his
study of the purple type from Dr. Calvin.

• In connection with our work with maize we have received numerous favors from Prof. R. A. Emerson
and Dr. E. G. Anderson, to whom we tender our best thanks.
^ ' Emerson, R. A. the genetic relations of generai, plant colors in maize. N. Y. Cornell Agr.
. ^ Exp. Sta. Mem. 39, 156, p., n col. pi. 1921.

or: _______

■* "Journal of Agricultural Research, Vol. XXII, No. i

j^ Washington, D. C. Oct. i, 1921

^^ zl. Key No. G-244

Journal of Agricultural Research

Vol. XXII. No. I

A few preliminary tests of the purple pigment convinced us that it was
an anthocyanin of which the nonsugar portion was of the same general
group as cyanidin, isolated by Willstatter ^ and his students from several
sources, including the cornflower, Centaurea. His proof of the easy
chemical transition from the anthocyanin to the flavonol series led us to
look for a member of the latter series in the brovvTi maize. In accordance
with expectations, we were able to isolate a glucosid of quercetin. This
discovery makes it seem exceedingly likely that the anthocyanin of the
purple type is a corresponding glucosid of cyanidin.

The brown color type is a still more unusual one than the purple.
It first appeared in Emerson's ^ cultures as a segregate in the second
generation of the cross purple X green and is unkno%vn outside this
series of cultures. Seedlings and young plants are wholly green. As the
flowering period approaches, a brown color appears in the lower sheaths,
and at flowering time the culm, sheaths, husks, and staminate inflores-
cences are brown. Light is not essential to the development of the
color. Our material of the brown type consisted of husks.

ISOLATION AND IDENTIFICATION OF THE FREE QUERCETIN

Ground husks were extracted in a large Soxhlet apparatus with redis-
tilled 95 per cent alcohol for about 72 hours, and the alcohol was evapo-
rated off in vacuo. The thin aqueous sirup was filtered from tarry
matter and the filtrate boiled with animal charcoal. By shaking with
ether it gave an etherial solution containing a free (nonglucosidal) fla-
vone which proved to be quercetin. The ether was evaporated off, and
the residue, after being dried in a desiccator, was extracted in a paper
thimble, first with benzene, to remove tarry colloids, oils, etc., and
finally, for a short time, with ether. The latter solvent dissolved part
of the quercetin but left the bulk of it in the thimble. This portion was
dried and acetylated for an hour with anhydrous sodium acetate and
acetic anhydrid. After purification the acetyl derivative was quanti-
tatively hydrolyzed with sulphuric acid in glacial acetic acid. The reac-
tion mixture was diluted and the recovered quercetin was washed with
cold water. The results are given in Table I.

Table I. — Data on hydrolysis , by sulphuric acid in glacial acetic-acid solution, of the
acetylated free flavone of brown maize husks

Sample I. Samples. Samples

Weight of acetyl quercetin (gtn.). . . .
Weight of recovered quercetin (gm.)
Percentage of recovered quercetin. ..

0.2521
.1484
58.86

0-3165
.1866
58.95

0.4908
.2902
59-13

1 Willstatter, Richard, and Everest, Arthur E. tjntersuchungen uber die anthocy.vne. i. user
DEN FARBSTOFF DER KORNBLUME. /« Licbig's Ann. Chem., Bd. 401, Heft 2, p. 189-232, 4 fig. 1913.
' Emerson, R. A. op. ax.

Oct. 1, 1921 Quercetin in Emerson s Brown-Husked Type of Maize 3

The mean of the three determinations is 58.98 per cent — in exact
accord with theory.

The entire yield of approximately i gm. of acetyl derivative was
divided to make the above determinations. The quercetin obtained
(0.6254 gin.) was again acetylated, yielding 0.8352 gm. of penta-acetyl-
quercetin. The acetyl derivative melted at 190° to 192° C. The recov-
ered flavone melted at about 305° to 306° with darkening. When
mixed with quercetin from Bscholtzia (melting point approximately
305° to 310°) the mixture melted at 306° to 307°. In other charac-
teristics the quercetin from maize was identical with a sample obtained
by the writers ^ from rutin, a glucosid of quercetin found in Escholtzia
petals.

Combustions of the free quercetin and of its acetyl derivative were
made, with the results shown in Table II.

Table II. — Combustions of the free quercetin of brown maize husks and of its acetyl

derivative

Quercetin.

Penta-acetylquercetin.

Sample i. Sample 2

Weight of sample (gm.)

Weight of carbon dioxid (gm.)

Weight of water (gm.)

Percentage of carbon

Percentage of hydrogen

0-1353
.2951
.0404
59-47
3-34

o. 1126
.2430
.0403
58-85

4.00

0.2026

•4338
.0691

58.39
3.82

Theory requires: For quercetin, carbon 59.59 per cent, hydrogen 3.34 per cent; for penta-acetylquercetin,
carbon 58.59 per cent, hydrogen 3.90 per cent.

PREPARATION OF THE GLUCOSID

After partition of the alcoholic extract of the brown husks between
ether and water, the aqueous solution, containing as one of its chief con-
stituents a quercetin glucosid, was treated with four successive portions
of lead acetate. The first fraction of the lead precipitate was discarded.
The second consisted largely of tarry matter and was therefore not used
for the preparation of pure glucosid but yielded quercetin on hydrolysis
after decomposition with hydrogen sulphid. The third and fourth frac-
tions were combined, suspended in hot alcohol, decomposed with hydrogen
sulphid, filtered, and evaporated to small bulk. A small quantit}' of
impure glucosid separated out on standing, but the greater part was got
by shaking the solution with ethyl acetate.

The glucosid was purified only with great difficulty, by fractional solu-
tion of the dry impure product in ethyl acetate and successive crystalli-
zation of the purer fractions from water. The yield of pure glucosid

' Sando, Charles E., and Bartlett, H. H. rutin, the flavone pigment of escholtzia californica
CHAM. /« Jour. Biol. Chem., V. 41, no. 4, p. 495-501, pi. 6-7. 1920.

Journal of Agricultural Research voi. xxii, no. i

obtained in this manner was insufficient for a thorough investigation,
which must be deferred until a new lot of material is extracted. It was
nearest in color to the "lemon yellow" of Ridgway's ^ color standards
and melted to a cherrj^-red liquid at 220° to 222° C. A\Tien hydrolyzed it
produced quercetin and apparently only one sugar, glucose, although the
latter point is to be more thoroughly investigated. The osazone of the
sugar melted at 204° to 206° and was evidently glucosazone. The
quercetin obtained by hydrolysis was identified by its general properties
and by combustions both of the free flavonol and of the acetyl deriva-
tive. The latter melted at 191° to 193.5° and had the properties of
penta-acetylquercetin. A sample weighing 0.4650 gm. gave 0.2735 &^-
of quercetin, or 58.81 per cent by quantitative hydrolysis; theory requires
58.98 per cent. The results of combustions are given in Table III.

Table III. — Combustions of the quercetin obtained by hydrolysis of the glucosid of brown
maize husks and of its acetyl derivative

Penta-
acetyl-
quercetin.

Weight of sample (gm.)

Weight of carbon dioxid (gm.).

Weight of water (gm.)

Percentage of carbon

Percentage of hydrogen

Quercetin.

1514

32«3

044S

0.1570

.3400

• 0537
59.06

3-83

Theory requires: For quercetin, carbon 59.59 per cent, hydrogen 3 .34 per cent; for penta-acetylquercetin,
carbon 58.59 per cent, hydrogen 3.90 per cent.

The glucosid is not one of the well-known ones but bears considerable
similarity to one which Heyl ^ recently isolated from the pollen of rag-
weed, probably Ambrosia artemisiijolia L., although he gives only the
common name.

SUMMARY

In accord \vith the expectation that the broAvn-husked t5'pe of maize
would be found to contain a flavonol, we have been able to isolate from
brown husks both free quercetin and a quercetin glucosid of which a
further investigation will be made.

The two compounds in question are both lemon yellow in color. If
they account for the truly brown color of the husks of this type, it must
be through their tinctorial quality, probably through their adsorption
on some colloid component of the brown tissues.

It is very probable that the quercetin glucosid is the counterpart in
the brown type of the anthocyanin of the purple type. The pigment
of the latter will probably be found to be allied to cyanin.

I RiDGWAY, Robert, color standards and color nomenclature. 43 p., 53 pi. (col.) Washington,
D. C. 1912.

' Heyl, Frederick W. the yellow coloring substances of ragweed pollen. In Jour. Amer.
Chem. Soc, v. 41, no. 8, p. 1285-1289. 1919.

BIOLOGICAL ANALYSIS OF THE SEED OF THE GEORGIA
VELVET BEAN, STIZOLOBIUM DEERINGIANUM

By Barnett Sure; and J. W. Read, Laboratory of Agricultural Chemistry, University

of A rkansas

The velvet bean, Stizolohium deeringianum Bort.,is annually becoming
more important in southern agriculture, and the acreage planted to this
crop in the cotton belt is continually mcreasing. From 1915 to 1917 it
is estimated that the area increased from less than 1,000,000 acres to
more than 5,000,000 acres. The acreage in 191 7 was 1 19 per cent greater
than in 19 1 6. It is the most vigorous growing annual legume in the
United States ; and on account of its very rank growth and the common
practice of cultivating it v/ith the corn crop it is chiefly used as a winter
pasture for cattle and hogs, although much larger quantities of the
beans are harvested from year to year and ground, either with or with-
out the pods, for market purposes. Harvesting with the com crop for
use as silage is also growing in favor.

Because of the rapidly increasing interest in this crop as a feed and
its very considerable promise in this respect, particularly to the South,
it occurred to one of the authors^ that a biological analysis should give
very fundamental information as to how the velvet bean might best be
utilized for feeding purposes. Accordingly the Georgia Velvet Bean,
commonly know as the Early Speckled, was chosen for our studies be-
cause of its early maturity, general popularity, and adaptability to the
more northern as well as to the other sections of the cotton area.

The Georgia velvet bean seed has a very tough, hard hull which con-
stitutes 12 per cent of the whole seed. In grinding the beans it was
found impossible to grind the hulls in a satisfactory manner; conse-
quently these were sifted out. The experiments reported in this paper
were conducted with hulled seed. It was later found, however, that
after the sifted hulls had been dried on a steam bath for from six to eight
hours they could be ground ; and experiments were later inaugurated,
introducing the hulls in the same proportions as they were found to
exist in the seed, the results of which will be reported later, together
with other data showing the supplementary relationships of the seed
to the leaf and the biological value of the whole plant. The nutritive
value of the seed and the whole plant in practical rations is also being
studied at the present time.

' Credit for the inauguration and outline of the velvet-been studies as approved under the Adams Fund
is due Prof. J. W. Read.

Journal of Agriculural Research, Vol. XXII, No.i

Washington, D. C. Oct. i, 1921

za Key No. Ark.-i

(5)

Journal of Agricultural Research voi. xxii. No. i

The experiments reported in this paper were conducted ^vith albino
rats, employing the standard technic adopted by the Department of
Agricultural Chemistry of the University of Wisconsin.

Preliminary experiments showed that young rats, 40 to 90 gm. in
weight, will exist only from 7 to 12 days on a diet composed solely of

Fig. I. — Gain in weight of lot 51 on ration of velvet beans, So per cent; butter fat, s per cent; No. 32 salts,
4 per cent; and dextrin, 1 1 per cent. The dextrin carried alcoholic extract of 10 gm. ether-extracted wheat
embryo. At point x 9 per cent of the velvet beans was replaced by 9 per cent casein.

the raw hull-less velvet bean seed and a liberal supply of distilled water.
The food consumption could be increased 50 to 60 per cent, however,
by feeding young animals the seed after it was autoclaved for one hour
at 15 pounds pressure. Consequently, autoclaved velvet beans were

Fio. 2. — G^a in weight of lot 52 on ration of velvet beans, 60 per cent; butter fat, s per cent; No. 32
salts, 4 per cent; casein, s per cent; and dextrin, 26 per cent. The dextrin carried alcoholic extract of
10 gm. ether-extracted wheat embrvO,

used in all- this work. In order to determine whether the water-soluble
vitamin was destroyed in the process of autoclaving, controls were
run with uncooked beans.

Even when fed upon autoclaved beans, ammals, although consuming
considerably more of the seed, existed only for a period of 17 to 21

Oct. 1,1921 Biological Analysis of Georgia Velvet Bean j

days when the diet was composed solely of the seed. On diets com-
posed of 80 per cent velvet bean with 20 per cent dextrin, and 60 per
cent velvet bean with 40 per cent dextrin, eight animals, having an
initial weight of 50 to 60 gm., maintained their weight for a period of eight
weeks but made no growth. Likewise, when the velvet bean formed
60 to 40 per cent of the ration, respectively, as a source of protein, in
the presence of all the other dietary factors, no growth resulted, but
all the animals maintained their body weight for a period of six weeks,
indicating that the proteins in the seed are deficient.

Since these experiments lasted for periods ranging from six to eight
weeks only, no charts were prepared illustrating the points mentioned.
All the rest of our findings are illustrated in figures i to 15.

When velvet beans formed 80 per cent as the source of protein in the
ration (fig. i), very little growth resulted. Altliough at point xg per
cent of the beans was replaced by 9 per cent casein, no appreciable
change in the character of growth ensued, lack of response to the addi-
tion of purified casein being due, as it will be noted from the following
graphs, to the injurious effect of this high plane of velvet bean intake.

Velvet beans fed at a 60 per cent level as a source of protein, supple-
mented with 5 per cent casein, produced a fair amount of growth (fig. 2).

When, however, 40 per cent velvet beans was the source of protein
and the ration was further fortified with 9 per cent casein, the two
females made normal growth for a period of four months and the two
males grew at a rate even beyond the expectation curve (fig. 3). Rat
211 was unable to rear her young, although her litter was reduced from
nine to four.

Figure 4 shows that young animals are unable to make any growth
on a ration composed of 80 per cent velvet beans as the source of salts.

When 40 per cent velvet beans served as the source of salts, some
little growth occurred during the first 10 weeks (fig. 5). It is evident,
then, that at least part of the failure of lot 61 (fig. 4) must be ascribed
to the harmful effect of the higher plane of velvet bean intake. A striking
change in the character of growth is apparent when at point x 4 per
cent of dextrin was replaced by 4 per cent of salt mixture No. 32.^

When I per cent sodium chlorid (NaCl) and 1.5 per cent calcium
carbonate (CaCOg) replaced salt mixture No. 32 in the ration, very
good growth was obtained for a period of three months (fig. 6) , indicating
that the calcium, sodium, and chlorid ions furnish the necessary mineral
supplements in the velvet bean seed.

Figure 7 shows that when 1.5 per cent calcium carbonate alone replaces
salt mixture No. 32 only a little growth results.

* Steenbock, H., and Gross, E. G. pat soLUBtE vitamine. n. the fat-soi,ubi,e vitamine con-
tent OP ROOTS together with some observations on their "WATER-SOtUBLB VITAMINE CONTENT. In
Jour. Biol. Chem., v. 40, no. 2, p. 505. 1919.

Journal of Agricultural Research voi. xxn. no. i

Figures i to 7 have indicated that velvet beans are detrimental to
young experimental animals when fed at an 80 per cent level. The
experiment on which figure 8 is based corroborates that fact. Although

Fig. 3. — Gain in weight of lot 53 on ration of velvet beans, 40 per cent; butter fat, s per cent; No. 3a
salts, 4 per cent; casein, 9 per cent; and dextrin, 42 per cent. The dextrin carried alcoholic extract of
10 gm. ether-extracted wheat embryo. Y indicates point at which young were littered.

when 80 per cent velvet beans served as a source of the fat-soluble
vitamin, two animals made a fair amount of growth for a period of four

SO
60
SO

Fig. 4.— Gain in weight of lot 61 on ration of velvet beans, 80 per cent; butter fat, 5 per cent; casein, s
per cent ;and dextrin, 10 per cent. The dextrin carried alcoholic extract of 10 gm. ether-extracted wheat
embryo.

months; two rats died after six weeks. The failure of these two ani-
mals can not be ascribed to the low concentration of the fat-soluble
vitamin in the seed, since lot 70 (fig. 11) made normal growth for a period

%244

-*^

A24/

,l>^

^242

Oct. 1,1921 Biological Analysis of Georgia Velvet Bean

of over five months when only 20 per cent velvet beans served as a
source of that vitamin.

200

/80

/60

/20

/OO

Fig. 5. — Gain in weight of lot 69 oa ration of velvet beans, 40 per cent; butter fat, 5 per cent; casein, 9
per cent; and dextrin, 46 per cent. The dextrin carried alcoholic extract of 10 gm. ether-extracted wheat
embryo. At point x 4 per cent dextrin was replaced by 4 per cent No. 32 salts.

A considerable improvement in the character of growth is obtained
when the plane of intake of velvet beans used to supply the fat-soluble
vitamin is reduced from 80 to 60 per cent (fig. 9) .

)

.>^

B—H-

0274

*Z7S

^76

Fig. 6. — Gain in weight of lot 75 on ration of velvet beans, 40 per cent; butter fat, s per cent; sodium
chlorid, i per cent; clacium carbonate, 1.5 per cent; casein, 9 per cent; and dextrin, 43.5 per cent. The
dextrin carried alcoholic extract of 10 gm. ether-extracted wheat embryo.

When the level of velvet bean intake was reduced to 40 per cent,
normal growth was obtained. Rat 270 failed to rear her young, although
her litter was reduced from eight to four (fig. 10).

Journal of Agricultural Research

Vol. XXII, No. I

Even when the seed was reduced to as low a plane of intake as 20 per
cent, it served as a very efficient carrier of tlie fat-soluble vitamin. It
is also apparent that autoclaving for one hour at 15 pounds pressure had

Fig. 7. — Gain in weight of lot 92 on ration of velvet beans, 40 per cent; butter fat, 5 per cent; calcium
carbonate, 1.5 per cent; casein, 9 per cent; and dextrin, 44.5 per cent. The dextrin carried alcoholic extract
of 10 gm. ether-extracted wheat embryo.

no deleterious effect on this vitamin. Although excellent growth was
obtained on this ration, mother rats No. 277 and 278 failed to rear their
young in every case even when their litters ranging from 7 to 10 were
reduced to only 4 (fig. 11).

Fig. 8. — Gain in weight of lot 63 on ration of velvet beans, 80 per cent; No. 32 salts, 4 per cent; casein,
5 per cent; and dextrin, 11 per cent. The dextrin carried alcoholic extract of 10 gm. ether-extracted embryo.
D indicates point at which rat died.

Figure 12 shows that on reducing the level of velvet bean intake to
10 per cent as a source of fat-soluble vitamin the character of growth
is considerably impaired.

When 80 per cent velvet beans was used to supply the water-soluble
vitamin very little growth resulted, one animal dying after three weeks
on this ration (fig. 13).

oct.i. I92I Biological Analysis of Georgia Velvet Bean

-^bl

Fig. 9. — Gain in weight of lot 67 on ration of velvet beans, 60 per cent; No. 32 salts, 4 per cent; casein,
5 per cent; and dextrin, 31 per cent. The dextrin carried alcoholic extract of 10 gm. ether-extracted
wheat embryo. At point x 4 per cent dextrin was replaced by 4 per cent additional casein.

GM.

240

220

Fig. 10. — Gain in weight of lot 67 on ration of velvet beans, 40 per cent; No. 32 salts, 4 per cent; casein,
9 per cent; and dextrin, 47 per cent. The dextrin carried alcoholic extract of 10 gm. ether-extracted wheat
embryo. Y indicates point at which young were littered.

Journal of Agricultural Research voi. xxn, no.

Sixty per cent of velvet beans used to supply the water-soluble vitamin
allowed only a very small amount of growth (fig. 14).

G/^.

Fig. II. — Gain in weight of lot 70 on ration of velvet beans, 30 per cent; No. 32 salts, 4 per cent; casein,
12 per cent; and dextrin, 64 per cent. The dextrin carried alcoholic extract of 10 gm. ether-extracted wheat
embryo. Y indicates point at which young were littered.

In order to determine whether the water-soluble vitamin was destroyed
during the process of autoclaving, 40 per cent velvet beans was fed un-
cooked. Very little growth resulted, nor was there any improvement

SA7.

Fig. 12. — Gain in weight of lot 90 on ration of velvet beans, 10 per cent; No. 32 salts, 4 per cent; casein,
16 per cent; and dextrin, 70 per cent. The dextrin carried alcoholic extract of 15 gm. ether-extracted wheat
embryo.

in the character of growth when, at point x, lo per cent dextrin was
replaced Avith lo per cent of an alcoholic extract of ether-extracted wheat
embryo. It will be noted that after point x this ration is identical with

Oct. 1,1921 Biological Analysis of Georgia Velvet Bean

that given to lot 53 (fig. 3) with the exception that lot 86 received the
beans raw while lot 53 received the beans cooked. The striking dif-
ference in the character of growth obtained in these two experiments
must be attributed to the fact that the velevt bean seed uncooked is
either toxic or indigestible at a concentration as low as 40 per cent. The

/20
/OO

Fig. 13. — Gain in weight of lot 62 on ration of velvet beans, 80 per cent; butter fat, s per cent; No. 32
salts, 4 per cent; casein, s per cent; and dextrin, 6 per cent. D indicates point at which rat died.

cause of the deleterious effect of the raw seed is being studied and will
be reported later.

DISCUSSION

The Georgia velvet bean, Early Speckled variety, has been found to
be injurious when fed in the raw condition at as low a level as 40 per cent
intake. This has been evidenced from an experiment where 40 per cent

^..^

(

Si^"'

245

i-..^

--^

WeEKS

Fig. 14. — Gain in weight of lot 74 on ration of velvet beans, 60 per cent; butter fat, 5 per cent; No. 32
salts, 4 per cent; casein, 5 per cent; and dextrin, 26 per cent. At point x 4 per cent dextrin was replaced
by 4 per cent additional casein.

velvet beans uncooked formed the source of water-soluble vitamin
(fig. 15). Growth was inhibited during the first six weeks of experi-
mentation, after which time lo per cent dextrin was replaced by an
alcoholic extract of lo gm. ether- extracted wheat embryo. This addi-
tion of the water-soluble vitamin should have rendered the ration entirely
satisfactory, judging by the character of growth obtained in a duplicate
experiment where the beans were furnished cooked (fig. 3).

Journal of Agricultural Research

Vol. XXII, No. I

The nature of the possible toxicity of the velvet bean has been recently
suggested by Miller * to be due to dihydroxypehnylalanine.

Cooking the seed at 15 pounds pressure for one hour destroyed for
the most part its harmful effects, but there was still some injury when
fed cooked at as high a plane of intake as 80 per cent. When 80 per cent
of the velvet bean served as a source of protein, little growth resulted,
nor was there a response obtained after 9 per cent of the seed was replaced
by 9 per cent of casein, although when only 40 per cent velvet bean was
served as a source of protein, supplemented with the same amount of
casein, excellent growth was obtained. Unpublished data in this labo-
ratory show that the better growth on the lower level of seed intake is
not to be attributed to the higher intake of dextrin. Additional evidence
is apparent from the fat-soluble vitamin experiment that when cooked

/GO

/20

/OO

So
60

Fig. 15. — Gain in weight of lot 86 on ration of velvet beans (uncooked) 40 per cent; butter fat, 5 per
cent; No. 32 salts, 4 per cent; casein, 9 per cent; and dextrid, 42 per cent. At point x 10 per cent dex-
trin was replaced by 10 per cent of an alcoholic extract of ether-extracted wheat embryo.

velvet beans are fed at an 80 per cent level some injury is still produced.
Reduction of the plane of intake from 80 to 40 per cent results in con-
siderable improvement in growth.

That the velvet bean seed is very rich in the fat-soluble vitamin is
evident from the fact that normal growth was obtained for a period of
over five months when only 20 per cent of the seed served as the source
of this syndrome. Reduction of the plane of velvet-bean intake to 10
per cent resulted in inferior growth. The fact that considerably inferior
growth was obtained on lower levels of seed intake with larger amounts
of casein and dextiin precludes, we believe, the possibility that our
casein and dextrin might have furnished appreciable amounts of the
fat-soluble vitamin at the higher levels of seed intake, where we had
remarkable success. It is also apparent from these experiments that
autoclaving the seed for one hour at 15 pounds pressure has no deleterious
effect on the fat-soluble vitamin.

-s

.^"^

rs<

^llr^

'-3

2
meAS

1 Miller, Emerson R. DrHVDRoxYPHENYLALANiNe, a constituent of the velvet bean.
Biol. Chem., v. 44, no. 2, p. 481-486. 1920.

In Jour.

Oct. 1,1921 Biological Analysis of Georgia Velvet Bean 15

The hull-less seed contained 27.5 per cent protein and therefore furnished
16.5 per cent protein when fed at a 60 per cent level ; however, this amount
of protein was inadequate for growth even though all the other factors
in the diet were rendered satisfactory by the addition of isolated purified
food substances.

Recently Johns and Waterman ^ have isolated two globulins and an
albumin from the Georgia velvet bean and have reported analytical data
on their composition, using the Van Slyke ^ method of protein analysis.
Their results show that, with the exception of the albumin, which is low
in histidin, the three proteins of the Georgia velvet bean are quite satis-
factory for their diamino-acid content. However, since we have insuffi-
cient chemical data on the amino-acid content of the Georgia velvet bean
no correlation can be made at present between the chemical composition
and the biological response of this seed. The nature of the amino-acid
deficiencies is being investigated.

The velvet-bean seed has also been found to be deficient for growth in
the character of its salts ; however, sodium chlorid and calcium carbonate
seemed to replace salt mixture No. 32 satisfactorily.

The concentration of the water-soluble vitamin in the seed has been
found to be low. Unpublished data show that the addition of the
ground hulls in the same proportions as they occur in the whole seed
does not improve the water-soluble vitamin content. It is not apparent
from these experiments whether this vitamin was in any way destroyed
during the process of autoclaving, since the seed was extremely injurious
when fed uncooked.

SUMMARY

(i) The Georgia velvet bean seed, Early Speckled variety, when fed
raw was found injurious to young rats even when constituting only 40
per cent of the total ration.

(2) Autoclaving the seed for one hour at 15 pounds pressure destroys
most of this injury, so that it is possible to include 60 per cent of the
bean in a ration. A ration composed of 80 per cent velvet bean cooked
still shows some harmful effects.

(3) This seed, unlike most seeds so far studied biologically, is very
abundant in the fat-soluble vitamin. The fat-soluble vitamin as it exists
in this seed is quite stable after the seed is autoclaved for one hour at 15
pounds pressure. The water-soluble vitamin, however, is of low con-
centration in the hulled seed.

(4) Both the proteins and salts of the velvet bean have been found to
be of deficient character for growth.

1 Johns, Carl O., and Waterman, Henry C. some proteins from the Georgia velvet bean, stizo-
LOBIUM deeringianum. In Jour. Biol. Chem., v. 42, no. i, p. 59-69.

2 Van Slyke, Donald D. the analysis of proteins by determination of the chemical groups
CHARACTERISTIC OF THE DIFFERENT AMINO-ACIDS. In Jour. Biol. Chem., V. 10, no. I, p. 15-SS, 2 fig. 1911.

EFFECT OF SOIL TEMPERATURE UPON THE DEVELOP-
MENT OF NODULES ON THE ROOTS OF CERTAIN

LEGUMES

By Fred Reuel Jones, Pathologist, Office of Cotton, Truck, and Forage Crop Disease
Investigations, Bureau of Plant Industry, United States Department of Agriculture,
and W. B. Tisdale, Instructor in Plant Pathology, University of Wisconsin

INTRODUCTION

During a search for the cause of a diseased condition of alfalfa under
observation in 191 7 and 19 18 the senior writer was led by observations
contributed by H. L. Westover, of the Office of Forage Crop Investiga-
tions, to believe it likely that soil temperature, within the range which
occurs in cultivated fields, affects the initiation and the development of
nodules on the roots of alfalfa and perhaps all other legumes to such a
degree that the assimilation of nitrogen by these plants is greatly modi-
fied by this lactor during the summer. The probable importance of such
an effect of temperature, should it be demonstrated, upon the develop-
ment of alfalfa and other legumes and especially its possible relation to
the disease in question seemed adequate reasons for making a beginning
at the experimental determination of the facts. Experimental methods
suitable for the performance of this work had already been highly devel-
oped in the course of the study of soil-inhabiting plant parasites at the
University of Wisconsin. Thus it came about that the collection of the
following data was begun at Madison by the junior author in 191 7 and
continued by both authors in 191 9 and 1920. A temporary suspension
of the work is the immediate reason for the publication of this prelimi-
nary report.

In the beginning, interest was centered upon ascertaining to what
extent soil temperature determined the number of nodules which any of
a selected group of legumes might develop. Later the size and composi-
tion of the nudoles appeared more significant than the number. Finally,
it is seen that soil temperature probably affects profoundly the rate of
nitrogen fixation within the nodules of the legumes studied and its
assimilation by the plants. A complete demonstration of such an effect
and a quantitative determination of its amount remains for the future.
During the progress of the work certain striking effects of soil temperature
upon the development of the plants quite apart from any relation to
nodule formation have been noted.

Journal of Agricultural Research, Vol. XXII, No. i

Washington, D. C. Oct. i, 1921

zo Key No. G-24S

54817°— 21 2

(17)

1 8 Journal of Agricultural Research voi. xxii. No. x

ENVIRONMENTAL FACTORS WHICH HAVE PREVIOUSLY BEEN FOUND
TO MODIFY THE FORMATION AND DEVELOPMENT OF NODULES

In the extensive studies which have been made of the conditions
which may favor or hinder the development of nodules, no one, so far
as the writers are aware, has concerned himself with the factor which
is considered here — ^namely, the temperature of the soil. There are a
number of factors, however, which are known to have very much influence
upon nodule development, and two of these which may have become
modified by our experimental methods for controlling soil tem-
perature must be considered. These are soil moisture and the concen-
tration of nitrates.

With regard to soil moisture, there appears to be both observational
and experimental data which indicate that high soil moisture tends to
increase nodule formation. Gain * notes that peas grown in wet soil
have far more nodules than those grown in drier soil close by. Wilson ^
in his experimental work reports that wet soil induces the formation of
a greater number of nodules on soybeans. Fortunately, in experimen-
tal work with controlled temperatures, it is comparatively easy to main-
tain soil moisture at a predetermined point with very slight fluctuation.
In the preliminary experiments, although no attempt was made to
control soil moisture exactly, it is not believed to have fluctuated suffi-
ciently to affect results appreciably. In the later work, soil moisture
was maintained in each series at one-half the moisture-holding capacity
of the soil used (14 per cent of the wet weight) by weighing the pots
each day, if necessary, and restoring the water lost by evaporation and
transpiration. It is believed that this method kept variation in soil
moisture within such very narrow limits that this factor could not have
produced appreciable variation in nodule formation.

That the amount of nitrate present in the soil affects nodule develop-
ment, completely inhibiting it when high concentrations have been
reached, has been demonstrated by several investigators. Wilson ^ has
added a considerable number of nitrates to soils in different amounts to
determine the concentration at which nodule formation is inhibited by
each of the compounds. Although complete inhibition is effected only
at concenti"ations which are not likely to occur in normal soils, the
marked effect of variations is so well attested that any differences
arising unavoidably during an experimental series must be taken into
account in the consideration of results.

The control of the concentration of nitrates in the soil solution in a
soil held at different temperatures offers difficulties which can be over-

1 Gain, Edmond. influence de i.'humidite sur le developpement des NODosixfes des l^gumineu-
SES. In Compt. Rend. Acad. Sci. [Paris], t. ii6, no. 24, p. 1394-1397. 1S93.

2 Wilson, J. K. physiological studies of baollus radiocola of soy bean (soja max piper) and
OF factors influencing nodule production. N. Y. Cornell Agr. Exp. Sta. Bui. 386, p. 363-413, fig.
80-94. 1917.

Oct. 1. 1921 Effect of Soil Temperature on Development of Nodules 19

come only within certain limits. As was expected in advance, the rate
of nitrification in soil differs greatly at the different temperatures,
producing greatly different concentrations within a short time after a
series of plants have been started. In addition, there is soon consid-
erable difference in the size of the plants at the different temperatures
and a consequent difference in ability to absorb nitrates. The varia-
tions which arise from these causes can be limited somewhat by the use
of soil low in total nitrogen, thus making impossible the accumulation
of large amounts of nitrates in any case. Variations in the concentra-
tion of nitrates which have been observed in the experimental work
described here will be noted later, and their possible effect upon the
results will be discussed.

APPARATUS AND METHODS

The apparatus used for the control of soil temperature in these experi-
ments is that which has been used in the Laboratory of Plant Pathology
at the University of Wisconsin for several years and needs no new
description.^ In all cases plants were grown in metal cans 6 inches in
diameter and 10 inches deep. The number of plants which could be
grown in each can vnthout serious crowding of roots was 3 for soybeans,
5 for peas, and 10 for clover and alfalfa. The soil used was a sandy
loam from a pasture which had never been cultivated. To this was
added about an equal weight of sand in order that the total nitrate
content should be kept low and that the mechanical condition of the
soil should permit the easy removal of the roots. The temperatures
noted in the different series were those at which the water was main-
tained in the tanks in which the cans were set. Fluctuations of tempera-
ture did not often exceed 1° C. from those given in the tables, and
were of only a few hours' duration. Although record was made twice
daily of the actual temperatures, it is not believed that a computation
of the mean temperature from these figures would give a figure more
significant than tlie convenient even numbers used here. It should also
be noted that although the surface of the soil was insulated to some
degree from loss of heat and moisture by the use of mineral wool, never-
theless at the higher temperatures the surface soil to the depth of about
I inch was usually cooler by i to i}4° than the water. However, it is
believed that the larger part of the roots and nearly all the nodules were
sufficiently deep in the soil below this cooler layer, so that error arising
from this source is not considerable.

Water was supplied through a glass tube which passed to the bottom
of the metal can where it entered an inverted unglazed flower pot 3
inches in diameter, which acted as a reservoir. In the last series the

> Jones, 1,. R. soil temperatures as a factor in phytopathology. In Plant World, v. 20, no. 8,
p. 229-237, 2 fig. 1917. lyiterature cited, p. 236-237.

20 Journal of A gricultural Research \o\. xxii. No. i

inverted pot was placed about 3 inches below the surface instead of at
the bottom in the hope of maintaining a more uniform and rapid dis-
tribution of moisture. This appears to have been an unfortunate
change in method, since at the higher temperatures roots tended to col-
lect around these pots, where they apparently developed more ex-
tensively and produced more nodules than they had in the previous
series. Inasmuch as no accumulation of roots took place at lower tem-
peratures, it is not easy to explain this fact.

Attention should here be drawn to the fact that two distinctly dif-
ferent methods of securing data have been used. In the preliminary ex-
periments, the plants were first grown in the cans at ordinary greenhouse
temperature for about two weeks before inoculation was made, with
the suitable strain of Bacillus radicicola Beyr., by pouring a water sus-
pension of the organism around the base of the plants, and the cans were
placed in the tanks adjusted at the predetermined temperatures. In the
later series the plants were grown from seed in inoculated soil held at the
required temperatures from the beginning. Several reasons led to the
change of method. In the first place, one could not be certain that the
bacteria poured around the plant in the first instance would become
rapidly distributed through the soil at all temperatures. This inequality
in rate of distribution might affect the number of infections and hence the
number of nodules formed. At least it might tend to limit the formation
of nodules to the roots near the surface of the ground where temperature
is less exactly controlled. A second objection to this method appeared
when the marked effect of temperature upon the morphology of the roots
themselves was observed. The number of root hairs through which
infection has been found to take place is much greater at lower tempera-
tures than at higher. In view of the possible effect of this difference
it appeared preferable to grow the plants from the beginning in inoculated
soil at the designated temperatures, even though the plants thus produced
would necessarily vary considerably in size. Data obtained by each of
these methods will be presented.

MEASUREMENT OF EFFECT OF SOIL TEMPERATURE UPON NODULE

FORMATION

When the experiments were begim it was assumed that different tem-
peratures, if they are at all potent, would produce such a marked effect
upon the number of nodules that count alone would give a significant
expression of results. This expectation was fostered by the fact that
Wilson 1 and nearly all previous investigators have used numbers to
express similar experimental results. It will be seen from data given
later that this hope was early disappointed. Different temperatures
usually seemed to affect number not nearly so much as rate of develop-

» Wilson, J. K. op cit.

Oct. 1, 1921 Effect of Soil Temperature on Development of Nodules 2 1

ment and size. In fact, in some instances, volume of nodular tissue
seemed in inverse ratio to number. With most legumes it is not easy to
get an accurate dry-weight determination of small nodules, because these
occur as swellings so closely attached to the root that it is hardly feasible
to separate them from the true root tissue. The one species tried which
gave least trouble from this source by reason of the distinct separation
of its nodules from the root, even at early stages of development, was
the soybean. For this reason, it alone was used in the final series recorded
here.

Of course it was soon realized during the progress of the work that vol-
ume of production of nodules was only an easily observed index, significant
chiefly in so far as it revealed important eff"ects of temperature upon the
physiological processes which are dependent upon the nodular structures.
It would be of greater interest, for example, to measure the amount of
nitrogen fixed in these nodules produced at different temperatures and
that portion which becomes available to the plant for use in its vital pro-
cesses. The demonstration of an important limiting effect of temperature
upon nitrogen fixation would be of no inconsiderable importance. Such
an effect would probably be indicated by large differences in size of
nodules, though it might occur without the appearance of such difference.
In any case it seems easily possible to determine approximately the effi-
ciency of nodules in the fixation of atmospheric nitrogen by growing
parallel series of plants, inoculated and uninoculated, in the same kind
of soil and at the same soil temperatures. If the amount of nitrogen in
the inoculated plants (aside from that foimd in the nodules on those
plants) is greater than in the uninoculated, the gain must be credited to
the efficiency of the nodules. The gains thus found should be an accurate
measure of the effect of soil temperature upon the fixation of available
nitrogen in the legume used in the experiment, and a comparison of this
gain with the weights of the nodules found on the inoculated plants
should give an approximate idea of the relation existing between effici-
ency of fixation of available nitrogen and volume of nodules. In the
last series recorded an attempt was made to carry out this experiment
with the soybean plant. Unfortunately some of the uninoculated plants
in the series became inoculated during the experiment and developed a
few nodules, thus making it necessary to discard the data so far as these
controls are concerned. Thus an exact determination of the extent of
the effect of soil temperature upon the fixation of nitrogen in the nodules
of legumes remains to be made. For the present we can only ascertain
the dry weights of the nodules themselves as they are found at the end
of a period of time and determine the amount of nitrogen found within
them.

22 Journal of Agricultural Research voi. xxii. no. i

DISCUSSION OF THE LEGUMES USED AND THEIR BEHAVIOR UNDER
THESE EXPERIMENTAL CONDITIONS

Four legumes were selected for tlie soil temperature series requiring
four different strains of Bacillus radicicola for their inoculation. One of
these, the soybean, flourishes well at high soil temperatures; one, the
Canada field pea, requires a low soil temperature for good growth; and
red clover and alfalfa occupy intermediate positions.

A few of the more striking reactions of the plants in these series will be
noted. The Canada field pea does not flourish vigorously at a soil tem-
perature as high as 30° C. (Table I) and is intolerant of temperatures
above this point, maintaining roots only very close to the surface of the
soil. It is perhaps misleading to infer that the lower surface temperature
is alone responsible for the position of the roots, since in field plots in hot,
exposed positions the death of deeper roots and the formation of surface
roots has been noted in hot weather.

Perhaps the more striking effect of the series of soil temperatures upon
the soybean plants, aside from the fact of the wide range through which
it grows vigorously, is the effect upon the color of the foliage. After
the plants had become 5 or 6 inches tall, in both series the leaf color was
much darker at the two ends of the series, especially at 30° C. and above,
than at 2 1° and 24°. This difference persisted, tending rather to increase
as long as the plants were grown.

One striking difference in behavior between peas and soybeans on the
one hand and clover and alfalfa on the other was noted in this series.
The annuals formed a rather regular series of plants as judged by appear-
ance (PI. 1) and also by dry weights (Table I). But with alfalfa and
red clover the seedlings at the lower temperatures, 12° and 15° C, though
little delayed in starting, remained small Alpine plants with thick dark
green leaves and \vith much red color in the petioles; whereas at 18° the
plants were more nearly what may be termed "normal" plants, larger,
with fairly long petioles containing less red color. ^

EXPERIMENTAL DATA

EFFECT OF SOIL TEMPERATURE UPON THE NUMBER OF NODULES FORMED

As previously noted, in the fiirst two preliminary trials the seeds were
planted in soil in the metal cans and grown for about 10 days at green-
house temperature (about 22° to 23° C.) before they were inoculated
with the suitable strains of Bacillus radicicola and placed in the tanks
adjusted at the temperatures designated. When it was believed that
sufficient time had elapsed for infection at all temperatiu-es, the tops
were cut from the plants, dried, and weighed. The roots were carefully

1 Since this was written clover and alfalfa have been grown under similar conditions at controlled tem-
peratures. The marked dwarfing of plants at 15° and 12° C. was found to disappear when the plants became
older, and especially later in the spring when light intensity became greater.

Oct. 1. 1921 Effect of Soil Temperature on Development of Nodules 23

washed from the soil and the nodules were counted. The count obtained
is given in Table I. In the two later series the seeds were planted in
soil which had already been placed in the tanks adjusted to the tem-
peratures designated. The air temperature ranged from 14° to 18°.
The counts obtained in these series are given in Table 11.

Table I. — Average number of nodules produced on plants 26 days old grown at a soil
temperature of about 20° C.for 10 days, after which mociilation was made and the tem-
perature of the soil was maintained as indicated

Temperature.

"C.
10 to 12

Alfalfa.

plants.

IS
35
18
16
5

plants.

Red clover.

plants.

17
27
40
69

75
4

plants.

7
16

47
4

75
6

Soybeans.

6
plants.

46
61

37
35

6
plants.

14
25
33
28

Field peas.

plants.

27
31
37
43
128

plants.

21
24

o The plants did not survive.

Table II. — Average number of nodules produced on plants grown at the soil temperatures

designated

Temperature.

Alfalfa.

20 plants 63
days old.

Red clover.

10 plants 63
days old.

Soybeans.

6 plants 63
days old.

9 plants 55
days old. a

Field peas.

S plants 32
days old.

5 plants 52
days old.

I. 19
1.6
16. 7
8.0

3-4
II. 6

10.7
3-5

1.8

5-0
12.5
24. 6
17.4

II- 5
8.9

5-3

o. o
6. I

5-9
4.0

II- 3
8.0

5-8

8.8

13.6

13.0
14.7
19.7
18.8
16.4
20.0
12.4

3-6

8.0

14. o

25.8

30.0

4.0

2.6
9.0

27.2
23.8
13.0

58.0

o- The larger number of nodules on the plants 55 days old as compared with those on plants 63 days old
is believed to be due largely to the fact that this series of plants was grown in spring, when longer days
promoted a far more vigorous growth than was produced by the other plants, which were grown in winter.

Although it will be seen at once that the data in the two tables are not
strictly comparable, nevertheless some temperature effects upon number
appear. Most conspicuous of all is the greatly increased number upon
peas near the upper thermal limit. But this increased number is accom-
panied by a more than proportionate decease in size. No plant has been
found to produce large nodules at 30° C. or above. Clover and alfalfa
tend to produce their largest numbers of nodules in the middle portion
of the range. Soybeans show no decided temperature effect at all, so

Journal of Agricultural Research voi. xxii. No. i

far as number is concerned. However unsatisfactory these figures may
be from several points of view,' nevertheless they establish one important
fact beyond reasonable doubt : Modified soil temperature ^vithin the range
which these plants can be expected to encounter in tlie field and even
within which they can be grown \vith vigor under experimental conditions
does not prevent the infection of roots by Bacillus radicicola and the
formation of considerable numbers of nodules. In other words, B. radi-
cicola, considered as a parasite, does not show the strongly marked inhi-
bition of its ability to infect roots of plants that has been found in a
number of fungus parasites.^

/Of-

__A{oho^.€^.^

' ... %

/o\

/■5

2/ 24 27

■PiG. I. — Comparison of dry weights of tops, roots, and nodules of soybeans given in Table HI, grcwn
during November, December, and January.

EFFECT OF SOIL TEMPERATURE UPON THE PRODUCTION OF NODULES AS
MEASURED BY DRY WEIGHTS

Although it is clear that soil temperature does not, in most cases,
greatly modify the number of nodules produced, it was obvious from the
very beginning that the size to which they developed was markedly and
consistently affected. Such effect is shown graphically in Plate 2, where
nodules from an equal number of plants are shown placed in rows and in
Plate 3, where nodules from a larger number of plants are placed in tubes
of equal diameter. Dry weights of the nodules shown are given in Table
III. Data are given for the soybeans only for reasons already mentioned,
but, judging by visual evidence obtained in studying all four legumes
used, it is believed that all behaved in essentially the same manner.

I Johnson. James, and Hartman. R. E., influence of soii, environment on the root-rot of
TOBACCO. /» Jour. Agr. Research. V. 17, no. 2, p. 41-86, pi. 1-8. 1919. Literature cited, p. 84-86.

TiSDALE, W. H. RELATION OF TEMPERATtree TO THE GROWTH AND INFECTING POWER OF FUASRIUM
UNI. In Phytopathology, v. 7, no. s, p. 356-360, i fig., pi. u. 1917.

Oct. 1, 1921 Effect of Soil Temperature on Development of Nodules 25

Now it would be expected, and it is clearly true, that plants grown in
soils held at such widely different temperatures would show in the given
time considerable difference in size and degree of maturity. The first
question which will be asked regarding this difference in nodule develop-
ment will be whether it does not correspond more or less approximately
with corresponding differences in root or shoot development. Does it
show a trend distinctly different from that of other portions of the
plant ?

When the figures given in this table are presented in graphs, the
differences in trend become obvious. A comparison of the weights of
the nodules with those of the roots (fig. i, 2) will show that in both

2/ 24 27 30

36 <^

Fig. 2. — Comparison of dry weights of tops, roots, and nodules of soybeans given in Table III, grown

during April and May.

series the maximum development of nodules occurs at 24° C, with
very slight development at the extremes, 15° and 36°. Root develop-
ment, on the other hand, rises much more rapidly at the lower temper-
atures and is maintained at the the higher temperatures, reaching a
maximum in the second series at a point 9° higher than that of the
nodules. Root development is far more uniform at all temperatures
than is nodule development.

A comparison of nodule development with shoot development (fig.i, 2)
shows that the effect of temperature upon the development of the two
structures is quite different. As with root development, shoot develop-
ment is relatively more vigorous at 15° and 18° C. than is nodule develop-
ment, which increases greatly at 21° and reaches a maximum at 24°.
"When at 27° the v/eight of nodules is beginning to diminish, that of
shoots maintains its level or increases. Through the higher temperatures
weight of nodules falls off rapidly, while that of shoots remains at the
high level.

Journal of Agricultural Research voi. xxii, No. i

Table III. — Dry weight per plant of shoot, roots, atid nodules prodticed in 6j days in
the first series, grown in November, December, and January, and 55 days in the second
series, grown in April and May

Temperature.

°C

First series.

Shoot.

Gm.

o- '^33

. 410

• 432
.632

.875
.771
.818
.863
.996

Root. Nodules.

Gm.
O. 040
. IIO

•135
. 140

•131
.108
.096
.116
. 108

Gm.

O. 000
. 006
.008
•033
•043
.030
. 014
. 012
.005

Second series.

Shoot.

Gm.

0. 922
1.430

1. 710

1. 900

2. 620

2.540
2.440
2. 130

Root. Nodules.

Gm.

3. 184
.318
. 269
. 296
•342
. 296

•437
. 422

Gm.

O. 021
. 060
. 108

•145
.094
.089
. 076
. 042

3S^

5.<5.5

T"—

!:c.

. y

^ 0^

/ 'V/

"*^^^^'-

-„«»■— ""

^\1^

/S /3 2/ 24 27 30 33 36

Pig. 3. — Ratios ot weight of nodules to weight (A tops and of roots. First experiment.

In order to obtain a clearer view of the contrast between the effect of
temperature upon weight of nodules and that of roots and shoots, the
ratios of the weights of these parts of the plant have been determined and
plotted (fig. 3, 4). If there is a direct relation between development of
nodules and that of either the aerial or subterranean parts of the plants —
if nodule development is conditioned by top or root development quite
independently of the temperature factor which was varied in these
experiments-^then the ratio should be approximately constant, or at
any rate should be a straight line. A glance at the graphs shows that
this is not the case. The ratios when plotted produce curves which are
closely similar. No direct relation appears to exist between weight of
nodules and that of either tops or roots under the conditions of these
experiments. At 21° and 24° C. the weight of nodules is relatively
larger than at temperatures above or below this region. The wide dif-

Oct. I, I92I Effect of Soil Temperature on Development of Nodules 2 7

ference in the ratios and the consistent similarity of the curves can
hardly be explained otherwise than as a temperature effect upon nodule
development which is quite different from that upon development of
root or shoot.

EFFECT OF SOIL TEMPERATURE UPON THE COMPOSITION OF THE
INOCULATED PLANTS

In order that comparisons might be made of the amount of nitrogen
found in inoculated and uninoculated plants, total nitrogen determina-
tions were made of shoots, roots, and nodules of the plants grown in
each series. Since the uninoculated plants did not remain free from
nodules, the desired comparisons can not be made. Nevertheless the

\
%.03

y .

:5/ 4i

=^cc^

^^y^

cPy

'•x.^

/" \i

-, X

n^*^

"-.

sdQ.

12 /S /a 2/ 24 27 30 33 3$

0£G7?£SS C£A^r/S7?^£>£

Fig. 4.— Ratios of weight of nodules to weight of tops and of roots. Second experiment.

difference in nitrogen found were so marked in the series that the analyses
of one series, the last that was grown, is given.

Table IV. — Percentage of total nitrogen found in shoots, roots, and nodules of soybean
plants grown at a series of soil temperatures in April and May

Temperature

°c.

Shoots.

Roots.

2.42

2.89

2.86

3-40

4.27

2. 64

4. 22

2. 98

4-55

2.66

4.58

2. 62

3-98

2. 67

3-77

2.85

Nodules.

5-95
6.95
6.25

5-95
6.25
6.25
6. 00
5-70

The greatest differences in content of nitrogen are found in the shoots,
the roots being very uniform and the nodules hardly less so. It will be
seen that, generally speaking, the high nitrogen content of the top is

28 Journal of Agricultural Research voi. xxii, No. i

correlated mth the best development of nodules, though the curve
which would be produced by these figures when plotted in the manner of
the preceding data would not have the same shape. The largest amount
of notrogen is found at a higher temperature than the point at which
the largest dry weight of nodules was found. A rather sudden increase
in nitrogen at 21° C. as compared with 18° and a sudden fall at 33° as
compared with 30° has characterized the series obtained thus far. The
result of the analyses which have been made seems worth recording;
but whether the low nitrogen content of the plants grown at both ends
of the series is wholly due to the small nodules found on these plants,
and whether the high nitrogen content of plants in the center of the series
is due to large and presumably efficient nodules, likely as this connection
appears, remains to be determined by more refined methods.

DISCUSSION OF FACTORS THAT MAY HAVE HAD AN INFLUENCE UPON

THE DATA GIVEN

CONCENTRATION OF NITRATES IN THE SOII,

In view of the fact already discussed, that large amounts of nitrates
in the soil solution decrease nodule development and even inhibit it
before the concentration becomes great enough to injure the plant
directly, it is unfortunate that the soil used in these series should have
had as much nitrate as was found (Table IV), even though the largest
amounts are far below the inhibition point. There appears to be no
data available in literature whereby we may know what is the maximum
or the more usual amount of nodular structure formed on the roots of
any of the legumes. Although the amount of nodular structure which
peas may produce may be quite different from the amount which soy-
beans may produce under the most favorable conditions, yet it may be
worth while to record here that the writers have found in one instance
a variety of wrinkled peas producing at the blossoming stage nodules
whose dry weight was 2.2 times as great as that of the entire root system
(average of 25 plants) ; and in individual plants the ratio of weight
of nodules to roots was as high as 4.5 to i. However the ratio of weight
of nodules to tops in these plants was 0.085 to i , a ratio not much different
from that found under the best experimental conditions for soybeans
recorded here (fig. 3, 4).

However, the question of immediate interest here is whether or not
the nitrate content of the soil used in tliese series was greatly changed
at any of the temperatures at which it was held, and if there is any
evidence that this change was of sufficient size and in the right direc-
tion to indicate that it may have been responsible for the increased or
decreased nodule formation at this temperature. In order to obtain
information regarding the change which soil temperature maj'- have
produced in the series, nitrate nitrogen determinations were made by

Oct. 1, 1921

Effect of Soil Temperature on Development of Nodules 29

the colorometric method of a composite sample of the soil at the begin-
ning of the experiment and of a sample from two cans of soil at each
temperature when the plants were harvested. In addition, in order to
get some clue to intervening changes, an unplanted can of soil was kept
at each temperature from which a sample was taken at about the middle
of the period. The results obtained are shown in Table IV. The effect of
soil temperature does not appear to have been as definite and consistent
upon the concentration of nitrate nitrogen as was anticipated. Until
further data are available, it seems unwise to attempt to interpret the
results. However, the very absence of large and consistent modifica-
tion enables us to believe that this factor was not important in its effect
upon nodule development. The only point at which nitrate accumula-
tion became very large occurs in the second series where the unplanted
soil shows at the end of 26 days a very high nitrate content at 21°
and 24° C. If it is assumed that a similar concentration took place in
the planted pot at an early stage in the development of the plants be-
fore they were large enough to reduce it by absorption, it would be an-
ticipated that a reduction in nodule production would be found here.
In fact, however, this point of high nitrate formation is the point of
highest nodule production, just as it is in the first series where no evi-
dence of high nitrate content at any time was obtained.

Table IV. — Nitrate nitrogen in the soil in which the soybeans grew and also in

unplanted soil

First

series.

Seconc

series.

Temperature.

Planted soil.

Unplanted soil.

Planted soil.

Unplanted soil.

At start.

At end.

After 24
days.

At end.

At start.

At end.

After 26
days.

After 61
days.

°C.

P. p. m.

20. s
20.5

8.6

16.3
II. 4

14.4
8-3

3-3

20.0

20.5

8.6

16.5

10. 6

14. 0

20.5

8.1

19-3

20.5

18.0

124

20. 5

10. 6

II. 8

14.4

8.5

150

20.5

10. 6

13-9

9.2

14. 0

20. 5

16.9

13-9

8-3

4.8

20.5

3-3

15-1

3-6

4.8

5°

20.5

9-7

21.8

3-6

30.0

MOISTURK CONTENT OF THE SOU.

In view of the effect which high moisture content of the soil is reported
by Wilson ^ and others to have in increasing nodule production, the
moisture content of the soil in the later series was kept uniform at all

'Wilson, ]. K. physiologicai, studies of bacillus radiocola of soy bean(soja max piper) and
OP FACTORS INFLUENCING NODULE PRODUCTION. N. Y. Cornell Agr. Exp. Sta. Bui. 386, p. 363-413, fig.
80-94. 1917-

30 Journal of Agricultural Research voi. xxii. No. i

temperatures at one-half the moisture-holding capacity of the soil,
previously determined to be 14 per cent of its dry weight. In order to
get further evidence as to the extent of the effect of high moisture con-
tent, a single can containing three plants was placed at each temperature
in the last series with moisture content of 18 per cent of the dry weight —
a distinctly wet soil. Accidents which befell several plants in the series
produce irregularities in the figures which would require long explana-
tion. Suffice it to say here that though the tops were increased in size
there is no evidence that the nodules were increased either in number or
size. Apparently moderate differences in moisture content of the soil
were not large factors influencing results in the previous experimental
work where exact control of soil moisture was not accomplished.

HYDROGEN-ION CONCENTRATION OF THE SODv SOL,UTlON

Inasmuch as it was considered possible that the extreme temperatures
at which the soil was held might produce changes which would alter the
hydrogen-ion concentration of the soil solution, and hence the formation
and perhaps development of nodules, a determination of this environ-
mental factor was made toward the close of the last series described.
Samples of soil were taken from the unplanted pots at 15°, 24°, and
36° C. A determination of the hydrogen-ion concentration of the soil
solution of the three samples by the colorometric method gave identical
results, the Ph value being 6.3 in all three cases. Thus no evidence
was obtained that temperature had altered this important factor in this

series.

SUMMARY

(i) Preliminary studies have been made upon the effect of soil tem-
perature on the development of four legumes, alfalfa, red clover, field
peas, and soybeans, with special reference to its effect upon the infec-
tion of these plants by Bacillus radicicola and the subsequent develop-
ment of nodules. The larger part of the data were obtained by growing
plants in soil held at a series of temperatures 3° apart from 12° to 36° C.
The air temperature was uniform for all plants, ranging from 14° to 20°.

(2) As was anticipated, the four plants differed in their ability to
tolerate soil temperatures at the ends of the series. Peas were dwarfed
at 30° C, clover developed poorly at 36°, while alfalfa and soybeans
still grew very well at 36°. Soybean plants grown in the soils held at
12°, 15°, 33°, and 36*^ showed very dark green color of leaves, whereas
those toward the center of the series became progressively lighter, those
at 24° being lightest.

(3) With regard to the number of nodules formed on plants grown in
soil held at this series of temperatures, irregularities were found in each
series; but no large consistent differences were discovered, except that
at the extreme upper and lower limits at which a plant will survive the

Oct. 1, 1921 Effect of Soil Temperature on Development of Nodules 31

number is reduced, and that peas usually produced greatly increased
numbers at 30° C. All these species form nodules in soils at any tem-
perature at which the plant can make a growth that is at all vigorous.

(4) While variation in number was not consistent, size measured by the
average dry weight per plant of all those formed on a number of plants
was found to differ greatly and consistently within the series, at least so
far as the soybean plant was concerned. The maximum weight attained
on the soybean plant after a period of two months was found at a soil
temperature of 24° C. Examination of nodules on the roots of the other
legumes indicated that their maximum production occurred at about the
same temperature.

(5) Weight of nodules produced by soybeans was not found to be corre-
lated with the weight of tops or of roots through the series of tempera-
tures. Weight of tops was almost or quite as great at 30° to 36° as at
24° C, while weight of nodules declined rapidly at the higher tempera-
tures. Weight of roots likewise showed no such diminution at the higher
temperatures or even at the lower temperatures as did weight of nodules.
With the soybean plant, and to a much less marked degree with the other
plants, there was a correlation between weight of nodules and color of
plant, the largest weight of nodules occurring on plants with the palest
green color.

(6) Generally speaking, plants with large nodules had a higher percent-
age of total nitrogen in the tops, though this correlation is not exact.

(7) Factors of soil environment that are regarded as having an influence
upon nodule formation have been taken into account. Soil moisture has
been controlled within narrow limits. Concentration of nitrates and the
hydrogen-ion concentration of the soil solution have been recorded. It
is not believed that variations in any of these factors are to be regarded
as having produced the variations in nodule development recorded at the
different temperatures in these series.

PLATE I

A. — Alfalfa plants grown 63 days in soil held at the temperatures indicated.
B. — Red clover plants grown under exactly similar conditions with the alfalfa
plants shown in A.

Effect of Soil Tomporature on Development of Nodules

Plate I

Journal of Agricultural Researcli

Vol. XXII, No. 1

Effect of Soil Temperature on Development of Nodulos

Plate 2

^-n .>^ r^' •* ^

27"" 30° ^3°

Journal ot Agricultural Research

Vol. XXII, No. 1

PLATE 2

A. — Soybean plants grown 63 days in soil held at the temperatures indicated.
B. — Nodules from 6 soybean plants (only 5 plants at 30° C.) grown 63 days at the
temperattu-es indicated. One-half of the plants are shown in A.
54817°— 21 3

PLATE 3

A. — Soybean plants inoculated with Bacillus radicicola contrasted with uninoculated
plants grown 55 days in soil held approximately at the temperatures indicated. The
pots are grouped according to temperature, with the control on the left and the inocu-
lated pot on the right in each set.

B. — Nodules from 9 soybean plants grown 55 days in soil held at approximately the
temperatures indicated.

Effect of Gnil Temperature on Development of Nodules

Plate 3

15'

Journal of A_;ricultural Research

Vol. XXII, No. 1

INFLUENCE OF THE PERIOD OF TRANSPLANTING
WESTERN WHITE PINE SEEDLINGS UPON THEIR
BEHAVIOR IN NURSERY AND PLANTATION

By E. C. Rogers

Forest Examiner, Forest Service, United States Department of Agriculture

At forest nurseries in the northern part of the United States the work
is customarily crowded into three or four weeks in spring immediately
following the time when the soil can first be worked. Preferably, the
stock is lifted, packed, shipped early, and spring sowing and transplanting
are all usually crowded into this period. At the Savenac Nursery, Haugan,
Mont., this spring congestion has been keenly felt; and the experiments
outlined below have had for an object the determination of the safe
limits of the transplanting season. The results may or may not apply
beyond the local conditions prevailing in the region of western Montana
and northern Idaho.

FALL TRANSPLANTING

Work was commenced upon this problem at the Savenac Nursery in the
fall of 1913. By the use of the Mast trencher method, 600 i-year-old
seedlings of western white pine {Pinus monticola Dougl.) were transplanted
on each of the four following dates: August 15, September i, September
15, and October 10. In May, 19 14, these plants were examined and the
overwinter losses were recorded. Loss by frost heaving, as indicated
in the figures, includes not only plants completely thrown out but also
those lying prostrate on the ground, even though they were quite firmly
attached and still alive, because in that condition they would never
recover sufficiently to be fit to plant. A few individuals showed the
symptoms of winterkilling. These were about evenly distributed among
the four units, in no case amounting to i per cent of the total. Figure
I shows the loss by frost heaving. From one- third to one-half of the
plants were heaved out during the cold nights of late October, before
the coming of snovv^, and during the clear weather of late March and
early April after the snow left. The loss was greatest in the October 10
unit. This may be explained by supposing that, because of the warmer
soil temperature the individuals transplanted earlier had had time to
make sufficient root growth to render them more resistant to the frost
lifting, but that those transplanted latest were virtually heeled in.
However, precise evidence on this point is lacking.

As it had been suggested that possibly in the Mast V-shaped trench
a pocket of loose soil was formed around the lower roots and that this

Journal of Agricultural Research, Vol. XXII, No. i

Washington, D. C. Oct. i, 1921

z p Key No. F-6

Journal of Agricultural Research voi. xxii. No.

predisposed the plants to heaving, several rows of i-o western white pine
seedlings, transplanted September 25, 1913, in open plowed trenches
were likewise examined in May, 1914. From a total of about 12,000
trees the loss from frost heaving was 29.6 per cent and that from winter-
killing 1.2 per cent. Here, again, nearly one- third of the plants were
thrown out — a loss hardly 6 per cent less than that by the Mast trencher

soo

4S.0

^.o

3S.O

30.0

£S.O

g 20.0
k

^ /s.o

/o.o

s.o

/-OSS OP^/ia// /jrcZ/7S/0/c7/7/s

.__

/i^ay/7

Ja/7e2 Ji//7e/S /?uy./S S(sp^/ Se/:>A/S

Oc/:?

Fig. I. — I,oss by frost heaving of seedlings transplanted at different dates.

method, which was used 10 days earlier. Apparently the method mat-
tered little, the loss having been a necessary consequence of fall trans-
planting.

Lorey ^ found that fall transplants of Douglas fir and European larch
led spring transplants in height growth in the transplant rows. More
recently, Toumey ^ states that fall transplants lead in earliness of growth
and in size, provided they escape winter injury. While no data with

1 Lorey, Tuisko. mitteilungen aus dem forstgarten und KtaTURBETRiEB. n. forstgarten
INSBESONDERE. In AUg. Forst u. Jagd. Ztg., N. F., Jahrg. 70, p. 193-197. 1894.

' TouMEv. James W. seeding and planting: a manual for the guidance op forestry students . . .
xxxvi, 4SS p., 140 fig. New York, 1916.

Oct. 1, 1921

Infltience of Period of Transplanting

respect to those matters were collected, the heavy loss through heaving
at Savenac Nursery more than balanced any possible gains of that
kind. Fall transplanting is clearly so.unsafe that no further local experi-
ments with it are necessary.

SPRING TRANSPLANTING

NURSERY TESTS

Experiments in the spring of 19 13 had for their object the compari-
son of three lots of 600 i-o western white pine each, transplanted on
May 17, June 2, and June 16. The first summer's loss from drought
increased with the lateness of the transplanting period, as is shown also
by figure i. The June 16 lot looked less thrifty than the others at the
end of the season, and it was concluded that^ in case of necessity, trans-
planting could evidently be continued as late as June 15, though it is
not desirable.

As a control on the tests made in the spring of 19 13 it was arranged
to transplant 1,000 i-o western white pine at Savenac Nursery every
10 days during the spring of 19 14. This was actually done on April
24, May I, May 9, May 20, May 30, June 12, June 19, June 30, and July
14. On each of these dates 100 more were removed from the seed-
bed, of which the weights and measurements appear in Table I.

Table I. — Weights and measurements of i-o western white pine on different dates of
transplanting in the spring of IQ14

Date of

Average
length
of stem.

Average
diameter
of stem.

Average

weight.

Per-
centage

Average new spring
growth.

trans-

Lot

No.

of plants

with

buds

closed.

of plants
with
buds

swelling.

of plants

plant-
ing.

Top.

Root.

with
buds
open.

Needles.

Rootlets.

Inches.

Mm.

Gm.

Gjn.

Inches.

Apr. 24

1-54

I. 17

0. 140

0. IIS

100

0. 0

COS

May I

1-52

•i53

. 122

100

I. SO

•143

. 090

1.90

.186

. 120

1.99

.125

.oSi

June 12

3.30

.265

.150

2-43

.203

• 103

100

2. 30

•313

•139

100

(')

July 14

2. 60

1-53

.274

.130

100

1. 02

1 Not recorded.

In Table I stem length is the distance from the ground line to the tip
of the terminal growing point. The stem diameter was measured at the
ground line. The average new growth of rootlets was based upon the
longest rootlet noted in each plant examined and not upon all new rootlets.
This figiure is only relatively correct, because some slight root breakage
was unavoidable in taking up the plants from the seed bed. Root growth
data were omitted in the two latest lots, since the older portions of the

* Unpublished progress report.

Journal of Agricultural Research voi. xxii, no. i

new roots were assuming a brown, mature appearance, and this made it
difficult to determine the margin of growth.

- Figure 2 shows graphically tlie condition of the seedlings as to length
of stem and spring growth of needles and rootlets on the different dates
of transplanting.

Apr £4 May I May 9

Julijl-^

Maij£0 May 30 June 12 June IS June JO
Time of 7~ran^ p/a nilng

Fig. 2. — I^ength of stem and spring growth of needles and rootlets of seedlings transplanted at different

dates.

Table I brings out the following points :

I. Root growth began prior to April 24 during the spring of 19 14, or
over three weeks before visible stem gro^vth. Although not so indicated
by the table, it was found that the earliest visible root elongation took
place in the superficial soil layers, gradually progressing to deeper and
deeper levels as the season advanced, presumably in response to changes
in soil temperature.

Oct. 1, 192 1

Influence of Period of Transplanting

2. The swelling of the buds began a little before May 9, and two-thirds
of them were fully open by May 20.

3. Although individual variations existed — due largely, it is thought,
to a lack of uniform density in the seed bed — stem length, stem diameter,
and weight of top increased in general as the season advanced.

4. The proportion of the fresh weight of the plants contained in the
root was greatest early in spring and decreased as the growth pushed
ahead in May and June. Figure 3 brings out this point. Owing to the
possibility of variation in the water content of top and root, dry weights
would be of interest, but circumstances prevented their being obtained.

so. Or

4-50 =-

4-0.0

JSO

30.0

"~~\

Percsnta^s offrssh \A'eigh1r of plant-

in root at different dates of transo/anting

__-

A\/eraffe fresh we/g/it ofp/ant

■~^~-

' ■

/\pri/2'}. Maj'l MayS May 20 May JO June/ 2 June/3 June SO Ju/y/4-

77/77G' o/' 7rt:?/7S/>/c?/7//no

Fig. 3.— Proportion of fresh weight of roots of seedlings transplanted at different dates.

Transplanting was done in adjacent parallel rows, and these were
irrigated at intervals during the summer of 19 14. As early as August i
there had come to be a marked differentiation in size and color. The
April 24, May i , and May 9 units showed particular vigor and had a rich
green color. The June 30 and July 14 lots showed much the same
development as the earliest ones but had a yellow color, which gave the
July 14 lot an almost sickly appearance. The remaining intermediate
lots showed a healthier color than the later ones but lacked the size and
development of the latter. This differentiation was increasingly marked
at the end of the growing season. The loss from drought in these lots
during the summer was very slight, the heaviest loss, 2.4 per cent, being
suffered by the July 14 unit.

Journal of Agricultural Research voi. xxii. No. x

On September ii, 1914, the season's growth of stem and needles was
obtained by measuring every tenth plant in each lot, or 100 in each unit.
The terminal buds of 500 plants in each lot were examined as to their
maturity on this same date. Buds having a definite form, of a deep
brown color, and covered with a protective coat of fine hairs were classed
as mature. Plants without a single well-defined bud and those whose
growing point had a tender green color, without the coat of hairs, were
considered of immature development. For purposes of comparison, data
similar to the foregoing were obtained from 2-year-old western white
pine plants from a representative area of seed bed. These plants were
of the same age and seed lot as the nine transplanted units, differing
only in that they had been allowed to remain in the seed bed. These
data are given in Table II.

Table II. — Growth and development of western white pine during the first season in the

transplant bed

Date of transplanting.

Lot

No.

Average

seasonal

stem growth.

Average
seasonal
growth of
needles.

Percentage of

plants with

mature

buds.

Percentage of

plants with

immature

buds.

Apr. 24

May I

June 12

July 14

Not transplanted .

Inches.

0-757
729

763

734
825
874
918
974

951
063

Inches.
0.934
952
897
424
566

570
611

715
943
131

80.0
75-7
75- 1
69. I
70.1
69.6

56.4
80.6
92. 6
93-5

20. o
24-3
24.9
30-3
29.9

32-4

43-6

19.4

7-4

6.5

By average growth of stem and needle is meant the growth for the
entire season, regardless of whether that growth took place in the seed
bed, in the transplant bed, or in both. Needle measurement was made
in the middle of the sector of currently- grown stem.

Table II brings out the following points :

1. The later the transplanting after the buds open, the higher the stem
growth for the season. It appears that height growth practically ceases
for a time after transplanting, the plant's energies being directed toward
getting established in its new habitat. In other words, the height
growth is roughly proportional to the length of time the plant is left in
the seed bed. Hence, plants that were not transplanted made a higher
stem growth than any of the transplanted lots.

2. The longest needle groAvth at the end of the season had been made
by the first and last lots (Apr. 24 and July 14). The needle growth of
the first lot had been made entirely in the transplant bed and was accom-
panied by a deep green color, but that of the last lot had taken place in

Oct. 1, I92I

Influence of Period of Transplanting

3,9

the seed bed before transplanting and the needles of these looked much
less vigorous. As figure 4 shows, the season's needle growth commences
to fall with the May 9 lot, drops abruptly with the May 20 lot, then
climbs gradually until the last lot equals the earliest. The stock that
was not transplanted produced longer needles than any of the trans-
planted units.

3. The difference in dates of transplanting had a pronounced effect
upon the maturing of the fall buds. The earliest maturing lots were
the two that were transplanted latest (June 30 and July 14). The less

k.O

0.0
%^^

c
2.0

1.6

Height

growth erf
in 1316. pric

planfj

r +oJali/ 1.

Growth

\ of naedfe

3 during

planti
dif

fran^pi^ni^d at

__^

--^^

: 1

AprZ^ Mayl Maij9 May20 MayJO Junel2 JunelS JuneJO Ju/(//4 Mf

Time of Transpthntinq planted

Tig. 4.— Increase in height and growth of needles of seedlings transplanted at different dates.

favorable weather conditions appear to hasten preparations for winter
by stock transplanted in summer.

4. Seedling stock of the same age and source (2-0) led transplants
(i-i) in current stem growth and needle development no matter what
the period of transplanting. The shock of the treatment, expressed
quantitatively, resulted in a loss of 0.3 inch of stem growth and 0.2 inch
of needle growth, even when the transplanting was done at the most
favorable period.

At the end of the season in which the transplanting was done it ap-
peared that the plants lined out before the buds were open had suffered
the least shock, and, judging from their unhealthy appearance, those
transplanted in midsummer (July 14) seemed to have suffered most.
No single item of weight or measurement appears to be a consistent
indicator of the degree of severity of the shock.

Journal of Agricultural Research voi. xxii. No.

On July I, 1916, 100 plants were washed out from each of these nine
transplanted units by the aid of water under pressure. At this time,
the stock could be considered to be in the i-2>^ age class. Data from
these 900 plants are assembled in Table III.

Table III. — Weights and measurements of 1-2% -western white pine

Lot

No.

Average

stem

height

growth

(cur-

reiit).a

Average

stem
diameter.

Average number of laterals.

Date of
trans-

First order.

Second order.

Average

total

fresh

weight

of plant.

Percent-
age of
weight
in root.

planting.

2 inches
and up.

0.5-inch

to 2
inches.

2 inches
and up.

0.5-inch

to 2
inches.

Apr. 24

May I

9
20

30
June 12

July 14

I
2

3
4
5
6

7
8

Inches.

2-75
2-75
3.66
2.09
3-27
3-58
2.8s
2.74
1.82

Mm.
6.31
6.72
7.14
5-65
6.48
6.48
6.07
6.27
4-95

10.2
10.3
10.3

9.0
10. 5

8.1

9.1
10.2

2.6

5-4
6.7
6.1
6.1
4-5
5-2
5-3
5-1
5-3

3-4
6.3
4.1

3-2
2.9
3-6
3-7
3-8

2-5

15-3
27.4
17.2
13.0
13.0

II-3

10.5

12.4

9.9

Gm.

3-09
3.16

3-78
1.84
2.78
2.74

2.54
2.62
1. 41

43-4
45-6
36.2

45-7
41.4

36.9
39-4
42.7
48.2

o By current stem growth is meant the 1916 growth prior to July i.

7.0

§ s.o

J.S

ao
2.6

^ Diamett
(me, Juli

1
r of sfern af ground
/.ISI6, of stock trans-

plan-f-e
in the

d at differ
spring. of

enr dates

Apr. 2^ Maul

Maij9 Maij20 Mat/JO JunelS Juna/S JuneJO Julijl^
7~irrre of 7~ransp/aniing

"Bic. 5. — Increase in diameter of stem of seedlings transplanted at different dates.

Oct. 1,1921 Influence of Period of Transplanting 41

A study of the proportion of the fresh weight of plant in the root
system shows that the relation between this proportion and the time of
transplanting, which was so evident two years before, had entirely dis-
appeared. All the plants were washed off, and the surfaces were allowed
to dry in the air; but as the evaporating power of the air varied, it was
not possible to compare directly the average fresh weights obtained at
tlie time of transplanting with those obtained on July i, 19 16. All
the weights obtained on the latter date are, however, comparable with
one another. Figures 3, 4, and 5 illustrate further the current height
growth of stem, the stem diameters, and the total fresh weight of the
plants.

A striking similarity will be noted in the curves in figures 3 (weight),
4, and 5. All agree in shovnng two minima, the first for the May 20 lot,
and the second and still lower point for the July 14 transplanting. This
bears out the conclusion reached in the fall of 19 14 that the shock of
transplanting fell hardest upon the July 14 lot. But it now appears that
the transplanting on May 20 was almost equally lasting in its unfavorable
influence upon growth in the transplant bed. Between these two dates
there appears to have been a period of about a month in which trans-
planting worked less injury to the plant.

As the roots of these plants were removed by washing, and with a
minimum of breakage, it seemed worth while to compare the average
numbers of laterals of the first and second orders. Figure 6 shows
graphically the data obtained. In order to bring the curves close
together, and thus facilitate comparison, the actual average number of
lateral rootlets of the second order, between 0.5 and 2 inches in length,
has in each case been divided by 2 in plotting the curves, and the average
total number of lateral rootlets in the four classes has similarly been
divided by 5. Because of the difficulty involved in recording them, and
the limited time available, no records were obtained of the number of
laterals of higher orders than the second, nor, in any case, of laterals less
than 0.5 inch in length. The figures obtained are, however, considered
indicative of the general nature of the root system.

There is a close relation not only between the average number of
laterals in each of the two length classes of the second order but also
between these and the total number of rootlets of the recorded classes.
On the other hand, the curves for the two classes of the first order run
quite differently. Yet, so far as determining the total curve is concerned,
these two classes could obviously have been neglected. There is a con-
sistent decline in the fibrous development of the root system as the
transplanting season advances. Transplanting in early summer, and
even more so in late summer, holds back lateral root growth in the trans-
plant bed, the effect being strikingly noticeable two years afterwards.

Journal of Agricultural Research voi. xxii. No.

Although several seasons' observations had shown no noticeable loss
through winter frost heaving of i-o western white pine transplanted in
April and in early May, it was found in the spring of 1918 that of 2,922
i-i white pine transplanted June 15, 19 17, in connection with another
experiment, 1,152, or 39.4 per cent, were heaved out during the late
fall, winter, and early spring of 19 17-18. This is nearly as heavy a loss

Ma(/9 May20 Mat/30 Junel2 JunelS JuneJO Jultjl^

Time of Transplanfina

Apr 24 May I

Fig. 6.— Number of lateral rootlets on seedlings transplanted at different dates.

as had previously been recorded for fall transplanting. However, an
unprecedented snowless period in December probably contributed to
produce this result. A greater susceptibility to frost heaving on the
part of late spring and summer transplants is the natural result of the
poorer root development just referred to. The plant must rebuild its
entire root system late in the season and so has a relatively poor anchor-
age when the frost comes. Furthermore, Cannon * has found that the

1 Cannon, William Austin, root habits oP desert plants. 96 p., x7 fig., 23 pi. Washington, D. C„
igii. (Carnegie Inst. Wash. Pub. no. 131.)

Oct. 1, 1921 Influence of Period of Transplanting 43

formation of an abundant lateral root requires a favorable water content
in the soil and a sufficiently high soil temperature. Although summer
soils are warm, yet, in spite of occasional irrigation, the greatest loss of
transplants from drought at Savenac Nursery occurs during July and
August, indicating that there is less available soil moisture during that
period, or at least that there is a smaller balance for growth when the
transpiration loss of the plant has been met.

It seems, therefore, that in the foregoing series the plant organism
was most deeply disturbed by being transplanted in midsummer. This
appeared to be a consequence of the high evaporation and lack of moisture
in the soil, along with the greater topheaviness of the plant. The May
20 transplants gave evidence of having been most severely set back, a
result which must be attributed either to external conditions or to the
internal state of the plant. The Savenac Nursery weather records show
a precipitation of 2.16 inches in April, 1914, well distributed throughout
the month, with only seven clear days. In May, previous to the
twentieth, there fell 0.58 inch of rain, and 12 out of 19 days were cloudy
or partly cloudy. On May 20, the soil was well stored with water and
was favorable for the reception of plants. The maximum temperature
on that day was 72° F., and it and the eight days following were partly
cloudy. During the period from May 22 to 28, inclusive, 0.46 inch of
rain fell, every day yielding at least a trace. The weather and soil
conditions were, therefore, sufficiently favorable to convince the writer
that the reason for the marked checking of the growth of the May 20
lot lay in the developmental stage of the plant itself. One-year-old
western white pine seedlings, whose buds are just opening and whose
tiny new needle fascicles are less than i/io inch long, show a particular
sensitiveness toward removal and replanting.

FIEI/D TESTS

The influence of the season of transplanting upon the behavior of the
tree in the plantation is of special interest to the forester. One hun-
dred of the plants from each of the nine spring lots described above
were planted October 6, 19 15, on the Wallace experimental area, near
Wallace, Idaho. A northwest aspect — a typical white pine planting
site — ^was selected. One row was devoted to each lot, and the rows were
placed adjacent to each other and parallel, extending up and down the
slope. The place where each tree was to be planted was previously
marked by a cedar stake 16 inches long, whose top had been dipped in
white paint to make it conspicuous among native cover plants. Each
stake bore its lot number in black lumber crayon. The same man
planted all the rows, using a uniform method.

Journal of Agricultural Research voi. xxii. No.

On September 21, 19 16, and on November 7, 19 17, these plants were
examined, their condition was noted, and the average height growth of
stem was recorded, this average being based upon all vigorous living
plants. Table IV gives the principal data secured.

Table IV. — Date of transplanting, average current height growth, and percentage of trees
surviving at the end of the igi6 and I gi J field seasons on the Wallace area

Date of transplanting — 1914.

Apr. 24.

May I .

20.

30
June 12.
19.

30-
July 14.

Average of all lots.

Lot
No.

Average current
height growth.

1916

Inches.

0-57
.91

•95
.82
.67
.96
.82
I. 00
■65

Inches.
O. 62

83
96
81

75
95
84
IS

Percentage of
trees surviving.

Fall 1916. Fall 1917

95-9

90.9

95-7
93-6
94.6

92-5
92.8

93-7
98.9
96. 2

94-3

The fact that the unusually dry summer of 191 7 caused almost
negligible losses makes improbable any further changes of importance in
the survival standing of the nine lots. The percentage of living trees of
all lots in the fall of 191 7 was above 90. There is no superiority on the
part of the early lots, the April 24 units standing lowest. The later
lots have, on the whole, lived best. Both the May 20 and July 14
plants, while outclassed in the transplant bed, showed better than an
average survival in November, 191 7. In fact, the time of transplanting
had no apparent influence in the field.

Figm-e 7 further illustrates the height growth of these plants. There
is a marked similarity between the growth curves for 1916 and 191 7;
hence each resembles the total growth curve for the entire two seasons.
For instance, in each of the three curves the average point for the June
19 lot falls exactly upon the horizontal average line for that curve. To
facilitate comparisons, the height growth curve from figure 4 is plotted
in figure 7 also. This renders it possible to compare the growiJi made in
the plantation the first year after planting (1916) Avith that made the
same season prior to July i by individuals left in the transplant beds
(curves A and D, respectively).

There are certain points of resemblance between the curves of growth
in transplant bed and in field. The May i and June 19 lots stand upon
or very near the horizontal average line in both. The July 14 transplants
stand low, and the May 9 and June 12 transplants stand high in both.
But, on the other hand, the May 20 lot, which had a low growth rate in the

Oct. 1, 1921

Influence of Period of Transplanting

transplant bed, lias an average rate in the field; but the May 30 and June
20 units reverse their positions with respect to the horizontal average
lines. The planting out of the stock has evidently caused a somewhat

Apr so May/. Maj'9 MaySO Ma/JO June I2 June/3 JuneJO July l4
T/mci of Transplanting
Fig. 7. — Increase in height of seedlings transplanted at different dates.

general rearrangement of growth rates in which the later lots tend to
overtake the earlier ones, this rearrangement being accentuated after a
second season in the field. The inferiority of the May 20 plants has

46 Journal of Agricultural Research voi. xxii, no. i

disappeared; the July 14 stock slightly surpasses the April 24 lot; and
there is nothing to indicate that summer transplants are not fully the
equal of spring transplants, so far as growth after planting is concerned.

CONCLUSIONS

Transplanting western white pine seedlings at any time in the fall is
not a safe practice at Savenac Nursery, because the frosts of the following
late fall and early spring heave out the young plants.

Results in the plantation thus far indicate that, where 1-2 stock is
grown, the transplanting season may be extended from a date as early
in spring as the ground can be worked until early July. The shock of
removal from the seed bed is greatest when the transplanting is done in
midsummer, on account of the high evaporating power of the air, the
low water content of the soil, and the greater top-heaviness of the plant
with its considerably increased transpiring surface. A particularly
sev^e shock was also suffered when transplanting was done at the time
of bud opening and before the rudimentary needle fascicles had reached
■^^g-inch in length. Stock transplanted at either of these critical periods
lagged behind the other lots for at least two years in the transplant
bed. However, when they were planted in the field as 1-2 stock, the
plants survived as well as the others, with little, if any, inferiorit}'- in
growth on account of the considerable rearrangement of growth rates
following the planting.

There are, nevertheless, other reasons which make transplanting at
Savenac Nursery safer in spring than in summer. June and July trans-
plants suffer more from drought the first season and, because of their
poor root development at the end of the growing season, are more subject
to frost heaving the following winter. The first disadvantage can be
met by proper irrigation, but the second can not readily be prevented.

The safest practice, therefore, is to confine this work as much as
possible to April and early May.

The foregoing conclusions apply to i -year-old seedlings which are to
remain two years in the transplant rows, 1-2 stock being the only age
class of white pine transplants at present grown at Savenac Nursery.

Certain points brought out by this study may have an important
bearing upon the season for field planting. It is probable, for instance,
that subsequent lateral root development in the plantation may be
decisively influenced by the time of planting.

A DRYROT CANKER OF SUGAR BEETS

By B. L. Richards
Department of Plant Pathology, Utah Agricultural Experiment Station

What appears to be an undescribed rootrot of the sugar beet was first
called to my attention on August 5, 1920, by Mr. A. H. Bateman. Speci-
mens of the diseased beets collected at this date at Cornish, Utah, ex-
hibited numerous brown, circular lesions that varied from j^ inch to i
inch in diameter (PI. 4 ; 8, D ; 9, A, B) . The outer surface of the root cov-
ering these lesions, which in most cases remained entire, had so sunken as
to give a definite undulating contour of alternating light and dark brown
concentric areas or rings (Pi. 4). The removal of this outer layer of cells
of the older lesions exposed deep cankers or pockets filled with hyaline-
mycelium embedded in the dry remains of partially decayed host cells.
This accompanying mycelium, when exposed to the atmosphere through
the cracking open of the outer covering, appeared dark brown in color
and immediately suggested the typical mycelium of the sterile, or
"Rhizoctonia," stage of Corticium vagum B. and C. The general prev-
alence of black sclerotial bodies on the outside of the diseased beets
(PL 7, A), together with the microscopic examinations made at this time,
confirmed this initial suggestion.

An examination of the field from which these first diseased specimens
were taken revealed the trouble to be of considerable economic impor-
tance; at least 20 per cent of the beets in this field of 40 acres were
diseased. The disease appeared to be confined to definite areas wherein
every beet might be found infected. These diseased spots varied con-
siderably in size and appeared to be widening most rapidly in the direc-
tion parallel with rows. Three adjacent fields were found at this time
to be seriously diseased, but none to the same degree as the field first
visited.

The progress of the disease in these fields appeared of such ominous
character as to require immediate investigation. However, as the
season was well advanced, little more than preliminary experiments
were undertaken. The results to date, while definite, are not sufiiciently
extensive to warrant final conclusions, and many of the important rela-
tions of the disease remain obscm-e; nevertheless it is felt that the
apparent economic importance of the trouble justifies a preliminary
description at this time.

The disease is first detected in the field by abnormal wilting of the
leaves in the daytime with partial or complete recovery at night. Later

Journal of Agricultural Research, Vol. XXII, No. i

Washington, D. C. Oct. i, 1921

zm Key No. Utah-14

54817°— 21 4 (47)

48 Journal of Agricultural Research voi. xxii. no. i

the older leaves fail to recover, turn brown, and die. This dying of the
outer or older leaves continues with the progress of the disease in the
root until all the leaves on the affected beets may succumb. Localized
browning frequently occurs in the blade and petiole, but to date no
suggestion of a parasitic relation has been found. Neither the petiole
decay reported by Duggar (2) ^ nor the "western crownrot" described by
Edson (j) have been found associated with thedryrot canker in the field.
A peculiar type of crownrot, however, is found late in the season, usually
well toward harvest time (PI. 6; 8, B). A study of a number of these
crownrot specimens indicates definitely that the causal organism enters
the beet below the stu-face of the soil and works upward in the tissues,
eventually destroying the crown. The fungus has not been observed
to attack the beet above the soil line.

It is evident that the fungus is unable to destroy the outer corky cells
of the beet root, but gains entrance to the inner tissue at a definite
point and works tangentially just beneath this outer layer. As the
fungus eats its way from the point of entrance the outer tissues, due to
killing and subsequent drying out of the cells beneath, sink in such a
manner as to produce the circular lesion with its very definite undulating
contour of alternating raised and sunken concentric "rings" (PI. 4).
The lesions appear first as a small, brown, sunken spot with a minute
perforation in the center (PI. 8, D). The first definite concentric "ring"
which is considerably sunken below the central area and usually dark
brown in color is noted before the lesion reaches a diameter of >^ inch.
With continued enlargement a second and somewhat broader "ring,"
less sunken and much lighter in color, results. Similar concentric areas
are developed alternately until the fimgus reaches its limit of lateral
spread. Individual lesions resulting from a single point of infection
may obtain a size of from ^ to i inch in diameter and develop as many
as eight alternate "rings" (PI. 4). When, however, adjacent lesions
become confluent, as they frequently do (PI. 4; 8, D; larger
lesions result which may in severe cases cover a large part of the root
surface. In such cases large concentric rings are produced, which become
common to a number of centers of original infection (PI. 4; 8, D; 9, A, B).

Another characteristic feature of the disease results in cases where
infection occurs at or near the apex of the root. The root in such an
event is usually severed at the point of infection and the fungus advances
upward, producing the typical dryrot with resultant concentric rings
which may encircle the entire root (PI. 7, B). Again, cankers may occur
with such frequency as to girdle completely the root (PI. 8, A).

The distinctive feature of the contour, as shown in Plates 4 and 8, D,
is obtained usually before the fungus penetrates deeply into the tissue
of the beet and before a serious rupture of the outer layer occurs. With

1 Reference is made by number (italic) to "I,iterature cited," p. 52.

oct.i.i93i A Dryrot Canker of Sugar Beets 49

the drying out and final cracking of this outer covering the fungus,
possibly because of a better oxygen relation, eats radially into the beet,
producing deep cankers (PI. 5 and 6). The decaying tissues rapidly
dry out as the fungus advances inward, leaving the cavity partially
filled with a dry, pithy residue. Frequently the content of the canker
appears as a definite plug, which, upon wetting, may be removed intact
from the cavity of the canker (PI. 9, D).

Except for slight cracking, the outer layer of dead cells remains entire
and furnishes a definite covering until the lesion has reached approxi-
mately its limit of tangential spread. As the cells of this outer covering
finally dry out the central perforation enlarges and ultimately gives rise
to a definite crack which may extend the entire diameter of the lesion
(PI. 4; 8, D; 5). Frequently adjacent cracks become confluent,
resulting in large characteristic fissures, which in severe cases of the
disease may obtain from 2>^ to 3 inches in length and from % to 1%
inches in depth (PI. 5, 6). With numerous points of attack the beet by
harvest time is converted into a dry, brittle shell filled with a pithy mass
of host and fungous debris (PI. 6).

During the season careful study was made of a large number of the
beets taken from each of the different fields in which the dryrot had
been found. In all cases the characteristic cankers exhibited the pres-
ence of the sterile stage of Corticium vagum. This fungus, it was found,
may be obtained regularly in a pure form from any part of the typical
canker, provided the outer covering of the lesion is not previously de-
stroyed. The brown layer separating the normal from the diseased
tissue (PI. 8, A-C) has never failed to yield the fungus free from other
organisms, and even from the open lesions cultures have been obtained
with remarkable ease and regularity. The degree to which other organ-
isms are found to be excluded is phenomenal.

To determine the etiological relation of the fungus, inoculations were
made September 3 on partially grown beets. In the process of inocula-
tion the soil was removed to a depth of approximately 4 inches from 2 1
beets in each of five rows. Each of the 2 1 beets in the first row was
punctured a number of times with a sterile needle, and the inoculum,
consisting of the beet fungus, grown for several days on potato agar, was
then scattered throughout the soil as the latter was replaced about the
beet. Row 2 was inoculated exactly as row i except that in place of
needle punctures slight incisions were made in the beet by the use of a
sterile scalpel. The beets in rows 3 and 4 were wounded as in row i,
and the soil was inoculated with two different "strains" of Corticium
vagum,} Row 5 was left uninoculated, and the beets after wounding as
in rows i and 2 were covered and grown as controls. All the wounded

1 These "strains" were obtained from the surface of a potato tuber in 1918 and have proved virulent
on potato stems in both sterilized and unsterilized soil.

Journal of Agricultural Research voi. xxii. No. i

beets in the control row healed normally. Infection occurred on but
one beet in rows 3 and 4. The other beets in these two rows healed as
perfectly as in row 5. The results of inoculation with the sugar-beet
strain of the fungus in rows i and 2 are given in Table I. The types
of lesions produced as a result of atrificial inoculation are shown in
Plate 9, A. D.

Table I. — Number of lesions on sugar beets inoculated with the sterile stage of Corticium

vagum

Beet No.

Row I,

needle

puncture.

Row 2,
incision.

I .

13
8
8
17
14
II

17
6

17
13
17
20

19
10

15
II
12
16

3- •
4. .

\.A\^.^:^uu^^

8 :

10 .;.

4
6

I -J

7
8

3
2

4
4
0

21 - -

Total

285
13-5

96
4-5

Average

Instructions for the inoculating of sugar beets with the beet fungus
without puncture or incisions were not followed. As a result the question
as to the ability of Corticium vagum to attack the sugar beet independ-
ently of other agents remains unsettled. It is quite conceivable that
sugar-beet root aphis {Pemphigus betae Sloane) and other insects so
prevalent in the soil may serve an important function in the initial en-
trance of the fungus. Having once gained access to the lower tissue,
however, it appears evident from the results that this particular "strain"
of C. vagum is capable of producing the type of canker and dryrot with
which it is so constantly associated in the field.

The peculiar method of decay, together with the sharp line of demarca-
tion between the diseased and the normal tissue (PI. 8, A-C; 9, C-F), pro-
vide the most distinctive characteristics of the disease. A dark brown,
watery layer invariably separates the dry, decayed mass occupying the
cavity of the canker from the normal host tissue beneath. This layer

oct.i.igzx A Dryrot Canker of Sugar Beets 51

when examined under the microscope is found to be composed of masses
of hyaline, vigorously growing young hyphae ramifying through and
between the rapidly decaying host cells. It is in this advance layer
that the major portion of the tissue destruction occurs. The brown
layer advances uniformly inward by additions from the normal host
tissue, while the outer surface of the layer rapidly dries out and con-
stantly contributes its substance to the pithy mass occupying the resulting
cavity of the canker. The thickness of the layer is dependent largely
upon the rate at which the moisture is lost from it outer surface as the
fungus eats its way radially into the normal tissue. No evidence of direct
penetration of tlie normal cells by the fungus has been found. On the
other hand, it appears that dissolving enzyms precede considerably the
advancing mass of young hyphae (PI. 8, C).

This method of tissue destruction resembles in a very definite way that
described by Ramsey (5), by which Rhizoctonia solani Kiihn attacks
and produces a definite pitting of the mature potato tuber. A similar
process of decay is described by Atkinson (r) for the "sore shin" of
cotton. He states that —

the fungus {Rhizoctonia solani) never seems to penetrate far into the living tissues, but
kills as it goes, and the tissues become brown, depressed, and present the appearance
of a plant having a deep and ugly ulcer at the surface of the ground.

A type of decay most accurately resembling this particular beet rot is
described by Richards (7) for the potato stem-canker caused by Corticium
vagum.

The early production of definite cankers by a slow corroding of the
normal tissue, finally resulting in a complete dryrot of the beet, suggests
a possible name "dryrot canker" for the disease here described.

Various American workers. {2; 4, p. 243-^4; 3) have reported rootrots
of the sugar beet which they attribute to the work of Rhizoctonia solani
Kiihn. It appears difficult at this time, however, to determine the possible
relation of these to the particular type of dryrot described in this article.
The indefiniteness of the literature on the subject in fact does not justify
any general statement as to the possible distribution of the disease.

During September and October of 1920 a preliminary survey ^ was
made of the beet-growing districts in four counties of Utah — Cache,
Davis, Utah, and Salt Lake. The disease was found in 18 fields of the
51 visited in Cache County and in 3 fields of the 20 surveyed in Davis
County. Very serious damage occurred in a number of these fields.
No indication of the trouble v/as found in either Utah or Salt Lake
Counties.

The limited survey does not permit of an estimate of the loss to the
total sugar-beet crop of the State; nevertheless, the general prevalence

1 This survey was conducted in cooperation with the Office of Plant Disease Survey, United States
Department of Agriculture. The author wishes to express his indebtedness to Dr. G. R. Lyman for this
support.

52 Journal of Agricultural Research voi. xxii. No. i

of the trouble would indicate that under more favorable conditions the
disease may become a serious factor in beet culture. It is not improbable
that a thorough survey may discover the "dryrot canker" in every beet-
growing district in this and surrounding States.

Since the appearance of the author's abstract (6), Dr. George L. Peltier
reports in a letter to the author that he noted during 1920 in Nebraska
what appears to be the same trouble. Preserved specimens in the
plant-disease herbarium of the Utah Agricultural College show that the
disease was collected in Utah as early as 19 15.

LITERATURE CITED
(i) Atkinson, Leo. F.

1895. DAMPING OFF. N. Y. Cornell Agr. Exp. Sta. Bui. 94, p. 231-272, fig.
55. 6 pi.

(2) DUGGAR, B. M.

1899. THREE IMPORTANT DISEASES OF THE SUGAR-BEET. N. Y. Cornell AgT.

Exp. Sta. Bui. 163, p. 335-363, fig. 49-63. Some references to the lit-
erature of beet diseases, p. 361-363.

(3) Edson, H. a.

i915. seedling diseases of sugar beets and their relation to root-rot
AND CROWN-ROT. In Jour. Agr. Research, v. 4, no. 2, p. 135-168, pi.
16-26. Literature cited, p. 165-168.

(4) Pammel, L. H.

1891. FUNGUS DISEASES OF SUGAR BEET. lowa Agr. Exp. Sta. Bui. 15, p.
234-254. 6 pi.

(5) Ramsey, Glen B.

1917. A FORM OF POTATO DISEASE PRODUCED BY RHIZOCTONIA. In Jour. Agr.

Research, v. 9, no. 12, p. 421-426, pi. 27-30.

(6) Richards, B. L.

1921. a dry rot of the sugar-beet caused by corticium vagum.
(Abstract.) In Phytopathology, v. 11, no. i, p. 48.

(7)-

1921. THE POTATO STEM CANKER. Utah Agr. Exp. Sta. Bui. 178.

PLATE 4

Sugar beet showing typical lesions as a result of natural field infection. Lesions
as sho^v^ may become confluent and develop common concentric rings. Initial
stages in fissiu-e formation are also evident. Photographed August 8, 1920.

A Dryrot Canker of Su::ar Beets

Plate 4

Journal of Agricultural Research

Vol. XXII, No. 1

A Dryrot Canker of Surrar Beets

Plate 5

Journal of Agricultural Research

Vol. XXII, No. 1

PLATE 5

Sugar beet showing various stages in the rupture of the outer covering of the lesion
resulting in the formation of deep fissures. The lesions shown on this particular
beet have not reached the size normally attained before rupture occurs.

PLATE 6

Late stage in the development of the disease, showing the beet as a dry shell par-
tially filled with a pithy residue composed of mycelium and dead host tissue. The
decay of tlie crown of this beet is a result of the fungus working upward from the point
of infection below the surface of the soil. Remnants of the concentric rings of typical
lesions are clearly visible. The cracking of the outer surface of the beet at this stage
is shown to extend beyond the lesions.

A Dryrot Canker of Sugar Beets

Plate 6

Journal of Agricultural Research

Voi. XXII, No. 1

A Dryrot Cankor of Su':ar Beets

Plate 7

Journal of Agricultural Research

Vol. XXII, No. 1

PLATE 7

A. — Portion of a sugar beet showing the typical sclerotial masses commonly found
adhering to the beets in the infested areas.

B. — Sugar beet showing the result of natiu-al infection near the apex of the beet,
at which point the root has been completely severed. The dryrot advancing upward
from the initial point of attack has produced the typical undulating contour so char-
acteristic of the small lateral lesions.

PLATE 8

Sections of diseased sugar beets, showing the abrupt drying out between the dis-
eased and healthy tissue. The prominent "feeding surfaces" composed of recently
killed cells and the young hypha is clearly evident.

A. — Cross section, showing complete girdling of the beet by cankers resulting from
separate points of infection. In such cases the continued penetration of the fungus
may completely sever the root at the line of greatest infection.

B. — Longtitudinal section of diseased beet, showing various stages of decay and
the pulpy material partially filling the cankers.

C. — Sugar-beet crown, showing the definite type of crownrot caused by the fungus
worked upward from a point of infection below the soil surface (PI. 6). A small region
of healthy tissue is shown to which a few sickly leaves were attached.

D. — Section of beet stuface, showing progressive stages in the development of the
lesions resulting from natural infection. The earliest visible stage is shown to exhibit
a slight perforation of the outer surface at the center of the lesion. This small open-
ing, present in all lesions, gradually enlarges with age and finally results in the large
fissures (PI. 5). Various stages in the coalescence of lesions are especially evident.

A Dryrot Canker of Su^ar Beets

Plate 8

Journal of Asricultural Research

Vi.1. XXII, No. 1

A Dryrot Canker of Sugar Beets

Plate 9

Journal of Agricultural Research

Vol. XXII, No. 1

PLATE 9

A, B. — Beets showing typical lesions produced by artificial inoculation. Needle
punctures through which the fxmgus entered permitted of rapid drying out of the
diseased tissue and of an early ruptiure of the outer layer of cells at the margin of the
lesions. A number of the lesions, however, show the concentric rings so character-
istic of the disease produced by natural infection. Cross sections of these lesions
are shown in C and D.

C, D. — Cross sections of the lesions in A and B. The lesions in C disclose the more
advanced stage of the disease wherein the outer layers of cells are broken down. In
D the outer layers of the lesions are more or less entire.

E, F. — Cross sections of cankers resulting from natural infection in the field. A
more advanced stage is shown than in C and D; otherwise the lesion produced by
the natural and artificial method of inoculation appeared identical.

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Vol. XXII OCTOBER 8, 1921 No. 2

JOURNAL OF

AGRICULTURAL

RESEARCH

CONTENTS

Page

Comparative Vigor of Fi Wheat Crosses and Their
Parents - - -- - -- - - 53

FRED GRIFFEE

(Contribution from Minnesota Agricultural Experiment Station)

Temperature and Humidity Studies of Some Fusaria Rots
of the Irish Potato - - - -- - -65

R. W. GOSS

( Contribution from Nebraska Agriculttual Kzperiment Station)

Blackleg Potato Tuber-Rot under Irrigation - - - 81
M. SHAPOVALOV and H. A. EDSON

(Contribution from Bureau of Plant Industry)

Microscopic Study of Bacteria in Cheese - - - - 93

G. J. HUCKER

(Contribution from New York Agricultural Experiment Station)

Further Studies on Relation of Sulphates to Plant Growth
and Composition - -- - - - - - 101

HARRY G. MILLER

(Contribution from Oregon Agricultural Experiment Station)

Soybean Mosaic - -- - - - - -111

MAX W. GARDNER and JAMES B. KENDRICK

( Contribution from Indiana Agricultural Experiment Staition)

PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE'

WITH THE COOPERATION OF THE ASSOCIATION OF

LAND-GRANT COLLEGES

^W^ASHINQXON, D. C,

EDITORIAL COMMITTEE OF THE

UNITED STATES DEPARTMENT OF AGRICULTURE AND

THE ASSOCUTION OF LAND-GRANT COLLEGES

FOR THE DEPARTMENT

ElARL F. KELLERMAN, Chairman

Physiologist and Associate Chief, Bureau
of Plant Industry

EDWIN W. ALLEN

Chief, Office of Experiment Stations

CHARLES L. MARLATT

Entonwlogist and Assistant Chief, Bureau
of Entomology

FOR THE ASSOCIATION
J. G. LIPMAN

Dean, State College of Agriculture, and
Director, New Jersey Agricultural Etptri-
menl Station, Rutgers College

W. A. RILEY

Entomologist and Chief. Dtvisiott of Ento-
mology and Economic Zoology, Agricul-
tural Experiment Station of the University
of Minnesota

R. L. WATTS

Dean, School of Agriculture, and Direetor.
Agricultural Experiment Station. Th»
Pennsyhania State College

All correspondence regarding articles from the Department of Agricult^ire should be
addressed lo Karl F. Kellennan, Journal of Agricultural Research, Washington, D. C.

All correspondence regarding articles from State Experiment Stations should be
addressed to J. G. Lipman, New Jersey Agricultural Experiment Station, New
Brunswick, N. J.

JOIMALOFAGBiaiTllRALffiSEARCH

Vol. XXII Washington, D. C, October 8, 192 1 No. 2

COMPARATIVE VIGOR OF F^ WHEAT CROSSES AND
THEIR PARENTS '

By Fred Griffee ^

Instructor in Plant Breeding, Division of Agronomy and Farm Management, Depart-
ment of Agriculture, University of Minnesota

The comparative vigor of Fj crosses and their parents is a subject of
much interest to the plant breeder. In crops where the technic of cross-
ing is comparatively easy, the increase in vigor obtained in the F^ cross
often more than pays for the additional trouble of producing the hybrid
seed. In self-fertilized crops like the small grains where considerable
labor is involved in making artificial crosses, it is apparent that F^ crosses
can not be used commercially as a means of increasing crop yields. The
suggestion, however, has been made by Anderson (ly that the added
vigor of the heterozygous condition might be utilized in small grains by
making a large number of crosses between strains which, when crossed,
show a considerable increase in yield. Produce from Fj and F3 progeny
could be used for seeding the general field, and the crosses could be
repeated each year in order to keep up the supply of seed.

Several theories have been advanced to explain the phenomenon of
heterosis. The discovery of genetic linkage has led to the development
of an adequate Mendelian explanation of the vigor so often obtained in
Fi crosses. An excellent review of the development of this theory is
given by East and Jones (<?). The theory explains the increase in vigor
shown in the first hybrid generation as being due to the meeting in the
zygote of dominant or partially dominant growth factors some of which
are contributed by each parent. Linkage is given as the reason why all
dominant factors can not be combined in a homozygous individual. Ac-
cording to this hypothesis the maximum number of favorable growth
factors can be obtained only in the heterozygous condition.

In producing new varieties by crossing, forms may be obtained in the F3
generation which appear homozygous for botanical and agronomic char-

• Published with the approval of the Director as Paper No. 259 of the Journal Series of the Minnesota
Agricultural Experiment Station.

' The writer wishes to express his appreciation to H. K. Hayes, Head of the Section of Plant Breeding,
Division of Agronomy and Farm Management, for suggestions and criticisms during the progress of this
study.

• Reference is made by number (italic) to "Literature cited," p. 62-63.

Journal of Agricultural Research, Vol. XXIL No. 2

Washington, D. C. Oct. 8, 1921

ir Key No. Minn.-«

(53)

54 Journal of Agricultural Research voi.xxn.No. a

acters but which may be heterozygous for growth factors. There is the
possibility that this heterozygous condition may cause the Fg or F.
hybrid to give a high yield. After several further generations this hete-
rozygous condition may be lost, with a consequent loss in growth stimu-
lus. A knowledge of the amount of added vigor in the F, generation is
of value in determining whether heterozygosis in Fg and F^ lines would
modify their yields sufficiently to interfere seriously with a determina-
tion of their value as improved varieties.

With these points in view a study has been made in wheat of the imme-
diate effect of cross-pollination on seed weight and the increased vigor
of Fi crosses. Pure lines were used of seven varieties of Triticum vulgare
Vill. and one variety of each T. compactum Host. (Little Club) , T. dicoccum
Schr. (Spring Emmer) , and T. durum Desf. (Mindum). Varieties of T.
vulgare were crossed with each other and with Little Club, Spring Emmer,
and Mindum. Little Club was crossed also with Spring Emmer and
Mindum.

IMMEDIATE EFFECT OF CROSS-POLLINATION

Because of the phenomenon of double fertilization it is possible in
some cases to obtain an increase in weight of seed as an immediate eflfect
of cross-pollination. The increase is due principally to an increase in
weight of endosperm in such crops as com, where the proportion of endo-
sperm to embryo is large.

Collins (5) observed open-pollinated ears of Chinese maize in which the
size of seed was increased by cross-pollination. Seeds which showed by
their color the effect of foreign pollen averaged 0.178 gm., while white
seeds from the same portion of the ear averaged 0.153 g^. Roberts (14)
mentions a similar instance with Chinese maize. Collins and Kempton (6)
compared the average seed weight of com from intravarietal and inter-
varietal pollinations. The intervarietal crosses exceeded the intra-
varietal in seed weight by 8.8 per cent. In a similar experiment, Wolfe
{16) found that 23 of 31 com varietal crosses yielded more grain than in-
travarietal pollinations. Carrier (4) obtained an increase in yield of
grain in strains of com when grown in a mixture as compared with any
one of the strains grown alone.

That an increase is also obtained in the size of the embryo is clearly
shown by Lewis and Vincent {12) in a comparison of seeds of Newtown
apple from self- and cross-pollinations. The crossed seeds showed a
striking increase in weight over that of the selfed seeds. As there is
little or no endosperm in apple seeds, an increase in seed weight is due
largely to an increased size of the cotyledons.

Since artij5cially pollinated seeds of wheat are usually smaller than nor-
mally pollinated seeds, spikes of each variety were emasculated in the
same manner as for cross-pollination and then pollinated with pollen
from plants of the same pure line. Seed from this intrapollination is

Oct. 8, 1921 Comparative Vigor of F^ Wheat Crosses and Their Parents 55

termed "incrossed seed" and is used as a basis of comparison in deter-
mining the immediate effect of cross-pollination. The average weight of a
normally pollinated seed for all varieties used was 26.65 ±0.22 mgm./
and the average for an incrossed seeds was 18.13 ±0.24 nigm.

A comparison is shown in Table I of the hybrid seed and the incrossed
seed where the average dates of pollination are the same or approxi-
mately so.

Table I.-

-Weight of seed of the immediate crosses compared with weight of seed of the
incrossed parents

Seed parent.

Cross.

Difference

Name of cross.

Num-
ber of
seeds.

Average weight
of seeds.

Num-
ber of
seeds.

Average weight
of seeds.

between cross

and female

parent.

Marquis X Velvet ChafFa

Marquis X Penny

38
38
49
39
44
104

Mgm.

12. 6±o. 5
12. 6± . 5
17. 2± .8
10. i± .5
26. 4± .8
19. 9± . 6
26. 4± .8

48
24
26

50
24

Mgm.

15. 6±o.5
20. 2 ±1.0
23- 5± -7
9-4± .3
27- I ±1.3
15. 9± .6
25. o±i. 2

Mgm.
+3. o±o. 7
+ 7-6±l.2
+6.3±i.o
-0. 7± .6

Haynes Bluestem X Marquis . .
Little Club X Marquis

Emmer X Velvet ChafE

Velvet ChafE X Mindum

Emmer X Little Club

+0. 7±i. 5
-4. o± .8
— I. 4±i. 4

a In the discussion of crosses the seed parent is given first.

The varietal crosses in every case showed an increased seed weight as
compared with the female parent. The largest increase in seed weight
was 7.6 ±1.2 mgm., which was obtained from the cross Marquis X Penny.
This hybrid gave on the average over 50 per cent heavier seeds than
incrossed Marquis. Of the species crosses none gave a significant increase
in seed weight. Velvet Chaff crossed with Mindum produced seeds which
on the average were 4.0^0.8 mgm. lighter than the seeds of incrossed
Velvet Chaff.

F, GENERATION CROSSES COMPARED WITH THEIR PARENTS

Some of the earliest hybridization work affords good examples of the
vigor of Fi crosses. For an excellent review of this subject the reader is
referred to the publication of East and Jones {8) .

In the present experiment the Fi generations and their parents were
grown in the greenhouse under controlled conditions. Care was taken
to plant seeds at a uniform depth, and when the seedlings were about 4
inches tall they were transplanted to 7-inch pots, two seedlings to a pot
and only like seedlings together. Unfortunately an epidemic of stem-rust

1 The probable error of an average of averages was calculated according to the formula:

in which n is the number of individuals in a generation, e the probable error, and A" the total niunbcr of
individuals (is).

Journal of Agricultural Research

Vol. XXn.No. a.

started about heading time, and some plants were rusted badly
Measurements of height were taken on those plants which were not
attacked previous to heading, and yield data were taken only on plants
uninjured by rust.

Both incrossed and normally pollinated seeds of the parental varieties
were planted. Amy and Garber (2) have shown that in some cases there
is a positive correlation between weight of seed planted and the vigor of
resultant plants. In order to determine whether the size of seed planted
was of importance in an analysis of individual plant yields in the present
experiment, correlation coefficients were calculated for the weight of seed
planted as subject and length of culm and yield of grain per plant as
relative. (Table II.)

Table II.— Correlation coefficients for weight of seed planted and the vigor of resultant

plants

Variety.

Marquis

Velvet Chaff. ...

Barletta

Penny

H. B. S. 1-16-12

Bobs

Little Club

Emmer

Mindum

CoefBcient of correlation with weight of seed planted as subject.

Length of tallest
culm (relative).

— O. 084 ±0. 100

+ .o69± .065

- . I44± • 085

- . i97± .075

— . 242± . 071

— . ii6± . 079

- . ii8± .082
+ • 024± . 086

— . 121 ± . 076

Total culm length
(relative).

+0. i36±o. 098

+ • 078± . 065

.205± .083

— . I29± .077
+ . I2i± . 071
+ .i69± .078
+ . i85± .080

— .oo7± .085
+ .38i± .066

Yield of grain in

grams per plant

(relative).

+0. 047±0. 173
+ .o87± . 108

+ . OIO± . 112

- .I94± .095
+ . 205± . 084
+ .032± . 099

— . 425 ± . 080
+ .i36± .095

The only significant correlation was obtained with the Mindum variety.
A correlation coefficient of +0.381 ±0.066 was obtained for weight of
seed planted and total culm length. In the light of these facts it was
considered legitimate to use the plants from normal and incrossed seed
as a single parent population.

The Fi crosses and their parents were compared for height of tallest
culm and for total culm length. (Table III.)

Six of the 1 1 Fi varietal crosses showed an increase in length of tallest
culm as compared with the parental average, and 5 showed a decrease.
The two Fi crosses between Mindum and vulgare varieties were con-
siderably taller than either parent. Similar results were obtained from
crosses between Emmer and the same vulgare varieties. On the othef
hand, the Fi crosses of Little Club with either Emmer or Mindum did
not show a significant difference in average height of tallest culm when
compared with the average of the parents.

In Table IV the crosses and their parents are compared for total culm
length.

Oct 8, 192 1 Comparative Vigor of F^ Wheat Crosses and Their Parents 57

TablB III. — Height of tallest cuUn of Fi wheat crosses compared with parental average

Name of one parent.

Num-
ber of
individ-
uals.

Height.

Name of other
parent.

Num-
ber of
individ-
uals.

Height.

Aver-
age
height
of par-
ents.

Fi cross.

Niun-
berof
individ-
uals.

Height

Marquis

Velvet Chaff

Penny

Haynes Bluestem. .

Marquis

Velvet Chaff

Average. . . .

Emmer

Mindum

Emmer

Average. . . .

Inches.

5
5
5
5
7
7
7
4

77
77
77
62
62

50.8

Velvet chaff. .. .

Barletta

Penny

Bobs

Barletta

Penny

JBobs

Marquis

JLittleClub

iLittleClub

Marquis

jVelvet Chaff . . .

Marquis

Inches.

105
60

74
70
60

74
70
70
46
66
66

66
66
46
105
105
46

Inches.
45- 6
47. 6
41.9

43-3
46.7
41. o
42.4

38.7
48.6
47.8
46.9

64
38
45
65
38

108

24
62

44.6

Inches.

42.3
50.8

41. I
47-9
50-9

42. 6
40. 7

50.8
50-5
43-3

45- o

47.2

49. 2

46.8

14
17
28
18

51.0
48.5
53-2
54-5
55-5
55-6

53-1

TablU IV. — Total culm length of Fj wheat crosses compared with parental average

Name of one parent.

Num-
ber of
individ-
uals.

Height.

Name of other
parent.

Num-
ber of
individ-
uals.

Height.

Aver-
age
height
of pat-
ents.

Fi cross.

Num-
ber of
individ
uals.

Height

Marquis

Velvet Chaff ....

Penny

Hajmes Bluestem

Average . . .

Emmer

Mindum

Marquis

Velvet Chaff

Average. . .

loS

105

L 105

Inches.
195
19s
195
195
19s
149
149
149
149
90
215

Velvet Chaff .

Barletta

Penny

Bobs

Little Club. .

Barletta

Penny

Bobs

Little Club. ..

Bobs

Marquis

171

77
77
77
46

105

204
131
131
131
195
149

157

^Little Club. .

Marquis

Velvet Chaff .

►Emmer

105
60

74
70
66
60

74
70
66
70
46

Inches.
149

157

136

149

157
90
136
149
136
195

Inches.
172
176

143
166
172

153
120

143
149

113
205

64
38
45
65
62

49
108

52
60

Inches.
146
168
132
211
167
174
133
153
153
III
214

140

156

66
66
46

105
62

149
149

19s
149

204
204

17s

177
140
163
140
200
177

166

160

14
17
i8
28

169
189

151
123
210
149

165

Journal of Agricultural Research

Vol. xxn. No. a

For total culm length, 6 of the ii varietal crosses showed an increase
over the parental average and 5 showed a decrease. The averages for
culm length of the F^ crosses and of their parents are practically identical
when the results of all crosses are considered together. This makes it
doubtful whether the increases of the F^ crosses over the parental averages
are the results of the vigor due to crossing or are due to some other
experimental factor.

Table V. — Average yield of grain per plant of Fi wheat crosses and their parents

Niun-
berof
individ-
uals.

Yield.

Name of other
parent.

Num-
ber of
individ-
uals.

Yield.

Aver-
age
yield
of pa-
rents.

Fj cross.

Per

centage
of in-

Name of one parent.

Num-
ber of
individ-
uals.

Yield.

Gm.
2.7
3-3

2-5

2.9
2.8

2-5

2.3
2.5

crease
with
paren-
tal av-
erage
as basis.

Marquis

1 ^5
\ '5

; 38

I 38

IS
38

Gm.
1.9
1.9

1-5
1-5
2.4
2.4

1.9
1-5

Penny. . . .

Bobs

Penny. . . .

JBobs

Marquis. .. .

JLittle Club.

36
59

I 59
15

f 46
I 46

Gm.
2.4
30
2.4
30

30
1.9

2. 2
2. 2

Gm.
2. 2

2-5
2. 0

2-3
2.7
2. 2

2. I
1.9

65
28

92
23
18

45
37

Velvet Chaff ....
Penny

32
25
26

4
14

Ha)mes B 1 u e-

stem.
Marquis

Velvet Chaff. ...

Average . .

1.9

2-5

2. 2

2.7

Little Club

Marquis

/ 46
I 46

1 ^^

I 38
IS

2. 2
2. 2
1.9
1-5
1-5
1.9

Emmer

[Mindum

[Emmer

48
f 49

49
I 49
/ 48
I 48

1. I

2. I
2. I
2. I
I. I
I. I

1-7
2. 2
2 0
1.8
1-3

1-5

13
8

23
18

•3
I. 0

•3
I. I

•5
.6

Velvet Chaff

Marquis

Average . .

1.9

1.6

1.8

For average yield of grain per plant, six of the eight variety crosses
yielded more than either parent, and all variety crosses jdelded more than
the parental average. Marquis X Bobs and Velvet Chaff X Little Club
exceeded the parental average 32 per cent in yield of grain per plant.

With the exception of crosses between common wheat and Little Club
the average yield of grain per plant of the species crosses was less than
that of the lower-yielding parent. This is due to the fact that the F^
plants had a high percentage of barren florets.

STERILITY IN SPECIFIC CROSSES

The occurrence of sterility in wheat specific crosses has been reported
by several workers. Tschermak {13), after several years of hybridiza-
tion work, found that hybrids of Triticum dicoccum and T. compactum or
vulgare varieties were only partially fertile. Hybrids of T. durum with
T. compactum or T. vulgare varieties were classed as fully fertile. Ster-

Oct 8. tgai Comparative Vigor of Fj Wheat Crosses and Their Parents 59

ility is mentioned by Kezer and Boyack (ii) as occurring in the F^ gen-
eration of the cross Fultz Mediterranean by Black Winter Emmer. In
crosses between Algerian Macaroni and Algerian bread wheats, Free-
man (9) reports that the F^ generation developed normally but in the
F2 generation all degrees of sterility appeared from complete sterility to
complete fertility.

Hayes, Parker, and Kurtzweil (10) crossed varieties of Triticum vul-
gare with varieties of T. durum and T. dicoccum. The parental varieties
showed an average of 4 per cent of barren florets. The F^ crosses of
varieties of durum with varieties of vulgare and the reciprocals showed
a barrenness of 47 per cent. The Fi crosses of T. dicoccum crossed with
varieties of vulgare showed 26 per cent barrenness and the reciprocal 29
per cent. The results are not in agreement with the conclusions of
Tschermak (13).

In the present experiment a count was made of the total number of
outer florets per plant and the number of these which were barren.
From these data the percentage of barren florets was computed. (Table

Table VI. — Barrenness of outer florets in wheat varieties and Fi crosses

Variety or cross.

Number Percentage

plants of barren

consid- outer

ered. florets.

Marquis

Velvet ChaJBf

Penny

Haynes Bluestem

Bobs

Little Club

Emmer

Mindum

Average

MarquisXPenny

MarquisXBobs

Velvet Chaff X Penny

Velvet Chaff X Bobs

Haynes Bluestem X Marquis

PennyXBobs

Marquis X Little Club

Velvet Chaff X Little Club. .

Average

MarquisX Emmer

Velvet Chaff X Emmer

Little Club X Emmer

Average

MarquisX Mindum

Velvet Chaff X Mindum ....
Little Club X Mindum

Average

15
38
34
47
59
47
49
44

18
21
IS

18
17
25
18

93
18

22
57
37

17
14
15
13
17
15
17
14

18
23

73
67
86

8
2

88
67

6o Journal of Agricultural Research vd. xxii. No. a

The parental varieties showed an average of 19 per cent barren florets.
Intercrosses of vulgare varieties and .crosses between Little Club and
vulgare varieties showed an average of 15 per cent barren florets. The
Fi crosses of Marquis, Velvet Chaff, and Little Club with Emmer gave
an average of 75 per cent barrenness. These same common varieties
and Little Club crossed with Mindum showed a barrenness of 70 per
cent. These data confirm the results of Hayes, Parker, and Kurtzweil (10)
and show conclusively that in some cases Fj crosses between varieties
of T. vtdgare and T. durtim or T. dicoccum are highly self-sterile.

DISCUSSION OF RESULTS

It has been pointed out by East and Jones (7) that the increase in
productivity of a cross is due to an increase in the number of growth
factors of which the maximum number can be obtained only in a hetero-
zygous condition. In a crop such as com, this heterozygous condition
is kept up by cross-fertilization. Selfing corn varieties reduces the hetero-
zygosity and consequently the vigor. In wheat the continued selfing
natural to the crop has brought about a condition of homozygosity.

In the present experiment all varietal crosses gave an increase in seed
weight as an immediate effect of cross-pollination. An increase is also
shown in the F^ crosses for average yield of grain per plant as compared
with the parental average. The increase ranged from 4 per cent in
Penny X Bobs to 32 per cent in Marquis X Bobs. Before attempting to
utilize the vigor of the heterozygous condition by growing Fg and F4
generation crosses as the commercial crop, it seems logical to combine
in one variety the maximum number of growth factors possible. When
the possibilities of combination have been exhausted and a variety, or
a series of varieties, has been secured which contains this maximum
number of growth factors, it may be desirable to follow out the sugges-
tion of Anderson (j). This method probably could not be used to
advantage except under intensive farming conditions. In case one
desired to use such a method it is logical to assume that the more desirable
crosses to make are those which show the greatest increase in yield of
grain in the F^ generation.

There is an indication that the increased productivity of the heterozy-
gous condition is a factor which must be considered in comparing F3 and
F4 lines for yielding ability. In the cross Marquis X Bobs the Fj genera-
tion showed on an average a 32 per cent increased yield of grain per plant
as compared with the average of the parents and a 10 per cent increase as
compared with the higher-yielding parent. In the Fj generation of such
a cross it is highly probable that some of the most vigorous plants will be
those with the greatest degree of heterozygosity. These heterozygous
individuals will produce F3 progeny the vigor of which likewise will be
partially due to the heterozygous condition. If the F3 lines are classified
on a basis of their yielding ability, some of these heterozygous lines will

Oct. 8,1921 Comparative Vigor of F^ Wheat Crosses and Their Parents 6i

be included as the best 3nelders. In subsequent generations as the lines
become homozygous their productivity may decrease. In generations
beyond the F5 the heterozygous condition of the population rapidly dis-
appears.

A method of breeding which, according to Babcock and Clausen (j),
has been used by the Svalof Station, seems worthy of wider application.
As self-fertilized crops approach homozygosis rapidly in generations follow-
ing a cross, it is suggested that a cross be made between varieties selected
because of the desirable characters which they possess. After 6 to 10
years have elapsed, during which time progeny of the cross has been grown
in bulk plots, selection of individual plants may be made with the assur-
ance that a high percentage of these plants will give homozygous progeny.
While this system requires some length of time before results are ob-
tained, it requires a minimum of labor.

When making crosses with the hope of increasing yield through a re-
combination of the desirable factors of both parents, the parents will
naturally be selected on the basis of their yielding ability. The chances
of favorable recombinations of yield factors in generations following a
cross will presumably be greater when dealing with a cross which shows
maximum increased yield over the parents in the Fj generation. Genetic
linkage, however, may make certain combinations difficult or impossible.

The sterility of the specific crosses, with the exception of crosses of
varieties of Triticum vulgare with Little Club, is partially or wholly
responsible for the low grain yield of the crosses as compared with the
parental averages. The fact that Little Club behaves in every way as a
variety of T. vulgare agrees with the view of Tschermak (75), who believes
that T. compactum and T. vulgare are closely related. Little Club
crosses readily with varieties of vulgare. When Little Club or varieties
of vulgare are crossed with Emmer or Mindum, the same high degree of
sterility is shown.

SUMMARY OF RESULTS

(i) An increase in seed weight was obtained in all varietal crosses as
an immediate effect of cross-pollination. The only significant differ-
ence shown by the immediate hybrids of specific crosses was a decrease
in seed weight obtained in Velvet Chaff X Mindum.

(2) In the Fj generation some of the hybrids exceeded the parental
average in height of tallest culm, and in total culm length others showed
a decrease. In all varietal crosses the F^ hybrid exceeded the parental
average in yield of grain per plant, and six out of eight crosses exceeded
the yield of the better parent.

(3) Crosses between Little Club and varieties of Triticum vulgare gave
results similar to those of crosses between vulgare varieties.

(4) The F^ generation of Emmer or Mindum crossed with varieties of
Triticum vulgare or with Little Club showed a high degree of sterility.

62 Journal of Agricultural Research voi. xxn.No. »

The average percentage of barren florets of tlie parental varieties was 19.
The average percentage of barren florets of the Fj varietal crosses, includ-
ing crosses of Little Club with vulgare varieties, was 15. The vulgare-
Emmer and Little Club-Emmer crosses produced 75 per cent barren
florets, while an average of 70 per cent of barren florets was obtained
from the durum-vulgare and durum-Little Club crosses.

LITERATURE CITED
(i) Anderson, T.

I919. THE IMI'ROVEMENT OF AGRICULTURAL CROPS BY SELECTION AND HYBRID-
IZATION. (Abstract.) In Scot. Joiir. Agr., v. 2, no. i, p. 10-20.

(2) Arny, a. C, and Garber, R. J.

I918. VARIATION AND CORRELATION IN WHEAT, WITH SPECIAL REFERENCE TO

WEIGHT OF SEED PLANTED. In Jour. Agr. Research, v. 14, no. 9,
p. 359-392, 8 fig. Literature cited, p. 391-392.

(3) Babcock, Ernest Brown, and Clausen, Roy Elwood.

1918. GENETICS in relation TO AGRICULTURE. XX, 675 p., 239 fig., 4 Col.

pi. New York, London. List of literature cited, p. 622-647.

(4) Carrier, Lyman.

I913. THE immediate effect ON YIELD OF CROSSING STRAINS OF CORN. Va.

Agr. Exp. Sta. Bui. 202, 11 p., 2 fig.

(5) Collins, G. N.

1909. A NEW TYPE OP INDIAN CORN PROM CHINA. U. S. Dept. Agr. BuT. Plant
Indus. Bui. 161, 30 p., 2 pi.

(6) and Kempton, J. H.

I913. EFFECT OF CROSS-POLLINATION ON THE SIZE OF SEED IN MAIZE. In U. S.

Dept. Agr. Bur. Plant Indus. Circ. 124, p. 9-15.

(7) East, E. M., and Hayes, H. K.

I912. HETEROZYGOSIS IN EVOLUTION AND IN PLANT BREEDING. U. S. Dept.

Agr. Bur. Plant Indus. Bui. 243, 58 p., 8 pi. Literature cited, p.

49-51-

(8) and Jones, Donald F.

1919. INBREEDING AND OUTBREEDING, THEIR GENETIC AND SOCIOLOGICAL

SIGNIFICANCE. 285 p., 46 fig. (in text and on 13 pi.). Philadelphia,
London. Literature cited, p. 266-277.

(9) Freeman, George F.

1919. THE HEREDITY OF QUANTITATIVE CHARACTERS IN WHEAT. In GcneticS,

V. 4, no. I, p. 1-93, 85 tab. (i fold.) Literature cited, p. 93.

(10) Hayes, H. K., Parker, John H., and Kurtzweil, Carl.

1920. GENETICS OP RUST RESISTANCE IN CROSSES OP VARIETIES OP TRITICUM

VULGARE WITH VARIETIES OF T. DURUM AND T. DICOCCUM. In Jotll.

Agr. Research, v. 19, no. 11, p. 523-542, pi. 97-102. Literature cited,
P- 541-542.

(11) KezER, Alvin, and Boyack, Breeze.

1918. MENDELIAN INHERITANCE IN WHEAT AND BARLEY CROSSES WITH PROB-

ABLE ERROR STUDIES ON CLASS PREQUENafiS. Colo. Agr. Exp. Sta.
Bul. 249, 139 p., 95 tab., 10 fig., 9 col. pi.

(12) Lewis, C. I., and Vincent, C. C.

1919. pollination of the APPLE. Oreg. Agr. Exp. Sta. Bul. 104, 40 p.,

14 pi. (in text).

Oct. 8, 192 1 Comparative Vigor of F^ Wheat Crosses and Their Parents 63

(13) Roberts, Elmer.

1918. FLUCTUATIONS IN A RBCESSIVB MENDELIAN CHARACTER AND SELECTION.
In Jour. Exp. Zool., v. 27, no. 2, p. 157-192, 3 fig., 2 pi. (in text).
Literattire cited, p. 176-177.

(14) Roberts, H. F.

i912. first generation hybrids of american x chinese corn. in ann.
Rpt. Amer. Breeders' Assoc, v. 8, p. 367-384, 5 fig. Literature cited,
p. 384.

(15) TsCHERMAK, Erich von.

1914. DIE VERWERTUNG DER BASTARDIERUNG FUR PHYLOGBNETISCHE

FRAGEN IN DER GETREiDEGRUPPE. /n Ztschr. Pflanzenziiclit., Bd. 2,
Heft 3, p. 291-312.

(16) Wolfe, T. K.

1915. FURTHER EVIDENCE OF THE IMMEDIATE EFFECT OF CROSSING VARIETIES
OF CORN ON THE SIZE OF SEED PRODUCED. In Joiu". Amer. Soc. Agron.,
V. 7, no. 6, p. 265-272. Literature cited, pp. 271-272.

TEMPERATURE AND HUMIDITY STUDIES OF SOME
FUSARIA ROTS OF THE IRISH POTATO '

By R. W. Goss
Assistant Plant Pathologist, Nebraska Agricultural Experiment Station

INTRODUCTION

The ability of Fusarium oxysporum Schlect. to cause a rot of the
potato tuber has been clearly demonstrated by a number of workers.
The influence of temperature on this disease has been reported in a
number o;f papers, but the experimental evidence as a whole is rather
meager, usually only extreme temperatures being vtsed. The effect of
moisture on the progress of the disease, except under conditions of
extreme dryness or saturation, has received practically no attention.
It was with the purpose of determining the relation of temperature and
humidity to the progress of potato tuber-rots caused by Fusaria that
the following work was undertaken.

HISTORICAL

The association of Fusaria with storage-rots of the Irish potato (Sola-
num tuberosum L.) has been a matter of common observation by most
workers in plant pathology from 1842 to date. Several species of the
form genus Fusarium Link have been described as causes of potato
tuber-rots, by Von Martins {12), Reinke and Berthold {18), Schacht
(jp), Pethybridge and Bowers (14)," Longman {10), and Sherbakoff {20).
The fact that Fusarium species could produce a rot of the tuber was
demonstrated by Pizzigoni {15) and Wehmer {24, 25), who described
the species they worked with as Fusarium solani (Mart). Frank (6),
De Bary (2), and others considered that the Fusaria were unable to
produce a rot of the tuber. In most of the earlier papers, F. solani, or
some species thought to be a synonym of it, was given as the causal
organism.

Owing to the absence of clearly defined species in all the literature
previous to Appel and Wollenweber's (i) monograph on the form genus
Fusarium in 191 2, no attempt will be made to review in detail the earlier
reports of potato tuber-rots caused by Fusaria.

Fusarium oxysporum was considered by Wollenweber (27) to be a
strictly vascular parasite producing a wilt of the potato vine but not

• Published with the approval of the Director of the Nebraska Agricultural Experiment Station. The
paper is based upon experimental -work undertaken at the Michigan Agricultural College in 1914-15, and
at the University of Wisconsin in 1916-17.

' Reference is made by number (italic) to "Literature cited," p. 77-79.

Journal of Agricultural Research, Vol. XXII, No. 2

Washington, D. C. Oct. 8, 1921

zb Key No. Nebr.-3

(65)

66 Journal of Agricultural Research voi. xxii. No. 2

causing a rot of the tuber. Carpenter (4) in 19 15 was the first to report
successful infections by inoculations with pure cultures of F. oxysporum.
He made these by dipping wounded tubers in a water suspension of
spores, wrapping in oiled paper and keeping them at controlled tem-
peratures ranging between 17° and 30° C. No detailed experiments
were reported except in this saturated atmosphere. He noted, however,
that either a dryrot or a wetrot was produced, according to the tem-
perature and humidity used. He concluded that a constant storage
temperature below 50° F. (10° C.) would prevent the action of F. radici-
cola Wollenw., F. eumartii Carp., and F. oxysporum. Previous to this
work of Carpenter's, Smith and Swingle {22), in 1910, described a bundle
blackening and a dry endrot of the tuber as two stages of the same
disease. They attributed this to a Fusarium for which they accepted the
name F. oxysporum as first applied to it by Schlechtendahl (2/, p. 139).
They noted that the disease continued in stored potatoes and that when
potatoes were stored in warm rooms, either moist or dry, they became
badly diseased, whereas those stored in cool places kept much better.
They did not differentiate this species of Fusarium from others occurring
on the potato, and no inoculation experiments were recorded. Manns
in 191 1 (//), working with the same disease, stated that the "dormant
internal infection" under improper storage conditions becomes so active
as to cause a high percentage of dryrot. He noted that the disease was
favored by high temperature and considerable moisture. At 36° to 40°
F. (2° to 3° C.) the disease made no progress, at 45° to 55° F. (7° to 12°
C.) it developed gradually and caused considerable rot, especially when
accompanied by high humidity. He made no mention of pure culture
inoculations on tubers or morphological studies.

Jamieson and Wollenweber in 191 2 {8) described a dryrot of the
potato tuber caused by a species of Fusarium which they named Fusa-
rium trichothecioides Wollenw. They made inoculation experiments and
found the most rapid penetration of the tuber to take place at 10° to 12° C
in an atmosphere of low humidity. Rotting took place at the high
humidities but not as rapidly. Wilcox, Link, and Poole {26) pubUshed
on a dryrot of the potato tuber caused by a Fusarium which they called
F. tuberivorum W. and L. but which was undoubtedly the F. trichothe-
cioides previously described by Jamieson and Wollenweber (<?), They
found that a temperature of 8° to 10° C. was only slightly inhibitive to the
growth of the fungus and that when potatoes infected with the organism
were stored at this temperature, the most rapid decay took place when
the humidity was high. Pratt (ly), working with the same disease,
found that temperatures ranging from 12° to 25° C. were favorable for
the progress of the disease and that dryrot did not develop at temperatures
below 2° C. He concluded from storage experiments that in a dry,
well-ventilated storage house losses would be very slight at temperatures
from 2° to 4° C.

Oct.8.i93i Temperature and Humidity Studies of Fusaria Rots 67

Link (9), making comparative studies of Fusarium oxysporum and
F. trichothecioides found that both were capable of producing a rot of
the potato tuber and that F. trichothecioides produced a typical dry-
rot. F. oxysporum produced a softrot of the whole tuber except under
cold, dry conditions, when a drjn-ot was produced. He ran his experi-
ments at controlled temperatures ranging from 1° to 30° C. in an
almost saturated atmosphere.

Pratt {16) found that Fusarium radicicola behaved much the same as
F. oxysporum, and he concluded from storage experiments that the
tuber-rot caused by this organism does not make any progress in storage
at a temperature of 48° F. (8.8° C.) or below.

In general, then, it can be said that a high temperature favors the
production of tuber-rots by all three of these Fusaria, although Fusarium
trichothecioides appears to be able to produce a rot at lower temperatures
than the other two. High humidities also appear to favor the produc-
tion of tuber-rot. With the exception of the paper of Jamieson and
Wollenweber {8) all the evidence points toward an increase in rotting
with an increase in humidity.

TEMPERATURE RELATIONS IN PURE CULTURES

A review of the literature shows a general conformity of results re-
garding the relation of temperature to the growth of Fusarium oxy-
sporum. Link (9) by making dry-weight determinations of growth in
liquid media found 30° C. to be the optimum for growth. Edson and
Shapovalov (5), working with Petri-dish cultures, obtained the same
optimum. They reported a maximum temperature of 37 C, where the
spores changed to chlamydospores ; they did not observe growth at 5° C.
Humphrey (7) gives 4° C. as the minimum temperature for certain
strains of F. oxysporum..

The writer, working with three strains of Fusarium oxysporum and
using the same methods for measuring growth, obtained somewhat
similar results to those reported by Edson and Shapovalov (5). The
minimum temperature for growth was 9.5° C, no growth taking place at
the next lower temperature of 7° C. The maximum temperature was
37.5° C, where there was a very slight growth.

Fusarium trichothecioides is apparently unable to grow at 30° C,
which is the optimum temperature for F. oxysporum. Link (9) found the
greatest growth of F. trichothecioides in liquid potato extract media at
the end of 20 days to take place at 12° C, with no growth present at 30°
C, although the organism was capable of living in the potato tuber at
that temperature. Edson and Shapovalov (5) obtained a much higher
optimum for F. trichothecioides; they found the greatest growth took
place at 25*^ C, with a sharp drop to the maximum temperature at 30° C,
where germination of spores took place but no growth of mycelium.

68 Journal of Agricultural Research voi. xxii.no. 2

The writer, working with two strains of Fusarium trichoihecioides
in Petri dishes, found 25° C. to be the optimum temperature, and with
one strain he was able to obtain sHght growth, 7 mm. in diameter, at the
end of one week at 30° C. At 5° C. germination took place and there
was slight growth.

The optimum temperature for Fusarium radicicola was 30° C, the
same as for F. oxyspornm. The minimum was at 5° C, where a very
slight growth was produced in 10 days. At 35° C. the growth was greater
than with F. oxysporum, although the rate of growth was slower. Edson
and Shapovalov (5) report a similar optimimi temperature, with germi-
nation but no growth at 5° C. They found that at 39° C. a transforma-
tion from normal spores to chlamydospores took place.

In general it can be said that at 25° C. the growth for all three species
is nearly equal, Fusarium oxysporum, and F. radicicola increasing in
growth up to 30° C. and F. iriclwihecioides decreasing. The minimum
temperature for F. oxysporum is higher than for the other two, and in
general F. trichoihecioides appears to be more tolerant of the lower tem-
peratures than the others.

Preliminary experiments, using liquid media and determining the
growth by dry weights, have been conducted with a number of strains of
these three species. While on certain media the results have in general
corroborated the foregoing cardinal points for growth, they indicated that
these cardinal points may vary with the medium used. For instance,
with an nutrient solution made up of ammonium nitrate (NH^NOj),
potassium phosphate (KHoPOJ, magnesium sulphate (MgSOJ, ferric
chlorid (FeCls), and sucrose, the results compared well with those ob-
tained on agar in Petri dishes. With a nutrient solution made up similarly
to the potato extract medium used by Link (9), the total growth at the
higher temperatures was considerably less than the growth obtained in
the first nutrient solution, while at the lower temperatures the growth
was much greater. The optimum temperature for growth of Fusarium
trichoihecioides in the first nutrient solution was 25° C, with no growth tak-
ing place at 5° C. With Link's potato-extract medium the optimum lay
between 15° and 20° C, and there was weighable growth at 5° C. These
results would possibly account for the considerable discrepancy between
the results obtained by Link (9) with liquid media and those obtained by
the writer and by Edson and Shapovalov (5) with agar cultures.

EXPERIMENTAL INFECTION OF TUBERS

The cultures used in the following experiments, with their origin, are
listed below. In practically all cases the various strains of the same
species behaved alike. Several other strains of Fusarium oxysporum, iso-
lated by the writer, were also used in the experiments in addition to the
ones listed below.

Oct. 8,1921 Temperature and Humidity Studies of Fusaria Rots 69

No. I. — Fusarium oxysporum, isolated by the author from browned
vascular bundles of potatoes and identified by H. W. Wollenweber and
numbered at Washington as 3377. '^-''^ r. 1 ;■;■.

No. 8. Fusarium oxysporum, obtained from C. W. Carpenter of the
United States Department of Agriculture, No. 3395.

No. 32. — Fusarium oxysporum, obtained from G. K. K. Link, of the
University of Nebraska, as No. 3345a.

No. 28. — Fusarium. irichoihecioides obtained from G. K. K. Link.

No. 31. — Fusarium irichoihecioides, obtained from A. C. Pratt of the
United States Department of Agriculture.

No. 29. — Fusarium radicicola, obtained from A. C. Pratt and numbered
716.

METHODS

In all inoculation experiments with tubers, potatoes which were of one
variety, of the same age, and had been kept under the same storage con-
ditions were carefully selected for uniformity of size, type, and freedom
from wounds. The stem ends always were cut and examined for natural
infection, and all tubers showing vascular discoloration were discarded.
The tubers were always treated with formaldehyde or mercuric chlorid
and washed in sterile distilled water.

The inoculations were made by wounding the epidermis, usually by
stabbing to a depth of 3 mm. with a sterile scalpel. The inoculum was
introduced in various ways as outlined in the experiments.

Experiment i, December, 1915- — Potato tubers of the Up-to-Date
variety were inoculated by wounding the tubers and then dipping them
in a water suspension of spores, wrapping in sterile waxed paper, and
placing in moist chambers at 25° C. Controls were treated in the same
way, being dipped in sterile water. Results were taken 18 days later.

Set No. I. Four tubers inoculated with Fusarium. oxysporum, No. i.
All tubers completely rotted. The two control tubers remained sound.

Set No. 2. Four tubers inoculated with Fusarium oxysporum, isolated
from infected tubers in storage. All tubers showed a complete wetrot; the
tissue was soft and of a light brown color; a large cavity was present in
each tuber containing masses of white mycelium. At the point of inocu-
lation there was a granular mass of hyphae and starch grains separated
from the rest of the tissue. Control tubers remained healthy.

Set No. 3. Four tubers inoculated with Fusarium oxysporum, isolated
from wilted potato vines. All tubers showed a dark brown dryrot pro-
gressing only a short distance from the point of inoculation. Controls
remained healthy.

Reisolations were made from all the rotted tubers, and Fusarium oxy-

sporufn was recovered in every case. No bacteria or secondary invaders

were found in any of the tubers. These results show that F. oxysporum

is capable of producing a rot of the tuber in a saturated atmosphere at

54818°— 21 2

yo Journal of Agricultural Research voi. xxn.No a

25° C. The characteristic rot under these conditions is a soft we trot with
no sharp line of demarkation between the healthy and diseased tissue.
The organism appears to be unable to attack whole starch grains, which
accumulate in a granular mass with the myceliimi, as in set 2. The
tuber-rot under these abnormal conditions is not typical of the rots
usually found in storage.

Experiment 2, February 23, 1916. — Further tests were conducted at
the same temperature but with a lower relative humidity to test the abil-
ity of the organism to cause a rot under conditions not so adverse for the
host as in the previous experiment. Tubers of the Up-to-Date variety
were inoculated by wounding and then placing a little of the fungus
mycelium and spores in the wound. The tubers were then placed in a
sterile moist chamber but were not wrapped in paper. Controls were
treated and wounded in the same way. The experiment was run at 25°
C. Twelve different strains of Fusarium oxysporum were used for the
inoculations, two tubers being used for each strain. Results were taken
after five weeks.

In only one case had the rot extended three-fourths of the length of
the tuber. In all the other tubers there was only a slight rotting extend-
ing for a short distance from the point of inoculation. The controls
remained sound in every case. The tubers were in a saturated atmos-
phere at the beginning of the experiment, gradually becoming drier until
at the end the tubers were considerably dried out. Compared to the
preceding test the amount of rotting was very slight, and its inhibition
may be directly attributed to the dryness of the air. The slight amount
of rot around the point of inoculation would indicate that the fungus
progressed a short distance into the tuber at the beginning of the experi-
ment when the humidity was high but was unable to advance further
under the drier conditions. This would indicate that the rotting of
tubers already started could be checked by submitting the tubers to
lower humidities.

Experiment 3, April, 26, 1916. — A further test on the relation of
humidity of the atmosphere to the rot of the tuber was started. The
inoculations were made as in the previous experiment, and the same
variety of potatoes was used.

Set No. I. The inoculated tubers were placed in sterile chambers, and
moist filter paper was placed in the chambers at the start of the experi-
ment to produce a favorable humidity for the initial penetration of the
tuber.

Set No. 2. The tubers were placed in moist chambers in which the
atmosphere was kept saturated throughout the experiment.

Both sets were kept at a temperature of 25° C. The results were taken
after seven weeks. (Table I.)

Oct. 8,1921 Temperature and Humidity Studies of Fusaria Rots 71

Table I. — Comparative amount of rot produced by Fusarium spp. under different con-
ditions of relative hum,idity

Strain.

Set No. I. Set No. i.

One-third rotted

2-mm. rot

One-third rotted

Healthy

Entirely rotted.
Do.

Half rotted.

Control

Healthy.

In all cases where rotting was present the starch grains were not
corroded. Culture No. 8 seemed to have a much slower initial growth
than the others, thus showing a greater difference between the two sets.
In general, it can be clearly seen that the rotting was much greater in
set No. 2, where the atmosphere was saturated throughout the experi-
ment. Although the organisms were capable of starting a rot under the
moist conditions at the start of the experiment in set. No. i, they were
later considerably checked imder the drier conditions.

Experiment 4, March 15, 19 17. — Further infection experiments were
started under conditions in which the relative humidity of the atmosphere
was controlled by the use of various concentrations of sulphuric acid.
Previous experiments conducted at the Michigan Agricultural Experi-
ment Station in 19 15 and described under experiment 5, in which the
relative humidities were carefully controlled, produced very good results
with Fusarium oxysporum.

The apparatus used in experiment 5 was not available in 191 7, so the
relative humidities used in experiments 4 and 4A were determined from the
tables given by Stevens (aj). One-quart Mason jars were used, in which
were hung small wire baskets containing the tubers, the acid being placed
in the bottom of the jar. Tubers of the Rural New Yorker variety were
inoculated as in the preceding experiments. They were then placed in
the baskets in the sterilized jars and were sealed with paraffin and placed
at the desired temperatures. The experiment was run in duplicate.
Three strains of Fusarium oxysporum, two of F. trichothecioides , and one
of F. radicicola were used for the inoculations. The temperatures used
were 5°, 9°, 16°, and 25° C. While these temperatures varied somewhat
during the experiment, the extremes did not in any case overlap. The
relative humidities obtained by using sulphm-ic acid remained fairly
constant throughout the experiment. One hundred cc. of each of the
acid solutions were used for each jar. At the close of the experiment the
specific gravity of the solutions was taken, and the calculated humidity
at this time was compared with that at the start, with the result that the
one having 1.5 per cent relative humidity had changed to 3.6 per cent,
the 33 per cent to 49 per cent, and the 66.5 per cent to 74 per cent. These
variations were not considered great enough to cause conflicting results.

Journal of Agricultural Research

Vol. XXII, No. 2

The results were taken after seven weeks, and the penetration of the
tubers was measured in millimeters, as shown in Table 11. The number
of individuals was so small that slight discrepancies in the tabulated
results are found. Fusarium trichothecioides produced a slight rot at
lower temperatures than F. oxysporum but did not produce as extensive
a rot at the higher temperatures. At 5° C. the only rotting found was
with one strain of F. trichothecioides, at 100 per cent humidity. No
rotting was found at the temperature of 9° at the lower humidities, but
there was slight rotting at this temperature at the higher humidities,
especially with F. trichothecioides. It is noticeable that at 9° with the
relative humidities of 66 and 100 per cent, the amount of rotting is greater
than at the increased temperature of 16°, with the relative humidities of
I and 33 per cent. The same comparative results are found between
the amount of rotting taking place under the several humidities at a
temperature of 16° and of 25°. The results do not conform with the
report of Jamieson and Wollenweber {8), that penetration of the tuber
by F. trichothecioides is favored by low humidities. The work by Link (9)
and Wilcox, Link, and Poole {26), however, would indicate that more
rapid rotting takes place in an atmosphere of high humidity, thus agreeing
with the results shown in this experiment. The results of F. oxysporum
accord well with those obtained in experiment 3.

Table II. — Extent of penetration of tubers in experiment 4

Tem-
pera-

Approxi-
mate
relative
hiunidity.

Fusarium oxysporum.

Fusarium
trichotliecioides.

Fusarium
radicicola.

ture.

Strain i.

Strain 8.

Strain 32.

Strain 28.

Strain 31.

Strain 39.

°C.

Per cent.

5......

100

3 mm.

0.3

I imn.

I mm.

100

I mm.

3 mm.

I mm.

2 mm.

3 mm.

I mm.

100

10 mm.

lo mm.

I mm.

T mm.

2 imn.

5 mm.

lo mm.

I mm.

10 mm.

15 mm.

3 mm.

2 mm.

6 imn.

20 mm.

Krot.

I mm.

25 mm.

6 mm.

100

Krot.

Complete rot.

Krot.

I mm.

Krot.

Complete rot.

Experiment 4 A, May 16, 191 7. — In order to check up the possible
error due to differences in the age of the tubers used in the various tests,
the following experiment was started. New tubers of the Bliss Triumph
variety were used in comparison with tubers of the same variety that had

Oct 8, 1921 Temperature and Humidity Studies of Fusaria Rots 73

been kept in cold storage from the previous year. The experiment was
conducted in the same way as experiment 4, and the same cultures of
Fusarium oxysporum, F. trichothecioides , and F. radicicola were used for
inoculations. Only two temperatures were used, 13.5° and 25° C, as
well as two humidities, 33 and 100 per cent at each temperature. The
results shown in Table III were taken after six weeks.

As in experiment 4 the rotting was much greater at the high tempera-
tures and the high humidities. At the lower temperature of 1 3.5 ■^ C. there
was no distinct difference between the amount of rotting in the old and
new tubers, due to the very slight penetration at this temperature. In
the old tubers at 25° the infection in every case had been rapid and the
rotting had progressed much further than in the new tubers. These
results support the statement of Bisby (j) that old tubers are more sus-
ceptible to rot than new tubers.

Table III. — Extent of penetration of old and new tubers

Ap-

Fusarium oxysporum.

Fusarium

Tem-

mate

Tubers.

Fusarium
radicicola.
Strain 29.

ture.

relative

hu-
midity.

Strain i.

Strain 8.

Strain 32.

StiainaS.

strain 31.

Per

°C.

cent.

/New .

2 mm.

5 mm.

I mm.

4 mm.

2 mm.

S mm.

TO - i

33 noid..

2 mim.

2 ram.

I mm.

7 mm.

10 mm.

S mm.

13-5

ijNew.

5 mm.

I mm.

2 mm.

5 mm.

lOld..

5 mm.

4 mm.

I mm.

5 mm.

6 mm.

5 mm.

[New.

20 mm.

2 mm.

3 mm.

4 mm.

5 mm.

Xrot.

2,3

Old..

Contami-

15 mm.

Xrot.

5 mm.

Xrot.

nated.

100

[New .

Contami-
nated.

Krot.

Contami-
nated.

2 mm.

Krot.

loid..

5 mm.

'A rot.

15 mm.

10 mm.

>^rot.

Complete
rot.

Experiment 5, 19 15. — The results of earlier experiments having
indicated that the influence of the relative humidity was nearly as great
as that of temperature, it was decided to run a more complete test on
the effect of the relative humidities at different temperatures. Since
no apparatus was available by which the relative humidity and tempera-
ture could be controlled at will, it was necessary to construct one.

The principle employed in experiment 4 of using sulphuric-acid solu-
tions of varying specific gravity in a closed chamber to obtain the different
relative humidities was not used in this test. In preliminary experi-
ments conducted in the same way as experiments 4 and 4A, the infection
usually resulted in a softrot which gave good comparative results, but
the type of rotting was not similar to that usually found in storage.
Cultures from these softrots invariably yielded the Fusarium sp. used
in the inoculation, and no bacteria were present in any case. Apparently

74 Journal of Agricultural Research voi. xxii. no. 2

the absence of any aeration was the cause of this abnormal type of rot-
ting, and the following method was devised to allow for aeration.

The principle j[inally decided upon was that of passing a current of
air, kept at a constant pressure, through sulphuric-acid towers and then
over calcium chlorid and sodium hydrate. This gave a constant stream
of dry, sterile air. The air was then passed over sterile water to bring
it to a desired humidity. The amount of water necessary for a given
humidity was determined by trials, and the air was then passed into the
jars containing the tubers. An outlet was provided at the bottom of
the jar. These jars were connected separately with the current of air
and not in series. Relative humidities were obtained and used through-
out the experiment as follows: i, 30, 70, and 100 per cent. These rela-
tive humidities were used at three different temperatures — 9°, 12.5°,
and 25° C. The set at 25° was placed in an incubator in the laboratory,
the set at 12.5° was placed in a special low temperature incubator, and
the set at 9° was placed in a well-insulated ice box. Each of these
temperatures was maintained within a variation of 2° throughout the
experiment. In this way four gradations of humidity at each of three
temperatures were obtained. The method provided the tubers with suf-
ficient aeration and secured sterile conditions throughout the experiment,
since the jars containing the tubers were not moved or opened until the
end of the period.

The humidity readings were taken by the wet- and dry-bulb method,
the thermometers being inserted into the stream of air at the entrance
to the jar. The readings were found to vary, and at least 10 trial read-
ings were taken for each jar after the preliminary determinations were
made and the apparatus was set up. These readings ranged as follows :
I to 10 per cent, 20 to 40 per cent, 60 to 80 per cent, and 90 to loo per
cent. These were the greatest extremes found; and since a knowledge
of the approximate relative humidity is all that is necessary in an experi-
ment of this kind, these readings were taken to be sufficient, inasmuch
as they showed a gradual gradation from approximate dryness to satura-
tion. The ranges given above simply denote the possible error due to
the method of taking the readings. The humidity necessarily remained
constant, since the temperature, water surface, and air pressure were
constant. It was found to be impossible to use the wet- and dry-bulb
method to determine the relative humidity at the lower temperatures.
The changes of temperature caused by opening the door to make the deter-
minations were found to change the readings. Therefore the sets at 9°
and 12.5° C. were installed temporarily at 25°, the preliminary deter-
minations were made, and the readings were taken at that temperature
and corrections made by the use of psychometric tables (13).

The large battery jars were fitted with wire screen supports, and six
tubers were used in each jar — four inoculated and two controls. The

oct.8.i92i Temperature and Humidity Studies of Fusaria Rots 75

control tubers were separated from the inoculated ones by a thin layer of
cotton. The entire apparatus was disinfected with formaldehyde gas
before the experiment was set up.

The tubers used were of the Up-to-Date variety. They had been
kept over winter in a cool cellar, and a few sprouts which had started
were removed. They were inoculated by wounding the epidermis and
placing several drops of spore suspension in the wound. They were then
placed in the jars which were closed with cork tops and paraffined. The
inoculations were made with Fusarium oxysporum No. 8. The jars were
opened up and the tubers examined after five weeks.

Set I (9° C). At 10 and 30 per cent humidity the tubers were all
healthy.

At 70 per cent the tubers were sound with no penetration, although
there was a slight growth of mycelium on the surface of the tuber at the
point of inoculation.

At 100 per cent the condition of tubers was the same as at 70 per cent,
except that the external growth of mycelium was greater. All the
control tubers of this set remained healthy, and both the controls and
inoculated tubers had sprouted.

Set 2 (12.5° C). At 10 per cent humidity the tubers were healthy.
There was no invasion of the tissues.

At 30 per cent, same as above with a slight external growth of mycelium
at the point of inoculation.

At 70 per cent the tubers were about the same as at 30 per cent.
(PI. 10, A.)

At 100 per cent invasion of the tissue had taken place for about 2 mm.
beyond the wound, causing a slight browning of the tissue. On the
surface there was a slight brown discoloration for several millimeters
surrounding the point of inoculation and a slight growth of aerial myce-
lium. All the control tubers in set 2 remained healthy, and both control
and inoculated tubers were sprouting normally. (PI. 10, B.)

Set 3 (25° C). The control tubers remained healthy and sprouted
at 10 and 30 per cent relative humidity, while at 70 and 100 per cent
there was a slight disorganization of the tissue around the eyes and the
sprouts were all dead. No actual rotting was present or any fungus
growth.

With infected tubers at 10 per cent humidity all inoculations were
successful and uniform. The fungus invaded the tissue for 2 cm. around
the wound. Immediately below the surface at the point of inoculation
there was in every case a cavity lined with a white mycelial growth.
The tissue surrounding the cavity was of a granular appearance. Exam-
ined under the microscope it appeared to be made up of a tangled mass of
mycelium and starch grains. A sof trot extended out from this area, the
tissue being light brown in color and completely invaded by mycelium
(PI. 10, C).

76 Journal of Agricultural Research voi. xxii. No. 2

At 30 per cent humidity the rotting took place in the same manner
as at 10 per cent, except that the cavity was larger and the rot extended
through about 50 per cent of the tuber (PI. 11, B).

At 100 per cent humidity there was a total rot of all tubers, most of
the surface being covered with a white mycelial growth (PI. 11, C).

The results of this experiment show very clearly that relative humidity
plays a very important part in determining the amount of rot produced
by Fusarium oxysporum. The only rot appearing at the low tempera-
ture of 12.5° C. was in an atmosphere of 100 per cent humidity. Even
at the high temperature of 25° complete rotting did not take place
at the lower humidities. A gradual increase in the amount of rot
corresponding to the increase in humidity was present in every case.
The fungus can live and sporulate at the lower temperatures and lower
humidities used in this experiment but apparently is not capable of
penetrating the tubers under these conditions. It can be safely con-
cluded that F. oxysporum under good storage conditions is not capable
of producing a tuber-rot of great importance.

CONCLUSIONS

(i) Fusarium oxysporum, F. trichochecioides , and F. radicicola are all
capable of producing a rot of the potato tuber.

(2) In pure culture the amount of growth of all three species is nearly
equal at 25° C, Fusarium oxysporum and F. radicicola increasing in
growth up to 30°, where they produce their maximum growth. The
growth of F. trichochecioides decreases above 25°, until at 30° very little
or no growth takes place. It is more tolerant of the lower temperatures
than the other two species.

(3) Preliminary tests with different liquid media would indicate that
the cardinal points for growth of these Fusaria vary to some extent
with the medium used.

(4) Experimental infection of tubers was produced with all three
organisms under various conditions of temperature and relative humidit}'.

(5) Preliminary tests with Fusariivm oxysporum indicated that the
relative hmnidity plays a very important part in determining the amount
of rotting.

(6) In comparative tests with new and old tubers there is a distinct
difference in the amount of rotting under the same conditions. The
rotting was much more rapid and progressed much further in the old
than in the new tubers.

(7) Comparative tests with all three species at controlled relative
humidities from i to 100 per cent and at controlled temperatures from
5° to 25° C. proved conclusively that —

(a) A temperature of 25° C. is favorable for the production of a tuber
rot by Fusarium oxysportim, F. radicicola, and F. trichothecioides.

Oct.8.i92i Temperature and Humidity Studies of Fusaria Rots 77

(b) Fusarium oxysporum grows more rapidly and produces a more ex-
tensive rotting of the tuber than the other two at a temperature of 16° C.
and above.

(c) Fusarium trichothecioides is capable of producing a rot at much
lower temperatures than the others, in some cases causing rotting at
5°C.

{d) The relative humidity plays a very important role in determining
the progress of tuber rots and has the same influence on all three species.
In every experiment it was noticeable that there was a gradual increase
in the amount of rot corresponding to an increase in relative humidity.
With a high humidity at a given temperature the rotting was always
greater than at a temperature 5° to 10° C. higher but with a low humidity.
The Fusaria used can all live and sporulate at the low temperature of 9°,
and with low relative humidities, but they are not capable of producing
a rot under these conditions.

Inasmuch as the three species of Fusaria used in these experiments
represent the common types causing storage-rots of potatoes, it is clear
that considerable attention should be given to moisture as well as tem-
peratures where incipient rot occurs in stored tubers. It is also en-
tirely probable that a rotting of the tubers initiated at high temperatures
and high relative humidities could be completely checked by submitting
the tubers to lower temperatures and lower humidities.

LITERATURE CITED
(i) AppEt, Otto, and Wollenweber, H. W.

I910. GRUNDLAGEN EINER MONOGRAPHIE DER GATTUNG FUSARIUM (LINK).

Arb. K. Biol. Anst. Land. u. Forstw., Bd. 8, Heft i, 207 p., 10 fig., 3
pi. (i col.) Verzeichnis der wichtigsten benutzten Schriften, p,
196-198.

(2) Bary, Anton de.

1861. DIE gegenwartig herrschende kartopfelkrankheit, ihre ursache
UND ihre verhutung. eine pflanzenphysiologische unter-

SUCHUNG . . . 75 p., I pi. I^ipzig.

(3) BisBY, G. R.

1919. STUDIES ON FUSARIUM DISEASES OF POTATOES AND TRUCK CROPS IN MIN-

NESOTA. TECHNICAL. Minn. Agr. Exp. Sta. Bui. 181, 58 p., 30 fig.
Bibliography, p. 40-44.

(4) Carpenter, C. W.

1915. SOME POTATO TUBER-ROTS CAUSED BY SPECIES OF FUSARIUM. In Joiir.

Agr. Research, v. 5, no. 5, p. 183-210, pi. A-B (col.), 14-19. Litera-
tiire cited, p. 208-209.

(5) Edson, H. a., and Shapovalov, Michael.

1920. TEMPERATURE RELATIONS OF CERTAIN POTATO-ROT AND WILT-PRODUCING

FUNGI. Ir„ Jour. AgT. Research, v. 18, no. 10, p. 511-524, 9 fig.

(6) Frank, Albert Bernhard.

1898. UNTERSUCHUNGEN USER DIE VERSCHIEDENEN ERREGER DER KARTOFFEL-

FAULE. In Ber. Deut. Bot. Gesell., Bd. 16, Heft 8, p. 273-289.

jS Journal of Agricultural Research voi. xxn. No. a

(7) Humphrey, H. B.

i914. studies on the relation of certain species of fusarium to the to-
MATO BLIGHT OF THE PACIFIC NORTHWEST. Wash. AgT. Exp. Sta. Bul.

115, 22 p., 5 pi.

(8) Jamieson, C. O., and WollenwebER, H. W.

I912. an external dry rot of potato TUBERS CAUSED BY FUSARIUM TRICHO"

THEcioiDES, WOLLENW. In Jour. Wash. Acad. Sci., v. 2, no. 6, p.
146-152, I fig.

(9) Link, George K. K.

I916. A PHYSIOLOGICAL STUDY OP TWO STRAINS OP FUSARIUM IN THEIR CAUSAL
RELATION TO TUBER ROT AND WILT OP POTATO. In Bot. Gaz., V. 62,

no. 3, p. 169-209, 13 fig. Literatiire cited, p. 207-208. Reprinted as
Nebr. Agr. Exp. Sta. Research Bul. 9. 1916.

(10) Longman, Sibyl.

1909. THE DRY-ROT OF potatoes. In Jour. Linn. Soc. (London), Bot., v. 39,
no. 270, p. 120-129, pi. 10. Literature referred to in the text, p. 129.

(11) Manns, Thomas F.

I91I. THE FUSARIUM BLIGHT (WILT) AND DRY ROT OF THE POTATO. PRELIM-
INARY STUDIES AND FIELD EXPERIMENTS. Ohio Agr. Exp. Sta. Bul.
229, p. 229-336, 15 pi. (in text).

(12) Martius, Carl Friedrich Philipp von.

1842. DIE KARTOFFEL-EPIDEMIE DER LETZTEN JAHRE ODER DIE STOCKFAULE UN
RAUDE DER KARTOFFELN, GESCHILDERT UND IN IHREN URSACHLICHEN

VERHALTNISSEN ERORTERT. 70 p., 3 col. pi. Miinchen.

(13) Marvin, C. F.

1900. PSYCHROMETRIC tables FOR OBTAINING THE VAPOR PRESSURE, RELATIVE
HUMIDITY, AND TEMPERATURE OF THE DEW-POINT ... U. S. Dept.

Agr. Weather Bur. Bul. 235, 84 p., 2 fig.

(14) Pethybridge, George H., and BowERS, E. H.

1908. DRY ROT OF THE POTATO TUBER. In Econ. Proc. Roy. Dublin Soc, v. i,
pt. 14, p. 547-558, pi. 48-

(15) PiZZIGONI, A.

1896. CANCRENA SECCA ET UMiDA dellE patate. In Nuovo Gior. Bot. Ital.,
n. s. V. 3, fasc. i, p. 50-53.

(16) Pratt, O. A.

I916. A WESTERN FIELDROT OF THE IRISH POTATO TUBER CAUSED BY FUSA-
RIUM RADicicoLA. In Jour. Agr. Research, v. 6, no. 9, p. 297-310,
Pl- 34-37-

(17)

1916. CONTROL OF THE POWDERY DRYROT OF WESTERN POTATOES CAUSED BY

FUSARIUM TRicHOTHEcioiDEs. In Jour. Agr. Research, v. 6, no. 21,
p. 817-832, pi. 108.

(18) Reinke, Johannes, and Bertholdt, Gottfried.

1879. DIE zersetzung der kartoffel durch pilze. 100 p., 9 pi. Berlin.
(Untersuch. Bot. Lab. Univ. Gottingen. Heft i.)

(19) Schacht, Hermann.

1856. bericht an das konigliche landes-oekonomie-collegium uber die

KARTOFFELPFLANZE UND DEREN KRANKHEITEN ... 29, II p., 10 pi.

(6, 8, 9 col.). Berlin.

(20) Shereakofp, C. D.

1915. fusaria op potatoes. N. Y. Cornell Agr. Exp Sta. Mem. 6, p.
87-270, 51 fig., 7 pi. (col.). Literatiu-e cited, p. 269-270.

(21) Schlechtendal, D. F. L. de.

1824. FLORA BEROLINENSIS, PARS SECUNDA. CRYPTOGAMIA. Berlini.

Oct. 8, I92I Temperature and Humidity Studies of Fusaria Rots 79

(22) Smith, Erwin F., and Swingle, Deane B.

1904. THE DRY ROT OF POTATOES DUE TO FUSARIUM OXYSPORUM. U. S. Dept.

Agr. Bur. Plant Indus. Bui. 55, 64 p., 2 fig., 8 pi. Literature, p.
61-62.

(23) Stevens, Neil E.

I916. A METHOD FOR STUDYING THE HUMIDITY RELATIONS OP FUNGI IN CUL-
TURE- In Phytopathology, v. 6, no. 6, p. 428-432. Literature cited,
P- 432-

(24) Wehmer, C.

1896. UEBER DIB URSACHE DER SOGNENANNTEN "TROCKENFAULE" DER KAR-

TOFFELKNOLLEN. hi Ber. Deut. Bot. Gesell., Bd. 14, Heft 3, p. loi-
107, 3 fig.

(25)

1897. UNTERSUCHUNGEN iJBER KARTOFFELKRANKHEITEN. 2. ANSTECKUNGS-

VERSUCHE MIT FUSARIUM SOLANI (DIE FUSARIUM-FAULE.) In Centbl.

Bakt. [etc.], Abt. 2, Bd. 3, No. 25/26, p. 727-743, pi. lo-ii (i col.).

(26) Wilcox, E. Mead, Link, George K. K., and Poole, Venus W.

1913. A DRY rot of THE IRISH POTATO TUBER. Nebr. Agr. Exp. Sta. Re-
search Bui. I, 88 p., 15 fig., 28 pi. (i col.). Bibliography, p. 85-88.

(27) WOLLENWEBER, H. W.

1913. STUDIES ON THE FUSARIUM PROBLEM. In Phjrtopathology, V. 3, no. i,
p. 24-50, I fig., pi. 5. Literatiu-e cited, p. 46-48.

PLATE lo

Tubers inoculated with Ftcsariwrn oxyspomm and kept for five weeks at the fol-
lowing temperatures and humidities:
A. — 12.5° C, 70 per cent relative humidity.
B. — 12.5° C, 100 per cent relative humidity.
C. — 25° C, 10 per cent relative humidity.

(80)

Temperature and Humidity Studies of Fusaria Rots

Plate 10

Journal of Agricultural Research

Vol. XXII, No. 2

Temperature and Humidity Studies of Fusaria Rots

Journal of Agricultural Research

Vol. XXII, No. 2

PLATE II

Tubers inoculated with Fusarium oxysporum and kept for five weeks at the fol-
lowing temperatures and humidities :
A. — 25° C, 30 per cent relative humidity.
B. — -25° C, 70 per cent relative humidity.
C. — 25° C, 100 per cent relative humidity.

BLACKLEG POTATO TUBER-ROT UNDER IRRIGATION

By M. Shapovalov and H. A. Edson, Pathologists, Office of Cotton, Truck, and Forage
Crop Disease Investigations, Bureau of Plant Industry, United States Department of
Agriculture .

OCCURRENCE AND GENERAL APPEARANCE ' '■

A bacterial field decay of the potato tuber, the real nature of which
has not heretofore been adequately explained, prevails in certain irri-
gated sections of the West. In early harvest, when the diseased tubers
are apparently free from fungous invasion, the trouble has sometimes
been assumed to be "sunscald"; during the winter months it has fre-
quently been taken for a form of freezing injury. In other instances
it has been confused with the so-called "jelly-end rot" and attributed
either to Fusarium radicicola Wollenw. or to F. oxysporum Schlecht. It is
probable, also, that on superficial examination some such material has
been classed as "leak" {Pythium debar yanum Hesse), when conditions
favored an extremely rapid progress of the decay, whether in the field
or in transit.

Specimens of this decay were received by the writers in 191 7 and
19 1 8 from Idaho, Nevada, and California. In material received in
August the decay was soft and mushy (PI. 12, A-C). The aflfected
tissues were in part brown to black, but mostly only slightly colored or
colorless, though with a darker margin on the border line between the
healthy and diseased portions. Disintegration, originating at one end
of the tuber, was advancing irregularly over the surface. In some
areas the decay was confined to the outer layer, just beneath the epi-
dermis, while in others the deeper tissues also were involved. As a
rule, the disease started at the stem end, but occasionally the eye end
became infected first (PI. 12, B). Decaying material usually possessed
a disagreeable odor. It is this soft type of the rot which some were
inclined to regard as sunscald injury.

Specimens received later in the season, during the months of Novem-
ber and December, presented an entirely different appearance. The
affected portions were not mushy, but more or less tough or dry and
shrunken (PI. 12, D). The diseased area was dark brown in color,
except when a fresh decay developed under favorable conditions deeper
in the tissues. In the latter case it was practically of the same color as
the normal flesh of the tuber, but soft and mushy in consistency. When
such tubers were cut open and the cut surfaces exposed to the air, the
diseased portions turned brown or even black. If the progress of the
decay is completely anrested, the trouble may readily be mistaken for

Journal of Agricultural Research. Vol" ^^^^ ^°- '

Washington. D.C. Key No'.'g-»46

(81)

82 Journal of Agricultural Research voi. xxii. no. 2

an inactive stage of jelly-end rot or for an after effect of freezing injury,
particularly if the disease has made but little headway. The true
nature of such obscure cases of the disease may be revealed with cer-
tainty only by a series of cultural studies, coupled with experimental
work and field observations.

CAUSAL ORGANISM

Isolations were made from every tuber of each of the four samples
received from the West in 191 7-18. The results were surprising. In
no case was Fusariu^n radicicola obtained; only one tuber yielded F.
trichothecioides Wollenw. (from Nevada), two yielded Rhizoctonia
(from Nevada), two F. oxysporum (from California), and a few gave
miscellaneous, apparently saprophytic, fungi. Bacteria, on the other
hand, were constantly present in the cultures, even when slightly acidu-
lated potato agar was used. Carpenter (2)^ noted the presence of
bacteria in jelly-end rot material, but he regarded these organisms as
saprophytic, as they probably were. In the writers' cultures, however,
the constant prevalence of one type of bacterial colony in the dilution
plates was significant and warranted a detailed study of this organism.
In the subsequent inoculation experiments with pure cultures it proved
to be strongly pathogenic and produced a progressive decay of the
tubers as well as a disease of the stems. A study of the cultural and
biochemical features of the organism showed them to be fully in accord
with the published description of the blackleg bacillus (5).

MORPHOLOGY

Short rod with rounded ends, also short chains; 0.5 to 0.9 Xi.c to
2.2 ;u, average 0.6 X i-S /x; flagella few, peritrichiate; no endospores and
no capsules; stains well in aqueous gentian violet, aqueous methylene
blue, aqueous fuchsin, anilin water gentian violet, alkaline methylene
blue, and carbol fuchsin.

CULTURAL FEATURES

Agar stroke. — Growth moderate, filiform, flat to slightly raised,
glistening, smooth, slightly opalescent; white, no odor; consistency slimy
to butyrous; one strain distinctly viscid at first, but after a few replat-
ings it lost its viscidity.

Potato. — Growth moderate to abundant; filiform at first then spread-
ing, slightly convex changing to flat, glistening, smooth to slightly ru-
gose, yellowish white or dirty white; a decided odor of decayed potatoes
on the third to fourth day at 22° to 25° C. ; consistency somewhat slimy;
medium slightly grayed at first, changing later to either plainly gray, or
purplish, or brown, or a combination of these shades.

' Reference is made by number (italic) to " Literature cited," p. 91-92.

Oct. 8, 192 1 Blackleg Potato Tuber-Rot under Irrigation 83

Agar stab. — Growth somewhat best at top, abundant, spreading,
filiform to slightly papillate.

Gelatin stab. — Growth best at top, filiform along the line of punctm-e,
liquefaction, beginning on the first day at 20° C, varying in shape from
crateriform or funnel-shaped to saccate and broadly infundibuliform,
complete in 7 to 12 days.

Nutrient broth. — Usually slight ring and slight granular pellicle in
young cultures, clouding moderate to strong, persistent; medium not
discolored, odor absent; sediment compact, granular, somewhat dirty
white; one strain decidedly viscid at first, but losing this character after
a few replatings. j-mi/ibtia Jrff-io

MiivK. — Coagulation and extrusion of whey at 25° C, beginning on the
fourth day; coagulum not digested; one strain extremely viscid at first,
but losing its viscidity in later replatings; medium not discolored.

Acid production in milk. — A slight increase of acidity in milk cultiu-es
was noticeable after 24 hours. Two series of tests were made at certain
intervals within the period of 20 days, two to three cultures being used
on each day for every strain. The average progress of the acidity of three
western strains was as follows :

AGE OP REACTION IN

CULTURE. fuller's SCALE.

1 day ,, +12. 28

2 days .'. .: '. +13-49

3 days +22. 13

5 days +28. 00

10 days +32. 90

20 days .,;..„ . .^,. , i: K-. +41- 12

The average reaction of the control tubes was +11. 8 Fuller's scale.
Two strains received from Dr. W. J. Morse, of the Maine Agricultural
Experiment Station, were tested along with the western strains and gave
similar reactions, one ("B. sol.") showing 37.0 and the other ("IIIA")
40.75 acidity on the twentieth day. The cultures were grown at 22° to
25° C.

Litmus milk. — ^At 22° to 25° C. bleaching was complete at the end of
three weeks; thorough reddening was accomplished in seven weeks.

Gelatin colonies. — Growth rapid, form round, edge entire, lique-
faction saucer-shaped.

Agar colonies. — ^Surface colonies; growth rapid, usually round, but
occasionally somewhat irregular, flat to slightly raised, entire to slightly
undulate, finely granular with an internal ring surrounded by radiate
striations; color pearly white, bluish opalescent by transmitted light;
maximum diameter of colonies after 2 days 2 mm., after 3 days 4 mm.,
after 7 days 7 mm., after 14 days 9.5 mm. Buried colonies lens-shaped
to nearly spherical, edge entire, color slightly yellow under hand lens.

Fermi's solution. — Moderate clouding in 2 -day cultures; later
growth becomes copious. ii^ mc*^":

54818*— 21 8

84 Journal of Agricultural Research voi. xxii, no. 2

Corn's SOLUTION. — No growth.

Uschinsky's solution. — Growth was somewhat irregular in the ordi-
nary Uschinsky's solution but was uniform and copious in the modified
Uschinsky's solution, clouding being very strong on the fifth day.

Sodium chlorid in bouillon. — Growth slightly inhibited by 3 per
cent and more so by 4 per cent; no growth appeared in 5 per cent tubes
until the third day, and only occasional tubes containing 6 per cent were
clouded after 5 days. Morse reports no clouding for Bacillus atrosepti-
cus Van Hall in concentrations higher than 5 per cent. At the end of
two months, when conditions remained unchanged, transfers were made
from 6 per cent and 7 per cent sodium-chlorid cultures of the western
strains to sterile broth. In 48 hours all the transfers from 6 per cent
solutions showed growth, and in three days clouding appeared in the
majority of the transfers from the 7 per cent solutions, the remainder
being dead.

Growth in bouillon over chloroform. — Growth somewhat re-
strained at first, but increasing gradually. On the fourth day there was
a strong and uniform clouding in all cultures.

Best medium for long-continued growth. — Morse considers that
in the case of Bacillus atrosepticus neutral beef bouillon is best for this
purpose. In the western strains the writers observed that the organisms
can live even longer on the agar than on the broth when grown at ordinary
laboratory temperature of 22° to 25° C. Their death on agar appears
to be primarily associated with drying of the medium, while in broth it
seems to be due to certain chemical changes in the substratum and takes
place sometime before the liquid dries up completely. Six series of
parallel broth and agar cultures were made and tested at diff"erent inter-
vals, from 8 to 36 weeks, by making transfers to tubes of sterile broth.
It was found that occasional broth cultures showed a somewhat weak-
ened vitality, as demonstrated by retarded clouding, at the age of 16
weeks; some died after the expiration of 20 weeks, and none lived beyond
26 weeks. On the other hand, in no case was the agar culture dead
before 26 weeks, and some remained alive even after 36 weeks. The
experiment was carried on with 10 cc. of medium in each test tube.

PHYSICAL and biochemical FEATURES

Fermentation tubes. — Gas and acid production as well as growth
in the closed arm was observed with dextrose, lactose, and saccharose.
No acid and no gas with glycerin in cultures 1,3, and 5 days old.

Ammonia production. — Feeble (tested by Folin's aspiration method).

Nitrates in nitrate broth reduced to nitrites.

Indol production. — Positive, but very feeble both in young and old
cultures.

Toleration of hydrochloric acid and sodium hydrate. — The
writers' western organism grew in tubes having an initial reaction before

Oct. 8, 1921 Blackleg Potato Tuber-Rot under Irrigation 85

final sterilization of +20 and —20, Fuller's scale, but not in those
adjusted to +30 or to —30. Uninoculated tubes held as controls and
titrated at the close of a 24-day incubation period showed marked
changes from the original reaction, due doubtless in considerable measure
to the absorption of gases with resulting chemical change. Tubes cal-
culated for an initial reaction of -f 30 showed a final reaction of from +20
to +25; those originally +20 were about +15; those —30 were about
— 10; and those —20 were about —6. Transfers from inoculated tubes
calculated for an initial reaction of + 30 and above and — 30 and below
made 24 days after inoculation developed growth in some cases in tubes
from + 30, but not in those from more acid reactions nor from the alka-
line broths.

Vitality on culture media. — ^Long on bouillon, but still longer on
agar.

Temperature relations. — In freshly inoculated broth cultures
exposed 10 minutes, occasional retardation of clouding began at 45° C. ;
occasional growth was noted at various points between 46° and 50°;
and in no case was growth present after heating above 50°. Optimum
temperature for growth about 25°. Maximum temperature for growth
between 33° and 35°. Minimum temperature for growth below 5°.

Effect of sunlight. — Thinly sown agar plates exposed on ice for 30
minutes the latter part of March in Washington, D. C, resulted in 100
per cent killed.

Cytase production. — Five-day-old 30-cc. broth cultures in 300-cc.
Erlenmeyer flasks were precipitated by 160 cc. of 80 per cent alcohol,
filtered, and the precipitate dried promptly in the air. The papers con-
taining the dried precipitate were washed with 30 cc. of water, and the
washings were received in a flask to which a few drops of toluene and three
raw Irish potato disks 15 by 2 mm. were added. The disks gradually
assumed a soft, cheesy consistency but did not entirely disintegrate.
Microscopic examination showed the cells had lost coherence through
softening of the middle lamella. The cellulose lamella and the starch
content of the cells showed no evidence of change. Controls with
uninoculated broth did not soften the disks.

GROUP number 22 1. 1 II 3033

The last three points in this group number differ from those given by
Morse (5) but coincide with the respective figures in Jennison's {3)
revision, as reported by him at the fourth annual meeting of the Pacific
Division of the American Phytopathological Society. The writers feel,
however, that this may be largely a matter of interpretation of certain
results and not necessarily an indication of actual difference in the
organisms. Jennison studied 12 different strains of the blackleg bacillus,
includingseveralof Morse's strains; but the results he obtained, apparently,

86 Journal of Agricultural Research voi. xxh. No. »

were identical for all strains. The writers regard their western strains
as nonchromogenic, although a certain yellow discoloration in cooked
potato cultures might be taken as a suggestion of yellow pigment. They
obtained no evidence of diastatic action on potato starch, nor of acid
production with glycerin.

It may, therefore, be concluded on the basis of the characters described
that the pathogenic bacillus isolated by the writers from a peculiar soft
decay of western potato tubers is essentially identical with the organism
causing the blackleg disease of potatoes for which Appel's binomial
Bacillus phytophthorus is regarded to be correct by Smith (7) . Morse (5) ,
who was unable to obtain an authentic culture of Appel's strain for his
comparative studies of various blackleg organisms, believed that
B. atrosepticus should be chosen in preference to other names he had
under consideration, but stated that —

There is nothing in the data here presented which bears on the relation between
the organism originally described by Dr. Appel (/) as B. phytophthorits and the other
strains of blackleg bacteria.

EXPERIMENTAL WORK

The pathogenicity of the bacterial organism described above was
established by means of the following laboratory, greenhouse, and field
experiments.

PLANTING OF THE ORIGINAL MATERIAL

Preliminary to the inoculation work some of the diseased western
material was planted in the greenhouse as soon as the isolations were
completed. Four tubers were selected and cut in halves so as to make
eight seed pieces. Each piece was planted in a separate large pot filled
with sterilized soil. Of these seed pieces one decayed completely in the
soil before germination, six produced diseased plants, and one produced
a plant considerably weakened though not clearly diseased. Some of
the affected plants decayed while very young, others grew up to practi-
cally normal size, developing blackening of the stem above ground and
brown to black lesions on the underground portions. The lower leaves
turned yellow, but the upper leaves wilted while green. In two cases
the blackening of the stems was very intense (PI. 13, A) while in the
remainder the appearance was less typical of the familiar field symptoms
of the disease as it occurs in the eastern sections of the country. Black-
ening of the pith of the stem developed to the very top in one case.
When an affected plant was removed from the pot and the soil was care-
fully washed off, it could be seen that the infection had spread from the
seed piece to the stem (PI. 13, B). No tubers were produced in this
experiment. Healthy sprouting Irish Cobbler tubers were replanted in
these pots, but no infection was contracted by this new set of plants.

Oct 8, 1941 Blackleg Potato Tuber-Rot under Irrigation 87

INOCUIrATlON OF HEALTHY TUBERS IN THE LABORATORY

Over 60 tubers both new and old of the Netted Gem as well as of the
Irish Cobbler varieties were inoculated in small lots at dififerent times
with three strains of the western decay bacillus. When inoculations
were made in wounds of any kind, whether on the side of the tuber or at
either end of it, the results were invariably positive. The progress of
the decay was much slower when uninjured potatoes were inoculated.
In these latter cases the organism penetrated either through the eyes or
through the young growing sprouts. If the infected potatoes are re-
moved from the moist chamber after the decay has made considerable
headway and are exposed to the dry air of the laboratory, the diseased
tissues become shriveled and folded, resembling very closely the original
specimens of natural infection (PI. 14, A, C). Ordinarily if the infected
material is kept in moist chambers the decay is soft, mushy, spreading
either equally throughout the tissue or sometimes more on the surface
of the tubers, and is not confined to their piths as is usual in typical
cases of blackleg. The color of the decaying areas ranges from that of
the normal flesh to light or dark brown, often with blackish streaks or
stripes in younger portions nearer to healthy tissues, but never black
throughout. The margin is usually well defined, and there is no gradual
transition from dead to sound tissues. This internal appearance changes
considerably when tubers are taken from the moist chamber and are
exposed to drying. The decay of the bark is then more or less arrested,
and the disintegration centers mainly in the pith, so that a more or less
sound shell surrounds the centers of the active decay. The diseased tis-
sue is brown to black, the older regions becoming slimy (Pi. 14, B, D).
In all cases the decay gives off a very strong putrefactive odor.

PLANTING OF ARTIFICIALLY INOCULATED TUBERS IN THE GREENHOUSE

Seven Irish Cobbler tubers inoculated with the western bacterial or-
ganisms and partly decayed were planted in sterilized soil in pots. Four
showed subsequently a stem decay and three remained apparently un-
affected. One plant became girdled and died early. The disease ap-
peared first on the remaining three plants in the form of black streaks in
various positions on the stems, particularly at the leaf petioles. Later
on in some instances the entire stalk became black at the base. Tuber-
rot did not appear except on one tuber in one of the diseased pots. In
this case it was a soft, watery decay, light in color, not typical for black-
leg. The causal organism, identical with the original strains, was, how-
ever, recovered from this area. Healthy sprouting Irish Cobbler tubers
were immediately planted in the same pots in which these specimens
were grown. The new plants were very vigorous, and none of them con-
tracted the disease.

88 journal of Agricultural Research voi. xxn. No. a

INOCULATION OF HEALTHY STEMS IN THE GREENHOUSE

The stems of four young healthy potato plants were inoculated with
24-hour-old broth cultures of the three western strains of the bacillus
injected by means of a hypodermic needle. A severe decay with an
accompanying blackening resulted in all cases.

FIELD EXPERIMENTS

These experiments were conducted for two successive years at Arling-
ton Farm, Va. In 1 9 19 Netted Gems and Irish Cobblers were used. The
tubers were inoculated with the western strains of the blackleg organism
a few days before planting. Six whole tubers and 20 halves of the first
variety and 9 whole and 19 halves of the second variety were planted.
In addition a number of uninoculated pieces of each variety were planted
for controls. Planting was done on May 5. One half-tuber seed piece
of each variety decayed in the ground. On July i one hill from the cut
seed of Irish Cobblers was noted to show secondary symptoms character-
istic of blackleg — ^namely, yellowing and rolling of the leaves. There
was no blackening of the stem above ground. The underground portions,
however, showed brown lesions and a brown rot of the stem at the point
of attachment to the seed piece and somewhat above it. None of the
remaining plants showed symptoms of the disease. At digging time, on
September 15, no decay of the tubers was found, with the exception of
one very small tuber of the Netted Gem variety which showed a soft bac-
terial decay at the stem end. The progress of the decay, however, was
checked, and the affected portion fell off, leaving only the sound part,
so that the recovery of the causal organism was not possible.

Since the hot weather after May 5 might have had something to do
with the slight progress of the disease in 19 19, two sets of plantings were
made on another piece of ground on the same farm in 1920 — one on
April 8 and the other on May 6. Only Irish Cobblers were used this time.
Twelve tubers were cut in halves through the inoculated wound so as to
make 24 seed pieces for each of the two series. Inoculations were made
a few days before planting. Eight tubers were inoculated with the three
western strains of the blackleg organism and 4 with the "B. sol." strain
received from Dr. Morse. Up to July 20 four hills out of 16 inoculated
with the western strains in the earlier planting and 2 out of 8 inoculated
with " B. sol." in the same series showed typical field symptoms of black-
leg, including an intense blackening of the base of the stem. On the
other hand, no hill of the series inoculated with the same organisms and
on the same plan, but planted one month later, showed any signs of
infection. At harvesting time, on July 20, a number of tubers in the
planting of April 8 showed blackleg-rot, and in the later planting only
2 tubers were found showing the same decay. It appears, therefore,
that the earlier planting, when the soil and the air temperatures were

Oct. 8, 1921 Blackleg Potato Tuber-Rot und^r Irrigation 89

lower and the soil moisture was more abundant, greatly facilitated the
development of blackleg.

FIELD OBSERVATIONS

The typical case of blackleg-rot on round varieties in the East has been
figured in publications and charts issued by several agricultural institu-
tions. As a rule, decay begins at the stolon end of the tuber with a
comparatively small amount of rot visible on the outside or often only a
small, black, circular opening. This opening leads to the interior of
the tuber, where a progressive decay develops in the form of an irregular
black, soft, or slimy hollow until nearly all of the tuber is consumed
(PI. 15, D, B). However, the development of the disease may deviate
from this type even in eastern and northern sections of the United
States, when conditions are abnormal and favorable to the disease, such
as those in moist places or in wet seasons. Morse stated (4) with refer-
ence to blackleg in Maine that —

When this disease occxirs on a field it doubtless is responsible for much of the soft
rot of the tubers observed in wet seasons.

It appears from certain observations made by the writers that under
conditions of excessive soil moisture the bacteria in stems or seed tubers
may be carried at least to the adjoining tubers of the same hill. The
latter then become infected from the outside, or, if they are already
infected through the stolons, the infection spreads in moist surround-
ings more rapidly on the outside over the surface of the tuber, or evenly
throughout the flesh. Specimens of this sort were observed on the
Eastern Shore of Maryland and Virginia (PI. 15, G) and in Wisconsin,
Minnesota, and Washington (PI. 15, F) on various round types of pota-
toes. More accentuated symptoms of this order were found in irrigated
sections of Colorado. The most peculiar manifestations of the blackleg
tuber-rot were seen in the Snake River Valley of Idaho, where the
Netted Gem variety is grown on a large scale. The following forms were
observed there dm-ing a field survey arranged by the Office of Cotton,
Truck, and Forage Crop Disease Investigations in 1920.

I. The stem-end rot of pointed-end Netted Gems. The external
appearance of this form is extremely misleading (PI. A, 1-4). It becomes
prevalent in southeastern and eastern Idaho during the latter part of
the season, shortly before the harvest. The relatively low temperature
prevailing at this time of year is, no doubt, an important factor in the
rapid progress of the disease. If the soil has plenty of moisture, freshly
dug affected tubers show no shrinkage and preserve their natural shape
(PL 15, A-C). In the course of two weeks the shrinkage is evident and
the decay takes on an inward trend (PI. A, 4). By another two weeks,
drying and folding of the decayed tissues become very pronounced, and
the external appearance at this stage of the decay may well pass as an

90 Journal of Agricultural Research voi. xxn, ko. n

illustration of any of a number of stem-end tuber-rots (PI. i6, A, B).
When a Netted Gem tuber of pointed shape affected with this type of
the disease is cut open longitudinally as soon as it is removed from the
ground, four distinct regions of decay may, as the rule, be seen: (a)
The extreme stem-end region is usually decayed throughout ; it is mushy
or slimy in consistency and dark brown to black in color ; in the field this
seldom extends deeper than the outer demarkation line of the decay,
but in storage under favorable conditions the disintegration advances
more rapidly in the inner tissues of the tuber, taking on a cup-like shape
and leading ultimately to the formation of a slimy cavity (PL A, 4; 14, B,
D; 16, B). (6) An area of fresh decay appears within the core just
beneath the first region; it is practically colorless, though it occasion-
ally contains dark or black streaks, and in the very early stages it has
the consistency of hardened butter (PI. A, 4). (c) The cambium layer
shows a brown discoloration extending sometimes close to the eye end;
in advanced stages a portion of this region nearest to the stem end is
more or less disintegrated and forms a channel attentuating toward the
eye end until it gradually transforms into a mere browning of the vas-
cular network which also gradually loses its intensity and finally disap-
pears altogether; this condition is very distinct with some freshly dug
tubers, but later on with the inward progress of the decay it becomes
less pronounced (PI. A, i, 4; 16, B), {d) The decay of the outer layer
develops in the bark region, is soft but not mushy in consistency and
more or less dark-brown in color; it frequently extends over the tuber
much farther than the pith decay, but not always as far as the cambium
discoloration; its progress is checked after tubers are dug and exposed
to drying (PI. A, i, 2; 16, B).

2. The shallow stem-end rot of round-shaped tubers. This type was
observed mostly on the Idaho Rurals. Under conditions of abundant
moisture the bacterial infection spreads from the stem end over the
surface of the tuber and penetrates into the bark region, though not
very deeply. When such tubers are taken out of the ground and exposed
to the sun, as happens at digging, the infected areas dry up very promptly
and form hard, black, shallow patches (PI. 16, E). The condition may
easily be mistaken for the black fieldrot described by Pratt (6) and
attributed to Fusarium radicicola. If, however, the tubers are again
transferred to a moist place with a moderate temperature, a soft, mushy
bacterial decay is likely to develop beneath these dry areas. On the
contrary, under conditions unfavorable to the blackleg decay the status
may either remain unchanged or become complicated by the entrance
of various Fusaria and other rot-producing fungi. In the latter case it
is impossible to determine the original cause of the disease.

3. Siderot of either round or long potatoes. It may penetrate inside
of the tuber to a considerable depth, and when a freshly dug diseased

oct.8.r«>4i Blackleg Potato Tuher-Rot under Irrigation 91

tuber is cut open it reveals a colorless buttery or mushy decay with
black streaks, usually on the border line of the diseased and healthy
tissues. If exposed to drying the decayed areas may become spongy
and very much resemble the texture which is usually observed in Fusa-
rium rots (PI. 16, D). In many instances, however, if the decayed region
is sufficiently deep to prevent complete drying, sections through such
tubers may show an inner layer of active bacterial decay. As is the case
with the other forms of blackleg-rot this form, too, may become further
invaded with various rot-producing or saprophytic fungi. The writers
had under their observation a tuber of this type with a copious growth
of Rhizoctonia on the outside all over the diseased area, while soft
bacterial decay was still progressing within the tuber even in the dry
laboratory atmosphere (PI. 16, C).

SUMMARY

(i) An organism isolated from western stem-end rotting potatoes is
identical with Bacillus phytophthorus Appel in all the essential charac-
ters commonly considered in the determination of bacterial species.

(2) It is pathogenic to the potato, and inoculations of healthy stems
or tubers with pure cultures produce, respectively, a rapid, soft decay
of stems or a tuber-rot.

(3) Blackleg tuber-rot under the field conditions in certain irrigated
sections of the West, particularly in pointed-end Netted Gems, takes on
a form atypical of the familiar manifestation of this disease in the East.

(4) The external appearance becomes especially confusing when the
affected areas dry up and shrivel in storage, but usually the trouble
may be identified by cultural work or by planting diseased tubers under
control conditions.

LITERATURE CITED
(i) Appel, Otto.

1903. UNTERSUCHUNGEN USER DIE SCHWARZBEINIGKEIT UNO DIE DURCH
BAKTERIEN HERVORGERUFENE KNOLLENFAULE DER KARTOFFEt. In
Arb. K. Biol. Anst. Land. u. Forstw., Bd. 3, Heft 4, p. 364-432, 15 fig.,
pL 8 (coL).

(2) Carpenter, C. W.

1915. some potato tuber-rots caused by species op fusarium. in jout.
Agr. Research, v. 5, no. 5, p. 183-210, pL A-B (col.), 14-19. Litera-
ture cited, p. 208-209.

(3) JENNISON, Harry Milliken.

192 1. BACILLUS ATROSEPTICUS VAN HALL, THE CAUSE OF THE BLACKLEG DIS-
EASE OF IRISH POTATOES. (Abstract.) In Phytopathology, v. 11,
no. 2, p. 104.

(4) Morse, W. J.

1910. CERTAIN DISEASES OF MAINE POTATOES AND THEIR RELATION TO THE

SEED TRADE. Maine Agr. Exp. Sta. [Misc. Publ.] 375, 12 p.

92 Journal of Agricultural Research voi. xxn. No. 2

(5) Morse, W. J.

19x7. STUDIES UPON THE BLACKLEG DISEASE OF THE POTATO, WITH SPECIAL
REFERENCE TO THE RELATIONSHIP OF THE CAUSAL ORGANISMS. In

Jour. Agr. Research, v. 8, no. 3, p. 79-126. Literature cited, p. 124-126.

(6) Pratt, O. A.

I916. A WESTERN FlELDROT OF THE IRISH POTATO TUBER CAUSED BY FUSARIUM

radicicola. In Jour. Agr. Research, v. 6, no. 9, p. 297-310, pi. 34-37.

(7) Smith, Erwin F.

1920. AN INTRODUCTION TO BACTERIAL DISEASES OF PLANTS. XXX, 688 p., 453

fig. [pi.] Philadelphia and London. Literature at end of most of the
chapters.

Blackleg Potato Tuber-Rot under Irrigation

Journal of Agricultural Research

Vol. XXIi, N

rivATE A

Types of blackleg potato tuber-rot on pointed-end Netted Gem from Idaho, showing
external as well as internal appearance. The photograph was taken two weeks after
the tvibers were removed from the ground. The same tubers are shown in Plate 15,
A, C, on the first day after digging.

PLATE 13

Forms of blackleg tuber-rot in the West.

A-C. — Specimens received in August, 1918, from Fresno, Calif.

D- — Specimen received in December, 1917, from Fallon, Nev.

Blackleg Potato Tuber-Rot under Irrigation

Plate 12

Journal of Agricultural Researcii

Vol. XXII, No. 2

Blackleg Potato Tuber-Rot under Irrigation

Plate 13

Journal of Agricultural Research

Vol. XXII, No. 2

PLATE 13

Blackleg on stems resulting from planting the diseased western material.

A. — Appearance of plant above the grotmd.

B. — Spread of the infecion from the diseased seed piece to the new stem.

PLATE 14

Result of inoculation of healthy tubers with the bacterial organism isolated from
the western diseased material.
A, B. — Netted Gem variety.
C, D. — Irish Cobbler variety.

Blackleg Potato Tuber-Rot under Irrigation

Plate 14

Journal of Agricultural Research

Vol. XXII, No. 2

Blackleg Potato Tuber-Rot under Irrigation

Plate 15

'^t

\ \

Journal of Afrricultural Research

Vol. XXII, No. 2

PLATE IS

Different types of blackleg tuber-rot.

A-C— Characteristic appearance on fresh specimens of the Netted Gem variety
from Idaho.
D, E. — T3rpical development on round varieties in the East.

p^ G.— Other forms occurring on round varieties in various sections of the coimtry.
Arrows in A, B, and C indicate the border line of decay.

PLATE i6

Confusing forms of blackleg potato tuber-rot in the West (all specimens collected
in Idaho).

A, B. — ^Netted Gem variety one month after digging. Tuber B as it appeared on
the first day after digging is shown in Plate 15, B.

C. — Long Idaho Rural, showing secondary growth of Rhizoctonia on the outside
and active bacterial decay in the inside.

D. — Idaho Rural with deep side infection of blackleg decay which became dry
and spongy on exposure to the sun.

E. — Shallow surface infection which became dry and black when exposed to the
sun after digging.

Blackleg Potato Tuber-Rot under Irrigation

Plate 16

Journal of Agricultural Researcli

Vol. XXII, No. 2

MICROSCOPIC STUDY OF BACTERIA IN CHEESE

By G. J. HucKBR

Associate in Research, New York Agricultural Experiment Station

INTRODUCTION

Heretofore cultural methods have ordinarily been used in the study
of cheese flora, but the usual routine technic has given only an inade-
quate conception of the number and types of bacteria present. Although
these cultural methods were employed in order to secure an idea of the
number and varieties of organisms occurring in cheese, they have failed
to establish the relative abundance of each type of microorganism in
the cheese. This objection remains valid in the light of both quantita-
tive and qualitative studies.

The so-called "dilution technic," which involves the mass action of
the organisms, has proved valuable in determining the type or group of
organisms which predominates in a given sample ; but it does not furnish
information regarding the general flora. This method often tends to
give erroneous results, especially when the dilution medium used favors
the growth of special groups. Under such conditions the results are
influenced by the selective action of the medium. This has been true
where milk was used as a dilution medium in examining cheese. The
milk favored the growth of the lactic acid group; while the inert and
nonlactose fermenting types or slow-growing cocci were overgrown, due
in a large measure to the selective action of the medium. This procedure
has well served its purpose in assisting to isolate the organisms for
which it has a special adaptation.

In general, cultural methods are preferable to a microscopic examina-
tion because cultures can be isolated and studied independently — a
feature which will always remain the outstanding advantage of these
methods.

Although subject to the same limitations as any microscopic method,
the following method has been successfully used in this laboratory and
has been employed in a routine way in determining the number of bac-
teria in cheese.

HISTORY

Johan-Olsen {Sy, working with the molds which ripen "Gammelost"
(a Norwegian cheese), mentions a sectioning method and implies that
it resembles the usual histological technic but does not outline the
procedure in detail.

' Reference is made by number (italic) to "Literature cited," p loo.

Journal of Agricultural Research, Vol. XXII, No. i

Washington, D. C Oct. 8, 1921

(93)
54818°— 21 4

Key No. N. Y. (Geneva) ,-6

94 Journal of Agricultural Research voi. xxii. no. 2

Troili- Peterson {11), in discussing tlie bacterial flora of Swedish
"Giiterkase," mentions the microscopic examination of cheese as a
control for the cultural procedure but does not give the technical details.
She presents photomicrographs of cheese sections and states that some of
the preparations were stained in methylene blue and that a few were
examined unstained.

Gorini (j), in studying the distribution of the bacteria in Grana cheese,
presents the details of a method by which he prepared sections for
microscopic examination. In his procedure he fixed and dehydrated
samples of cheese by passing them through a series of alcohols of increasing
concentrations until a strength of 95 per cent was reached. The usual
histological methods of sectioning were followed, and the sections were
stained in an aqueous solution of methylene blue.

In the following year Rodella (9) reported a method used in his labo-
ratory for preparing sections used in the direct examination of cheese
samples. With his technic the samples were dehydrated and fixed by a
method similar to that of Gorini and sectioned in the usual way. He
found, however, that carbol-thionin gave better results as a stain than
did methylene blue.

Harrison (<5) outlined in detail a method for embedding and sectioning
cheese which is similar to the common histological method, but like his
predecessors he made no estimate of the number of bacteria present.

During the year in which Rodella (9) presented his paper, Troili-
Peterson {12) and Gorini (4) published notes discussing the question of
priority raised by the practically simultaneous publication of their
papers. It appears that the methods followed by Trioli- Peterson were
similar to those of Gorini, but that she did not feel the necessity of pre-
senting the technical details because of the universal knowledge of the
common embedding methods.

No results have been obtained in any of this work that permit a com-
parison between counts made by the plate method, so commonly used
in floral studies of cheese, and counts made by direct microscopic examina-
tion. Following the method outlined below, comparatively accurate
counts have been made by the direct method, and the number of the
different types of bacteria have been determined as they actually exist
in the cheese mass.

TECHNIC

EMBEDDING AND SECTIONING

The samples of cheese were embedded by the usual histological technic
and sectioned with a Minot rotary microtome. In sectioning, the micro-
tome was so adjusted as to give sections 5 ^i thick. The sections were
stained by the Gram method and with an aqueous solution of methylene
blue.

Oct. 8,1921 Microscopic Study of Bacteria in Cheese 95

In order to determine the effect of the embedding process upon the
cheese, small measured cubes of cheese were subjected to the routine
procedures. Only a slight shrinkage was found, indicating that the
volume of embedded cheese when examined is approximately the same
as that of the fresh sample.

MICROSCOPIC EXAMINATION

The preparations were examined with an oil immersion lens and a
high power ocular, the most satisfactory combination being a 1.9-mm.
fluorite objective with a numerical aperture of 1.32. Where a thick
coverslip was used it was necessary to have a 3-mm. apochromatic
objective with a numerical aperture of 1.4. Greater depth can be secured
with compensating oculars than with the ordinary Huygenian oculars.

The method, although at first used only for determining the types of
organisms present in the samples and as a check on the usual plate
method, was found useful as a means of determining the number of
organisms present. In order to make such a computation the micro-
scope was so standardized as to allow an estimate of the number of
organisms per gram when only a small amount of the original section
was examined. This computation is similar to that used in the direct
method of counting bacteria in milk described by Breed and Brew (2).
This was accomplished by measuring both the diameter of the micro-
scopic field and the thickness of the section from which the amount of
cheese actually seen in each field examined was determined. Knowing
the volume and specific gravity of the cheese examined, the total number
of organisms per gram can readily be computed. With the diameter
of the field measuring 0.14 mm. (140 fx), the microtome so adjusted as
to cut sections of a thickness of 0.005 nim. (5 ij.) , and a specific gravity of i,
the amount of cheese examined per microscopic field would be 1/13,000,000
gm. — that is, each organism observed in a single microscopic field repre-
sents 13,000,000 per gram.

This factor may be computed by the following formula, in which any
measure may be substituted :

1,000 , . ,
— -2 — 0 = factor per gram.

In the above formula,

r = the radius of the field examined in millimeters as determined by
actual measurement.

a = the thickness of the section in millimeters.

b = the specific gravity of the cheese.

The radius of the field, as has been stated, is determined by measure-
ment with a stage micrometer and varies with the magnification and with
the tyoe of ocular used. However, it was found advisable to adjust the

96 Journal of Agricultural Research voi. xxii. no. ,

draw tube of the microscope so that the field would be of the greatest
possible diameter without losing definition, as the greater the diameter
of the field the less the increment of error in the total counts.

The thickness of the section is controlled by adjusting the microtome
to cut sections of a desired and known thickness. If all the adjustments
on the microtome are firm and a sharp knife is used, sections can be cut
of uniform thickness with surprising accuracy. The thickness of the
sections can also be remeasured with the fine adjustment screw on the
microscope. Although not perfect, this method of measurement serves
as a check upon the accuracy of the sectioning. The measurement is
accomplished by focusing with the graduated fine adjustment screw on
both the upper and lower surfaces of the section and noting the differ-
ences in the readings between the two levels. The difference can be
read in microns where graduations are given on the fine adjustment
screw.

To convert the per-cubic-centimeter counts into numbers per gram,
the specific gravity of the cheese must be considered. As the specific
gravity of all samples has been assumed to be approximately i, the
counts are interchangeable. This assumption in regard to the specific
gravity is arbitrary, but the variations in the specific gravity of cheddar
cheese are so slight that the total count is not affected to any appreciable
degree. Accurate determinations did not seem practicable, as the speci-
fic gravity varies with the fat content and with the moisture and general
consistency of the cheese.

With the measurements and adjustments used in this laboratory the
per-gram formula resolves itself into the following:

r = 0.07 mm. (70 fx).

a = .005 mm. (5 /x).

6 = 1.0.

1 ,000

7—- X I = approximately i-; ,000,000.

3.1416 X 0.0049 X 0.005

APPLICATION OF THE METHOD

It is evident that this microscopic technic is subject to the limitations
of any direct method of examination, many of which are unavoidable
and are due to mechanical limitations or to the human error, which
enters in when counts or estimates are made.

QUALITATIVE EXAMINATION

As previously stated, cheese has been examined microscopically by
many investigators. The possibility of error is not as great when samples
are examined to determine the types of organisms present as when
total count is made, which is true of any microscopic work. Our present
staining methods make possible a direct visualization of the microorgan-

oct.8, igji Microscopic Study of Bacteria in Cheese 97

isms together with their morphological and other general characteristics,
but an attempt to enumerate these types involves other difificulties.

The direct examination of cheese in the different ripening stages is
advantageous and important, since the different groups of organisms
can be studied as they actually occur in the cheese mass, and their
groupings and relative relationships noted. The grouping may be
especially important when considered in relation to the number present.
For example, an organism may be present in large numbers during the
early stages of ripening, but appear in scattered and isolated groups
containing only a few individuals. In some instances only single bac-
teria were found through the mass. In such cases the total number of
this group by the plate count may be large, but the grouping, as deter-
mined by direct examination, may demonstrate that they are not ac-
tively growing and playing a part in the ripening of the cheese. On
the other hand, the presence of large clumps of organisms, with the size
of the clumps increasing during ripening, indicates that such groups are
developing in the cheese mass and are probably playing an important
r61e in the changes involved.

That this grouping of the organisms actually occurs can be seen in
Plate 17, A. In this photomicrograph are shown the types and groupings
of organisms found in a very green cheese, showing that the Streptococcus
lactis-like organisms predominated and were scattered in pairs over the
field. Any migration of these bacteria through the cheese mass appears
to have been impossible, and one is impressed with the fact that growth
and reproduction could not have been taking place rapidly or the number
of individuals per group would have been larger. In Plate 17, B, which
represents a section from a cheese 5 months older than that shown in
Plate 17, A, the organisms are found in larger clumps with many of them
so massed that accurate counting is impossible. From the examination
of a series of sections from cheeses of varying ages, it has been found that
the clumps increase in size as the cheese ripens, reaching a limit after
seven to eight months. It is evident that the organisms in the clumps,
mostly cocci and a few rods, are thriving and reproducing and must,
therefore, change the surrounding medium as they utilize it for food.
It is not within the scope of this paper to discuss the significance of this
occurrence but only to point out that such variations are found when
samples are examined directly.

QUANTITATIVE EXAMINATION

An objection often made to counting organisms in microscopic prepa-
rations of dried liquids is the uneven thickness of the resultant dried
film. This objection is eliminated when parafiin sections are used, as
such sections are uniform in thickness and the organisms remain in their
natural relationships. Boekhout and DeVries (z) at one time endeavored

98 Journal of Agricultural Research voi. xxii. no. 2

to show that the scattered organisms in cheese sections were due, in a
large measure, to the breaking up and scattering of the clumps by the knife
edge. This explanation will hardly appear plausible to anyone familiar j
with the perfection of delicate histological sections prepared with a sharp
knife.

The grouping and clumping of the organisms often cause difficulty in
accurately determining the number of organisms in the cheese sections.
This is especially true in sections of old cheeses in which the bacteria
tend to clump in large masses. The error can be overcome to a large
degree by counting or estimating a large number of fields, the larger the
number examined the smaller being the error in the final estimate.

In a sample of green cheese where the organisms appear in large num-
bers, but are evenly scattered, it is impracticable to count the entire
field, and an ocular disk divided into quadrants may be inserted in order to
facilitate accurate counting.

In all cases 20 or more fields should be counted, and especially where
the organisms are unevenly distributed. In such instances, typical
fields which represent the general flora should be located by studying the
entire section.

COMPARISON OF DIRECT AND PLATE COUNTS

Table I gives a few representative comparisons between direct micro-
scopic and plate counts made from cheese samples in various stages
of ripening. The plate counts average approximately one-twelfth the
direct count, but no common ratio has been found to exist between the
results obtained by the two methods. Wide variations in the ratios be-
tween the counts were found, but in general the ratios from green cheese
appeared to be larger than those from cheese more advanced in ripening.

The above plate counts compare well with those found by other observ-
ers who have examined cheddar cheese, Russell (10) found from 62 to
665 million per gram. Harrison and McConnell (7) found the count to be
as high as 625 million per gram in the earlier stages of the ripening, while
Harding and Prucha (5) observed from 37 to 177 million per gram.

Several explanations may be offered to account for the apparent
discrepancy between the results obtained by the two methods. The
plate count is an estimate based on observations of the growth of organ-
isms on some particular medium which, in cheese investigations, usually
contains lactose. Lactose has been generally used because media con-
taining this particular carbohydrate have been found to allow the develop-
ment of a larger number of colonies than do sugar-free media. Investigators
have based their cultural methods upon media giving the largest counts
rather than upon media which might serve as an index to the relative
number of types present. In comparing the microscopic counts with
results obtained with the plate method, it may be noted that the types

Oct. 8, 1921

Microscopic Study of Bacteria in Cheese

present in the cheese, as seen by direct examination, are not present
in the same proportions on the plates, because those types which grow
abundantly in the presence of lactose have outnumbered all groups
which do not grow as readily on such media.

Table I. — Relation between microscopic and plate counts obtained from cheese

Approximate age
of cheese.

Miscroscopic count (millions per gram).

Plate coimt (millions
per gram).

Cheese.

Cocci.

Short
rods.

Yeast.

Strep-
tococ-
cus
lactis.
(Lister)

Total.

Strep-
tococ-
cus
lactis.

Miscel-
laneous.

Total.

OOCIIXII 2....

100
14,650
312
1,690
962
338

600
S, 200

780
260
26

26"

4.500
21,450
143
6,760
988
3.029

5,200

41,300

481

9.230

2,210

3.393

46
264
132
251
306

284
90
41

121

55
548

OOCI3 XII 2 . . .

6. 4,11

BCII

292

427
702

6. 26, II

5 months, 23 days.

Unknown; a p -

peared giecn.

OO38I8

Results of plate counting may also be lower because of insufficient
grinding and emulsifying of the cheese sample previous to plating. This
appears to be especially significant in cases where investigators grind the
sample with sterile quartz or sugar and suspend the ground mass in
sterile water in preparation for plating. If the sample is not well ground,
small particles of cheese remain in the emulsion, and the individual bacteria
are not separated so as to allow them to grow into separate colonies on
the artificial medium. Emulsions examined under the microscope often
show comparatively large masses of cheese which have not been affected
by the grinding process.

CONCLUSION

The microscopic examination of cheese embedded and sectioned by
the usual histological method is a valuable and satisfactory method for
studying the different stages of cheese ripening. Such a direct method
of examination may be used to determine the number of organisms
present in the sample. It also serves as an index to the types of organisms
present and makes possible a study of the organisms as they actually
exist in the cheese mass, allowing observations on the groupings and
relationships during cheese ripening.

The cultural methods do not yield as high a count as the microscopic
method, due primarily to the selective action of the medium used and the
difficulty of liberating the organisms from the cheese mass previous to
plating.

A combination of microscopic and cultural studies yields a far more
complete picture of what takes place in cheese ripening than can be
obtained by the use of either method alone.

loo Journal of Agricultural Research voi. xxii, no. a

LITERATURE CITED
(i) BoEKHODT, F. W. J., and Ott de VriBS, J. J.

1899. UNTERSUCHUNGEN t)BER DEN RElFUNGSPROZESS DES EDAMER KASES.

In Centbl. Bakt. [etc.], Abt. 2, Bd. 5, No. 9, p. 304-307.

(2) Breed, Robert S., and Brew, James D.

I916. COUNTING bacteria BY MEANS OP THE MICROSCOPE. N. Y. State AgT.

Exp. Sta. Tech. Bui. 49, 31 p., 5 fig., i col. pi. Bibliographical
footnotes.

(3) Gorini, Costantino.

1904. SULLA DISTRIBUZIONE DEI BACTERl NEL FORMAGGIO DE GRANA. In

R. 1st. Lombardo Sci. Let. Rend, ser, 2, v. 37, fasc. 2, p. 74-78,
2 fig. (on I pi.).

(4)

1906. ZUR PRIORITAT DER METHODE DER KASEUNTERSUCHUNG DURCH MIKRO-

SKOPISCHE SCHNITTPRAPARATE. In Centbl. Bakt. [etc.], Abt. 2, Bd.
16, No. 1/3, p. 66.
(,5) Harding, H. A., and Prucha, M. J.

1908. THE BACTERIAL FLORA OF CHEDDAR CHEESE. N. Y. State Agf. Exp.

sta. Tech. Bui. 8, p. 120-193.

(6) Harrison, F. C.

1906. the distribution of lactic acid bacteria in curd and cheese of
THE CHEDDAR TYPE. In Rev. G6n. Lait, v. 5, no. 18, p. 409-415, 9
fig. (on 4 pi. ) References, p. 413.

(7) and CoNNELL, W. T.

1903. A COMPARISON OP THE BACTERIAL CONTENT OF CHEESE CURED AT DIF-
FERENT TEMPERATURES. In Rev. Gen. Lait, v. 3, no. 4, p. 80-85;
no. 5, p. 103-111; no. 6, p. 126-137, 1903 • "O- 7> P- i50~i55; iio- 8,
p. 173-180, 1904.

(8) Johan-OlsEn, Olav.

1898. DIE BEi kasEREifung wirksamen pilze. In Centbl. Bakt. [etc.],
Abt. 2, Bd. 4, No. 5, p. 161-169, 17 fig. (on pi. 4-9, 4 and 5 col.).

(9) RODELLA, A.

1905. EINIGES t)BER DIE BEDEUTUNG DER DIREKTEN MIKROSKOPISCHEN PRA-

PARATE FtJR DAS STUDIUM DES KASEREIFUNGSPROZESSES. In Centbl.

Bakt. [etc.], Abt. 2, Bd. 15, No. 4/5, p. 143-153- 5 ^g- (o" i pl-)-

(10) Russell, H. L.

1896. THE RISE AND FALL OF BACTERIA IN CHEDDAR CHEESE. In Wis. Agr.

Exp. Sta. 13th Ann. Rpt. i^g^jgb, p. 95-111, fig. 25-26 (26 on col.
fold, pi.)

(11) Troili-Peterson, Gerda.

1903. STUDIEN t;BER DIE MIKROORGANISMEN DES SCHWEDISCHEN GCTERKASES.

In Centbl. Bakt. [etc.] Abt. 2, Bd. 11, No. 4/5, p. 120-143; No. 6/7,
p. 207-2151, 5 fig. (on 3 pi.).

(12)

1905. BEMERKUNGEN ZUR DER ARBEIT VON A. RODELLA " EINIGES t)BER DIE
BEDEUTUNG DER DIREKTEN MIKROSKOPISCHEN PRAPARATE FCR DAS

STUDIUM DES KASEREIFUNGSPROZESSES." In Centbl. Bakt. [etc.],
Abt. 2, Bd. 15, No. 13/14, p. 430.

PLATE 17.

A. — Section of cheddar cheese i month old, stained with an aqueous solution of
methylene blue, showing isolated pairs of Streptococcus lactis Lister throughout the
field. X 500.

B. — Section of cheddar cheese 6 months old, stained as in A. X 800.

Microscopic Study of Bacteria in Clieese

Plate 17

Journal of Agricultural Research

Vol. XXII, No. 2

FURTHER STUDIES ON RELATION OF SULPHATES TO
PLANT GROWTH AND COMPOSITION

By Harry G. M1L1.ER
Chemistry Department, Oregon Agricultural Experiment Station

This is a continuation of an investigation, part of the results of which
were reported in a former pubHcation {4}} As stated in the earher paper,
the addition of the different forms of sulphur caused a marked increase
in the dry weight of red clover, and beneficial results were obtained with
oats and rape. One very noticeable result observed in the former work
was the high nitrogen content of the clover grown on soils in the green-
house receiving sulphur fertilizer, compared to that of clover receiving
only the residual sulphur of the soil.

This great increase in nitrogen assimilation by the clover where sul-
phates were applied, and under the conditions described, led the writer
to believe that the sulphates favorably influenced the activity of the
legume bacteria. Especially did this appear true where the beaverdam
soil was used. This soil contained o. 18 per cent sulphur with appreciable
quantities of sulphate sulphur in the soil extract, and no beneficial result
from sulphur fertilizer was expected. Oats did not respond to sulphur
with this soil, although the sulphur content of oats (j) and the amount of
sulphur removed by one crop of oats is as large as with a red clover crop.
From present data, the responses of red clover so often obtained with
gypsum compared to cereals can not be explained through a difference
in sulphur requirement. With alfalfa the amount of sulphur removed is
so large compared to the cereals and red clover that the addition of
sulphates would apparently function directly as a plant food where in-
creased growth results. An example of the latter would be the enormous
increases in the yield of alfalfa obtained in southern Oregon (7) where
sulphur fertilizers were applied to soils with a very low sulphur content.
These authors, however, mention the favorable action that sulphur ferti-
lizers had on the root development and nodule production of alfalfa.
Duley (2) reports increase nodule production on red clover where sul-
phiu- was added to soils. Pitz (5) observed increased nodule production
and root development with red clover by applying gypsum to soil cultures.

As far as the writer can ascertain, no correlation has been shown be-
tween nodule production and nitrogen content of the plant, by influencing
the development of the former, with ordinary sulphur fertilizer com-
pounds. In this paper a study has been made of the effect of different

' Reference is made by number (italic) to "Literature cited," p. no.

Journal of Agricultural Research. Vol. XXII, No.

Washington, D. C. Oct. 8, 1921

zt Key Na Greg. 7

(lOI )

I02

Journal of Agricultural Research

Vol. XXII, No. 2

concentration of sulphates on growth and nitrogen assimilation, and also
the relation of total sulphur content of the plant as influenced by available
nitrogen. The red clover and rape were used in this work. With clover,
the initial concentration of legume bacteria has been varied by inocu-
lating certain cultures, while others were not inoculated.

Table I. — Analytical results with red clover on Medford loam soil

Treatment.

Weight
of air-
dried
clover.

Total

in-
solu-
ble in
acet-
ic-
acid.

Total
S.

Sul-
phate
S.

Or-
ganic
S.

P.cl.
2. 88

P.ct.

2- 13

P.ct.
0. 20

P.ct.
0.06

P.ct.

0. 14

^■l'^

.04

It)

2.8l

.07

3-31

. 02

3- 30
3-36

2
2

15
22

. 10

. 12

16
16

3-58
3-48

31
19

•25

• 14

3-46
3-38

2
2

28
34

• 13

•IS

15
19

3-34

2
2

.27

•IS

14
18

Sin-
S in soluble
acetic- in
acid acetic-
solu- acid
tion. I solu-
tion.

Weight
of air-
dried
roots.

Total
S in
roots.

Total
Nin
roots.

Ratio
of

tops

roots.

2. 64

2. 77

2. 74
2- 73

Control:

Uninoculated . .

Inoculated

NaNOs:

Uninoculated . .

Inoculated

NaNOa and HaSOt:

Uninoculated . .

Inoculated

Na!S04:

Uninoculated . .

Inoculated

CaSOi:

Uninoculated . .

Inoculated

NaNOs and CaSOi:

Uninoculated . .

Inoculated

Gm.

15-84

31. 24

17. 70
21- 29

18.82

23-35

15. 00
20. 31

16.49

16. 6s

14. 18
16.89

.08
.07

• 15
-17

Gm.

6. 00

7- 6s

6.45
7.80

7. 60
7. 10

8.60
8.20

6. 10
6. 20

P.ct.

50
61

2
2

•55

-55

2. 39

3. 92

In the first experiment Medford loam soil, designated as soil B in the
previous publication (4), was used. This soil was heated in an electric
oven where the temperature was gradually raised to 120° C. and main-
tained for six hours. This was to destroy the legume bacteria present
in the soil. Four kgm. of soil, after being mixed with the different fer-
tilizers, were placed in paraffined clay pots and carefully seeded to red
clover. One series was inoculated with Bacillus radicicola Bey. The solu-
tion for inoculation was prepared by removing the growth of organisms
from a culture and mixing with water. Each inoculated soil culture re-
ceived a definite number of cubic centimeters of the bacterial solution,
concentrated where the seeds were placed. The amount of different fer.
tilizers added per pot was as follows : Sodium sulphate (NajSO^) , 3 gm. ;
sodium nitrate (NaNOj), 2 gm. ; calcium sulphate (CaS04.2 H3O), 3.75
gm. ; and calcium carbonate (CaCOg) , 3 gm. The cultures were placed
in the greenhouse on October 16 and harvested on March 18. Ten plants
were allowed to grow in each pot. The cool temperature in the green-
house did not permit rapid growth, and the plants were cut before ma-
turity was reached. The treatment and analytical results are given in
Table I. The cultures were weighed every other day, and the moisture
was maintained at 20 per cent. In removing the roots the soil was
shaken out of the pot and carefully loosened. The roots were then sepa-
rated out and washed. After drying they were weighed, and the non-

Oct. 8,1921 Relation of Sulphates to Plant Growth and Composition 103

volatile matter was determined by ashing a ground sample representing
each cultm-e. This was done to correct for any excess weight due to
adhering soil particles.

The total sulphur was determined by the sodium-peroxid method.
The sulphate sulphur was extracted by taking 2 gm. clover and 150 cc.
of water and digesting on the steam bath for three hours. It was then
slightly acidified with hydrochloric acid, and after standing for an hour
the extract was filtered. Five cc. of 10 per cent barium-chlorid solution
was used to precipitate the sulphate sulphur in the hot solution. After
standing overnight the barium sulphate settled to the bottom of the
beaker in all cases, and no particles of precipitate could be detected in
the supernatant liquid. This liquid was carefully decanted off; and the
white precipitate was washed on a Gooch crucible, dried, and weighed.
Several of these precipitates were ignited, but since no appreciable loss
in weight was detected this method appeared perfectly reliable for
comparable results on sulphate sulphur in the different samples of plant
material. For the determination of total nitrogen insoluble in acetic
acid, i-gm. samples were digested with about 150 cc. of water on the
water bath for two hours. The extract was then acidified with dilute
acetic acid and filtered after standing about 30 minutes. The total
nitrogen was determined on the precipitate by the Kjeldahl method.
The filtrate was made alkaline with sodium carbonate, evaporated to a
few cubic centimeters in volume, transferred to a nickel crucible, and
total sulphur determined by the sodium-peroxid method.

The dry weights of plant material produced show no increase in pro-
duction that can be attributed to presence of sulphates. This result is
different compared to the noticeable increase reported with the same
soil before (4). As mentioned above, conditions were very unsatisfactory
for growth, and the plants were cut before maturity five months after
planting. In the former work reported, conditions permitted rapid
growth, and the plants, though not mature, were harvested two months
after seeding. As the soil used in this later work had been heated, there
was perhaps some change in degree of solubility of soil minerals and in
the biological flora. The concentration of added mineral salts was also
greater in this work.

Examination of the roots showed that all plants had become infected.
In the uninoculated series, roots from cultures i and 2 contained very
few nodules compared to the roots grown in the soil receiving sulphate
fertilizer. This remarkable difference in nodule formation no doubt
accounts for the low nitrogen content of the clover plants in pots i
and 2. That these plants became infected without any artificial inocu-
lation is not surprising. Wilson (8) found that —

of fifteen legumes grown in Volusia silt loam soil, only one, Trifolium pratense,
developed nodules without artificial inoculation.

I04 Journal of Agricultural Research voi. xxii. No. a

During the growth of the plants the inoculated series showed greater
development, which is apparent upon examining the dry weights.
Cultures i and 2 of the inoculated series show no effects from lack of
sulphates, and all the sulphur-fertilized pots contained numerous well-
developed nodules.

The total nitrogen insoluble in acetic acid was no greater in some
of the cultures receiving added sulphates than in the controls, so no
statement can be made that sulphate addition caused this fraction to
become larger. The nitrogen content of the fraction soluble in acetic
acid is lower in i and 2 of the uninoculated series. The increase in
percentage of sulphur caused by fertilizer treatment is accounted for
generally by higher sulphate content. Although the organic sulphur
is apparently higher in some, the results are not consistent with the total
sulphur, to state that the former results from increased sulphur assimi-
lation in this experiment. Total sulphur in the acetic-acid extracts
runs parallel with sulphate sulphur results and is slightly higher, showing
that there is some sulphur in the organic form not accounted for in the
precipitate from acetic-acid solution. This was also found to be true
with clover grown in other pots which was harvested while in blossom.

The ratio in weight of tops to roots is greater in the inoculated sulphur-
fertilized cultures than in the uninoculated sulphur-fertilized cultures.
This difference does not appear to be due to inoculation alone, for this
does not hold true in comparing i and 2 of both series, while the ratio of
tops to roots in i and 2 of the uninoculated series is greater than the
remaining four where there is heavy nodule growth. Amy and
Thatcher (/) report a greater ratio in weight of tops to roots where
inoculation was made with alfalfa and sweet clover.

The sulphur content of the roots is larger than in the other portion
of the plant, whereas the opposite is true in percentage of nitrogen.

The second part of this work was carried on with beaverdam soil and
red clover. Each pot contained 7 kgm. of soil, and the following amount
of fertilizers were added as indicated in Table II: 12 gm. of calcium
sulphate, 10 gm. of sodium sulphate, 2 gm. of sulphur, and 6 gm. of
sodium nitrate. Two gm. of potassium chlorid and 10 gm. of calcium
carbonate were added to all the soil cultures. Twenty red clover plants
were allowed to grow in each pot, and the moisture content was kept at
40 per cent. The first crop grew at the same time as the clover on the
Medford loam soil and was also cut before the blossoming stage. Three
other crops were grown on these same cultures. The first was harvested
on March 24, the second on May 20, the third on July 9, and the fourth
on August 17. The last three crops were cut during the blossoming stage.
The results are given in Table II.

Oct. 8,1921 Relation of Sulphates to Plant Growth and Composition 105

TablS II. — Analytical results with red clover on beaverdam soil

Treatmeat.

Control

CaSOi and NaNOs.

CaS04

NaN03

NasSOi

Na2S04 and NaNOs

SandNaNOs

Crop I.

Weight
of air-
dried
clover.

Gm.
46

Total

P. a.

3-28
3- SO
3-41
3-25
3-47
3- 12
3-42
3-58

Total
S.

P.ct.

Crop II.

"Weight
of air-
dried

clover.

Gm.

102
91

80
83

Total

P.ct.

2. 70
2. so
2. 40
2-53
2.49
2.47
2. 56

Total
S.

P.ct.

Cropni.

Weight
of air-
dried,
clover.

G.m

69
66

Total

Sul-
phate
S.

P.ct.

o. 02

Or-
ganic
S.

P.ct.

O. 12

•15
. 12
•13

Treatment.

Control

CaS04and NaNOs..

CaS04

NaNOs

Na2S04

Na2S04 and NaNOs.
S.

S and NaNOs.

Crop IV.

Weight
of air-
dried
clover.

Gm.

Total N.

Per cent.

3- 16

Total S.

Per cent.
18

Sulphate
S.

Per cent.
03
15
12
06

Organic
S.

Per cent.
IS
IS
IS
16

.16
16
14

.16

Bxammation of the foregoing data shows no result from sulphate
application in the first two crops. There is no increase in dry weight
in the pots receiving sulphur fertilizers, and in some crops the yield on
the control is greater. In the third and fom'th crops, one distinctive
difiference appears in the lower nitrogen content of the clover grown on
the control soil cultures. The fact that the clover grown under condi-
tions supplying more sulphate sulphur has a higher nitrogen content
adds to the data already obtained pertaining to the favorable influence
that sulphates have upon legume bacteria, the action of which results
in a higher nitrogen content of the clover. According to experimental
evidence, sulphates do not increase nodule production on all legumes.
Wilson (8) reports that certain sulphates depressed nodule formation
on the soybean. On the other hand, Prucha (6) mentions magnesium
sulphate and calcium sulphate as exerting a beneficial influence on nodule
development of the Canada field pea.

The low nitrogen content did not appear to be due to the absence of
sulphates in the plant tissue, thus limiting protein synthesis, as sulphates
were present in all samples. However, the percentage of sulphate
sulphur was lower in clover grown on the control pots.

That the presence of available nitrogen or nitrogen assimilation by
the plant tends to control or limit the total sulphur assimilation is illus-
trated by data in Table III,

io6

Journal of Agricultural Research

Vol. XXII. No. a

In this experiment sea sand was used which had been washed with
dilute hydrochloric acid and large volumes of distilled water. The sand
still contained sulphur compounds, but no sulphates soluble in dilute
hydrochloric acid. Six kgm. of sand were mixed with lo gm. of calcium
carbonate and placed in paraffined clay pots. Where elemental sulphur
was used, 0.75 gm. was added at the same time. The other nutrients
added were applied in solution form through a period of 70 days; the
growing period was 80 days. The total amount of sodium sulphate
which was added varied in the different cultures. Concentration i
denotes 1.55 gm., concentration 2, 3.10 gm., etc. The same figures apply
to calcium sulphate too. All cultures, with the exception of No. 3,
13, 20, and 21, received 3.9 gm. of sodium nitrate, and they each re-
ceived 0.75 gm. Every culture received 2.6 gm. of potassium dihy-
drogen phosphate and 1.3 gm. of magnesium chlorid. Twenty plants
grew in each pot, and the moisture content varied fron 15 to 20 per cent
in the different cultures. The weights recorded are the average of
duplicates, and the analyses were made on a sample obtained by mixing
the duplicates. The 22 cultures from 11 to 21, inclusive, were inocu-
lated with legume bacteria and 5 gm. of beaverdam soil added to the
same culture.

Table III. — Analytical results of clover grown on sand receiving a nutrient solution

Uninoculated series.

Cul-
ture
No.

Form of sulphur
added.

NasS04

....do

Na2S04 and CaSO^ . .
NajSOi

Control

CaS04

....do

NajSOi and CaSO* . .

Sul-
phate
con-
cen-
tra-
tion.

Weight
of air-
dry
clover.

Gm.
9.65
10. 7

3-95

8.7s
6.9
4- IS
6.62
7. 02
S-I

Total
S.

Per ct.
o. 26
.42
•30
•30
•34

Total

Per ct.
3-76
3-77
2. 00
3- 50
3-69
3-90
3-75
3-8s
3- 80

Inoculated series.

Cul-
ture
No.

013
14
tS
j6
J7
18
19
a 20
a ji

Weight
of air-
dry
clover.

Gm.

10. 00
7. 00
4.80
6.4s
6.8s
7-3
7- 77
7-3°
5.90
7-5°
S-07

Total
S.

Perct.

o. 27
30

Total

Sul-
phate
S.

Perct.
3.62
3-71
3.49
3- 70
3.60
3-90
3- 69
3- 70
3- 90

2. 42
2.46

. 12
.08
.18

■ I.";
.08
• 17
.18

.09
.14

Or-
ganic
S.

Perct
IS

" Low nitrate.

In the foregoing data the low sulphur contents occur in the clover
grown in the pots receiving less nitrate nitrogen. In the inoculated series
the sulphur content of the clover does not appear to increase by increasing
the sulphate sulphur of the nutrient media. On the other hand, where
there is a reduction in nitrate added, there is an appreciable reduction in
the sulphur content of the clover. In the other series, where the concen-
tration of legume organisms was not as great at the start, the percent-
ages of sulphur generally run higher. In No. 3 the percentage of

Oct. 8,1921 Relation of Sulphates to Plant Growth and Composition 107

sulphur is not lower than in some of the others, but here the total yield
is small, and this often accounts for higher percentages of certain
elements. The jdelds in No. 3 and 6 are about the same; but the
sulphur content is higher in 6, although this culture depended only
upon the sulphur in the sand. No. 2 shows response in sulphur con-
tent to the higher concentration of sulphates in the media. The cor-
responding culture 12 in the other series does not show higher sulphur
content; and as the average sulphur content is lower in this uninoculated
series, it appears that the legume organisms might have some effect on
limiting the quantity of sulphur present in the clover hay.

Table IV. — Data showing the sulphur-nitrogen relation in the portion insoluble in

dilute acetic acid

CaS04

and
NaNOs.

In soil treated with-

NaNOa.

Na2S04.

NajSOi

and
NaNOs.

Average.

Ratio
of Nto
S in the
insolu-
ble por-
tion.

Aver-
age
ash
con-
tent.

Crop I :

Percentage of N insoluble in

acetic acid

Percentage of S soluble in

acetic acid

Percentage of S insoluble in

acetic acid

Crop II:

Percentage of N insoluble in

acetic acid

Percentage of S soluble in

acetic acid

Percentage of S insoluble in

acetic acid

Crop III:

Percentage of N insoluble in

acetic acid

Percentage of S soluble in

acetic acid

Percentage of S insoluble in

acetic acid

Crop IV:

Percentage of N insoluble in

acetic acid

Percentage of S soluble in

acetic acid

Percentage of S insoluble in

acetic acid

Inoculated series, Medford loam:
Percentage of N insoluble in

acetic acid

Percentage of S insoluble in

acetic acid

Uninoculated series, Medford loam :
Percentage of N insoluble in

acetic acid

Percentage of S insoluble in

acetic acid

Per a.
2. 26

.08
•13

I. 70
. II
.09

1.78

•13

.09

2.05
.19
. II

Per ct.

2. 24

.07
. 12

1-57
.06
.08

I. 76

•05
.09

1.97
.09
•13

Per ct.

2.58

.09

I. 64
.07
.09

1.87

.16
. II

Per ct.
2.15

.07
•15

1-73
. II
.09

1. 85
. II
.09

2. 12
.18
. II

Per ct.
2.31

• 13-
1.66

m-1

1. 81

>i9. 4

•93
2.03

•"5

2. 20
■125

I. 21
. II

ni-s

► 17. 6

Per ct.

9.94

7-31

6-95

54818°— 21-

io8

Journal of Agricultural Research

Vol. XXII, No. 2

To say that percentage of sulphur will not increase regardless of sul-
phate concentration in the nutrient media without increasing the avail-
able nitrogen would not be in accordance with data already obtained.
It does appear, though, that when the lack of nitrogen is sufficient to
lower the nitrogen content compared to the normal nitrogen content of
the clover there is a tendency toward decreased sulphur assimilation.
It is interesting to compare the rape plant with the clover in this respect.

The figures given in Table IV show that the clover cut before the blos-
soming stage not only contains a higher percentage of total nitrogen but
also a higher percentage of nitrogen insoluble in acetic acid. As the per-
centage of nitrogen removed by this fraction varies, so also does the per-
centage of sulphur. There appears to be a definite relationship between
the sulphur and nitrogen content in this insoluble portion, thus adding
more significance to this fraction in regard to quality and perhaps rep-
resenting the true protein of the clover hay. No difference in ash con-
tent caused by variation in fertilizer treatment was observed in the
diflerent pots. The ash content of the different crops did vary however,
as is sho\vn in Table IV.

EXPERIMENTAL WORK WITH THE RAPE PLANT

The Medford loam soil used in the first part of this work was used in
this experiment. After the clover roots were removed the soil was re-
turned to the pots and seeded to rape. Three gm., of sodium nitrate
were added to those cultures which had received nitrate nitrogen in
the clover experiment. The plants were harvested after a growing period
of 50 days. At the end of this time there had been a cessation of growth,
and the basal leaves dried up and fell off. The results appear in Table V.

Table V. — Analytical results obtained with rape

Treatment.

Control ,

NaNOg

NaNOg and Na, SO4. .

Na2 SO4

NaNOj and CaSO^
CaS04

Number

of
plants.

Weight.

Total N.

N insol-
uble in
acetic
ac'd.

Total S.

Sulphate Org
S. S

anlc
5.

Gm.

Per cent.

Per cent. Per

cent.

12. 0

1-93

0. 90

0.35

0. 16 0

S-o

2.88

•39

. 12

12 2

2-35

. 22

•03

10. I

4. 14

. 16

. 02

"•3

3-36

I. 46

I. 09

14. 0

2. 72

1.38

•97

2.4

2.57

3-^Z

2. 76

a 8

14-45

2-59
2.30

2,41
I. 26

2.05
■83

3b
43

0 6

16.35

2.27

1-31

•93

2.4

2. 69

3.28

2.98

1.8

2.66

2. 70

2-37

Ash.

Per

cent.
13.6
19.

IS-

3
9
o

18.3
17-7
22. o

19.4
14.7

16. 2
31. 9
21. 9

« Pots from the inoculated series.

The presence of added sulphates appeared to have retarded growth of
the rape, as there is a greater dry-weight yield on the controls. With

oct.8,i92i Relation of Sulphates to Plant Growth and Composition 109

the cultures receiving nitrate fertilizer the addition of sulphate sulphur
apparently caused increased growth or had just the opposite efifect.
If the concentration of the sulphates was great enough to produce a
toxic effect, the sodium nitrate may have counteracted this action.

The wjiter has observed just the opposite effect with clover seedlings
growing on agar agar-mineral salt nutritive media. One gm. of sodium
nitrate per liter had a noticeably toxic effect, while the same concentra-
tion of sodium sulphate produced no noticeably injurious effect. In the
cultures containing both the same concentration of sodium nitrate and
sodium sulphate there was an improvement in growth over the former
sodium-nitrate cultures.

Application of nitrates produced very good 5delds on a comparative
low sulphur assimilation by the plants. The question naturally arises
whether the rape does not absorb sulphur, if present, far in excess of that
required for carrying on the synthesis of its organic compounds. This
appears so noticeable in comparing the figures in Table V. Of course
it is realized that the optimum concentration of nutrients for plant
nutrition has always been a problem. The acetic acid-insoluble nitrogen
is higher in the rape grown on the soil receiving nitrate fertilizer only,
compared with that in the rape which received both nitrate and sulphate
fertilizer. There seems to be a tendency of the sulphates to decrease
this form of nitrogen. Sulphate application increased the organic
sulphur and total sulphur content of the rape, while at the same time the
presence of these sulphate compounds retarded growth where no nitrates
were added. The extremely high sulphate content is very obvious in
these samples of rape. This may account for the high ash content. The
percentage of ash in the samples of rape varies considerably, depending
upon the fertilizer treatment and magnitude of growth. Such a variation
did not occur with the clover.

SUMMARY

Sodium sulphate and calcium sulphate had a beneficial effect on
nodule development and nitrogen assimilation of the red clover grown
on previously sterilized soil. On a similar series which was artificially
inoculated with Bacillus radicicola at the time of seeding, sulphates
caused no increase in nodule development.

When a soil of high sulphur content was used, the nitrogen content in
clover of the third and fourth crops was lower on the control pots than
where either sulphur, calcium sulphate, or sodium sulphate was applied.
As sulphate sulphur was present in all plants, the low nitrogen content
could not be explained by a cessation in protein synthesis due to the
absence of sulphates.

This again shows the relation of sulphates to nitrogen assimilation and
the favorable influence of sulphates on the legume bacteria or on some
other agency controlling nitrogen assimilation.

no Journal of Agricultural Research voi. xxii, no. 2

The ratio of nitrogen to sulphur in the portion of the clover plant
insoluble in dilute acetic acid remains about the same, regardless of the
stage in the development of the plant. This gives further support to the
view that the nitrogen insoluble in acetic acid represents protein nitrogen.
The total nitrogen and total nitrogen insoluble in acetic acid wias higher
in those plants cut before the blossoming stage.

With clover growing on sand cultures, it was possible, by reducing the
available nitrate, not only to limit the growth and nitrogen content but
also to decrease the sulphur assimilation. So, while sulphates appar-
ently cause greater nitrogen assimilation through their beneficial effect
on nodule development, the amount of sulphur taken up by the plant is
limited by the total nitrogen absorbed.

The rape plant assimilated a large amount of sulphur, although the
presence of sulphates reduced the yield compared to the control soil
cultures. Sulphate plus nitrate caused increased yields compared with
those secured when nitrate was added alone. There does not appear
to be any direct relation between nitrogen and sulphur assimilation in
the rape plant.

LITERATURE CITED
(i) Arny, a. C, and Thatcher, R. W.

1915-17. THE EFFECT OF DH'FERENT METHODS OP INOCULATION ON THE YIELD
AND PROTEIN CONTENT OP ALFALFA AND SWEET CLOVER. In JOUT. Amer.

Soc. Agron., v. 7, no. 4, p. 172-185, 1915; v. 9, no. 3, p. 127-137.
1917.

(2) DULEY, F. L.

1916. THE RELATION OF SULFUR TO SOIL PRODUCTIVITY, /n JoUT. Amer. Soc.

Agron., V. 8, no. 3, p. 154-160.

(3) Hart, E. B., and Peterson, W. H.

191 1. SULFUR REQUIREMENTS OF FARM CROPS IN RELATION TO THE SOIL

AND AIR SUPPLY. Wis. Agr. Exp. Sta. Research Bui. 14, 21 p.

(4) Miller, H. G.

I9I9. RELATION OF SULFATES TO PLANT GROWTH AND COMPOSITION. In Jour.

Agr. Research, v. 17, no. 3, p. 87-102, pi. 9-12. Literature cited,
p. 100-102.

(5) PiTz, Walter.

1916. EFFECT OF ELEMENTAL SULFUR AND OF CALCIUM SULFATE ON CERTAIN

OF THE HIGHER AND LOWER FORMS OF PLANT LIFE. In JoiW. AgT.

Research, v. 5, no. 16, p. 771-780, pi. 56.

(6) Prucha, Martin J.

1915. PHYSIOLOGICAL STUDIES OP BACILLUS RADICICOLA OF CANADA FIELD PEA.

N. Y. Cornell Agr. Exp. Sta. Mem. 5, 83 p. Bibliography, p. 79-83.

(7) Reimer, F. C, and Tarter, H. V.

I919. SULFUR AS A FERTILIZER FOR ALFALFA IN SOUTHERN OREGON. Oreg.

Agr. Exp. Sta. Bui. 163, 40 p., 9 fig. Bibliography, p. 39-40.

(8) Wn^ON, J. K.

1917. PHYSIOLOGICAL STUDIES OF BACILLUS RADICICOLA OF SOYBEAN (SOJA

MAX piper) AND OF FACTORS INFLUENCING NODULE PRODUCTION.

N. Y. Cornell Agr. Exp. Sta. Bui. 386, p. 363-413, fig. 80-94. Bibli-
ography, p. 412-413-

SOYBEAN MOSAIC '

By Max W. Gardner, Associate in Botany, and James B. Kendrick, Assistant in
Botany, Purdue University Agricultural Experiment Station

In a small field of Hollybrook soybeans in West La Fayette a typical
mosaic disease was found August 25, 1920. A rather low percentage of
the plants were affected, and the disease was more or less confined to
one quarter of the field adjacent to which were several rows of garden
beans affected with mosaic to a considerable degree. In another larger
field of soybeans in the same locality no mosaic was found. Leafhoppers
were very prevalent on the soybeans. The impression was gained that
the disease might have spread from the garden beans to the soybeans,
but as yet no evidence to support such a theory has been obtained.

Clinton^ found soybean mosaic in 19 15 at Mount Carmel, Conn., and
under the name of chlorosis or crinkling has given an excellent account
of the leaf symptoms along with a good illustration. He found the
disease on the varieties Medium Green, Wilson, Swan, Kentucky, Wing's
Mikado, and Hollybrook, and states that the Hollybrook showed the
most marked symptoms. He found the chlorosis without the crinkling
on the varieties O'Kute, Ito San, and Manhattan. C. R. Orton ^ has
reported the occurrence of mosaic in a field of Ito San soybeans at
Girard, Pa., July 30, 1920.

SYMPTOMS

The mosaic symptoms on the soybeans were conspicuous and unmis-
takable, resembling those characteristic of mosaic diseases in general.
Affected plants were stunted, and petioles and intemodes were shortened
to some extent. The leaflets were stunted, greatly misshapen, and puck-
ered with dark-green puffy areas along the veins (PI. 18, A, C, D, B).
Between these puffy areas the leaf tissue was etiolated. Affected leaflets
tended to be asymmetrical, twisted, and curled downward about the
margins (PI. 18, D, E). As in other mosaic diseases, the young, rapidly
growing leaves showed the most severe effects, and in some cases whole
leaflets or portions thereof were extremely stunted or killed outright by
the disease (PI. 18, B). The mosaic symptoms were readily distinguish-
able from a uniform crinkling of the leaflets which was rather common
in this field and apparently attributable to insect injury.

The pods on mosaic plants were stunted and flattened, less pubescent,
and more acutely curved than those on normal plants (PI. 19, C, D).

' Contribution from the Botanical Department of Purdue University Agricultural Experiment Station,
I^a Fayette, Ind.

2 Clinton, G. P. notes on plant diseases of Connecticut. In Conn. State Agr. Exp. Sta. Ann,
Rpt., 1915, p. 446-447, pi. 23a. 1916.

3 Fromme, F. D. diseases of cereal and forage crops in the united states in 1920. In U. S.
Dept. Agr. Bur. Plant Indus. Plant Disease Bui., Sup. is, p. 173- 1921- Mimeographed.

Journal of Agricultural Research, Vol. XXII, No. a

Washington, D. C. Oct. 8, 1921

zu Key No. Ind. -10

(III).

112 Journal of Agricultural Research voi. xxii, No. 2

Those borne at the upper nodes were more severely affected. The yield
of seed was very materially reduced (PI. 19, A, B), since a considerable
proportion of the pods contained no germinable seeds and the remainder
as a rule not more than one or two seeds (PI. 19, D) . Even the germinable
seeds were in general undersized.

Observations made a month later showed that the mosaic plants were
remaining green longer than the normal plants, so the disease evidently
delayed maturity.

FIELD INOCULATIONS

In another field of soybeans in which no mosaic was present inocula-
tions were made August 2 7 by rubbing the young intemodes with cotton
soaked in the juice from crushed mosaic soybean leaves and then wound-
ing these intemodes with a needle. One hundred and fourteen plants were
thus inoculated, but no mosaic developed. Fifty- two plants were simi-
larly inoculated, except that the juice of leaves from mosaic garden beans
was used as inoculum, and none developed the disease. Forty-six garden
bean plants were also inoculated in a similar manner with the virus from
soybean mosaic, and none developed mosaic.

SEED TRANSMISSION

To determine whether or not the disease was seed-borne, a quantity
of seed was saved from mosaic and healthy plants early in October for
subsequent tests in the greenhouse. On October 25, 150 seeds from
mosaic plants were planted in 25 pots of sterilized soil, 6 in each pot.
By December 15, 124 plants had come up, and 18 showed unmistakable
mosaic symptoms. None of the 148 controls grown from seed from
normal plants showed mosaic.

In a second trial about 180 seeds from mosaic plants were planted
December 9 in 59 pots of sterilized soil. February 3, 192 1, 11 out of the
106 plants which were up showed mosaic. None of the 38 controls
grown from seed from normal plants showed the disease. As a result
of these two tests it is evident that about 13 per cent of the seedlings
from seed produced on mosaic plants developed the disease.

The mosaic seedlings were spindling (PI. 18, F, G), and the j&rst pair of
true leaves were characterized by downward, longitudinal curling or
rolling, a crinkling, and a faint etiolation or mottling. These leaves
turned yellow prematurely. The leaves subsequently formed were
greatly stunted and showed the mottling and crinkling more conspicu-
ously than the first leaves.

GREENHOUSE INOCULATIONS

From these mosaic seedlings the disease was transmitted to healthy
soybean seedlings. Several methods of inoculation proved successful.
A number of inoculations made early in January yielded only negative
results, but later better success was obtained.

Oct. 8,1921 Soybean Mosaic 113

On January 26, twenty-five plants were inoculated by pricking with a
needle at the nodes and rubbing the wounded areas with cotton soaked
in the juice from crushed mosaic leaves. Eight plants used as controls
were similarly treated, except that sterile water was substituted for
the mosaic virus. Because of the unfavorable greenhouse conditions
the plants made slow growth during the winter, so that the mosaic
symptoms were very slow in developing. On March 5 two plants
showed mosaic mottling on the young leaves. On March 25 two more
showed mosaic, and on April 7 seven out of the 25 plants had developed
the disease. The controls developed no mosaic.

A number of inoculations were made March 2. In one series crushed
mosaic tissue was inserted into slits made with a scalpel near the grow-
ing points and on the petioles. On March 15 two of the seven plants
thus inoculated showed mosaic symptoms on the young leaves, and on
April 7 five had developed mosaic.

In a second series of inoculations made the same date by cutting off
one leaf at each node and smearing these wounded surfaces with crushed
mosaic tissue, three out of eight plants showed mosaic symptoms on
the new leaves March 15, or 13 days after inoculation, and on April 7
six plants had developed mosaic.

In a third series five plants were inoculated by a combination of the
two methods above described. Thirteen days later three showed mosaic
mottling, and by April 7, or 37 days after inoculation, four had developed
the disease.

In a fourth series, five plants w^e inoculated by rubbing the under
surfaces of the leaves with slightly crushed mosaic leaves forcibly enough
to cause slight abrasions. On March 15, four of these plants showed
the disease, and on April 7 all showed typical mosaic.

None of the five control plants inoculated by one or the other of these
methods without the application of mosaic tissue developed mosaic.
At no time was there any spread of the disease in the greenhouse.

In these inoculations the symptoms became evident only on the
young leaflets. These in some cases developed distinct mottling, and
in other cases they exhibited a slight degree of etiolation and the char-
acteristic downward, longitudinal rolling. The incubation period
under the conditions of this test was 13 days.

Preliminary cross inoculations to garden beans and cowpeas have
given negative results. Further tests are being made.

Soybeans, therefore, are subject to a destructive mosaic disease which
greatly reduces the yield of affected plants. The disease is transmis-
sible from plant to plant and also is seed-borne.

PLATE i8

A. — Typical mosaic leaf showing darker green puffy areas along the veins,
B. — Mosaic leaf showing extreme sttmting of terminal leaflet.
C. — Normal leaflet.

D. — Mosaic leaflet showing longitudinal rolling.
E. — ^Typical mosaic leaflet.

F. — Mosaic seedlings from seed from a mosaic plant, showing stunting of the plant
and longitudinal rolling of first leaves.
G. — Normal seedlings from seed from a mosaic plant.

Soybean Mosaic

Plate If

Journal of Agricultural Research

Vol. XXII, No. 2

Soybean Mosaic

Plate 19

Journal of Agricultural Research

Vol. XXII, No. 2

PLATE 19

A. — Upper nodes of a normal plant, showing yield of pods.
B. — Upper nodes of a mosaic plant, showing effect of the disease on the yield.
C. — Normal pod.

D. — Type of pods produced by a mosaic plant.
54818° — 21 6

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Vol. XXII OCTOBER 15, 1921 No. 3

JOURNAL OP

AGRICULTURAL

RESEARCH

CONXKNXS

Page

Influence of the Plane of Nutrition on the Maintenance
Requirement of Cattle - - - - - - -115

F. B. MUMFORD, A. G. HOGAN, and W. D. SALMON

( Contribution from Missouri Agricultural Experiment Station )

Turnip Mosaic - - - - - - - - 123

MAX W. GARDNER and JAMES B. K^NDRICK

( Contribution from Indiana Agricultural Experiment Station )

Hydrocyanic Acid in Sudan Grass - - - - - 125

C. O. SWANSON

( Contribution from Kansas Agricultural Experiment Station )

Nutrient Requirements of Growing Chicks: Nutritive
Deficiencies of Corn - - - - - - - 139

F. E. MUSSEHL, J. W. CALVIN, D. L. HALBERSLEBEN
and R. M. SANDSUEDT

( Contribution from Nebraska Agricultural Experiment Station )

Aecial Stage of the Orange Leafrust of Wheat, Puccinia
triticina Eriks. - - --- -- - 151

H. S. JACKSON and E. B. MAINS

( Contribution from Bureau of Plant Industry and Indiana Agricultural Experiment Station )

A Transmissible Mosaic Disease of Chinese Cabbage,
Mustard, and Turnip ------- 173

E. S. SCHULTZ
(Contribution from Bureau of Plant Industry)

PUBUSHED BY AUTHORITY OF THE SECRETARY OF AGRICCLTDRE,

WITH THE COOPERATION OF THE ASSOCIATION OF

LAND-GRANT COLLEGES

WASHINOTON, D. C.

EDITORIAL COMMITTEE OF THE

UNITED STATES DEPARTMENT OF AGRICULTURE AND

THE ASSOCIATION OF LAND-GRANT COLLEGES

FOR THE DEPARTMENT

KARL F. KELLERMAN, Chairman

Physiologist and Associate Chief, Bureau
of Plant Industry

EDWIN W. ALLEN

Chief, Office of Experiment Stations

CHARLES L. MARLATT

Entomologist and Assistant Chief, Bureau
of Entomology

FOR THE ASSOCIATION

J. G. LIPMAN

Dean, State College of Agriculture, ottd
Director, New Jersey Agricultural Etperi-
ment Station, Rutgers College

W. A. RILEY

Entomologist and Chief, Division of Ento-
mology and Economic Zoology, Agricul-
tural Experiment Station of the University
of Minnesota

R. L. WATTS

Dean, School of Agriculture, and Direct,
Agricultural Experiment Station, The
Pennsylvania State College

All correspondence regarding articles from the Department of Agriculture should be
addressed to Karl F. Kellerman, Journal of Agricultural Research, Washington, D. C.

All correspondence regarding articles- from State Experiment Stations should be
addressed to J. G. Lipman, New Jersey Agricultiu-al Experiment Station, New
Brunswick, N. J.

JOmAL OF ACRiaiTIIAL RESEARCH

Voiv. XXII Washington, D. C, October 15, 192 1 No. 3

INFLUENCE OF THE PLANE OF NUTRITION ON THE
MAINTENANCE REQUIREMENT OF CATTLE'

By F. B. MuMPORD, Dean of the College of Agriculture, A. G. Hogan, of the Depart-
ment of Animal Husbandry, and W. D. Salmon, Graduate Student in Animal Hus-
bandry, College of Agriculture, University of Missouri.

In 19 1 4 an investigation was begun at the University of Missouri to
study some of the effects of underfeeding. Calves of beef-breeding
stock were secured, and they were placed on three planes of nutrition.
Group I was fed to grow rapidly, but not to become fat. Group II was
placed on a lower nutritive plane and was fed to gain about }4 pound per
day. Group III was placed on a still lower nutritive plane and fed to
gain about V3' pound per day. At the present time three animals remain
that were started on the investigation in 19 14. Seven others were added
in 191 7. The older animals, therefore, have been under observation for
seven years and the younger animals for four years. Under these cir-
cumstances it seemed desirable to make a study of the maintenance
requirement of steers at different ages and on different planes of nutrition.^

The ideal method of conducting an investigation of this kind would
require a respiration calorimeter. Since that was impossible, the alter-
native was to calculate the energy value of the feed consumed and cor-
rect this for the estimated value of the gains or losses in body weight.

The net energy of the feed consumed was calculated in accordance
with procedures developed by Armsby.^ The energy value of the changes
in body weight were calculated from the composition of steers that had
been analyzed at this Station by the Department of Agricultural Chemis-
try. So far as possible steers were selected as controls for this purpose
that were of similar age, measurements, and weight and that had received
similar treatment.

' The data for this paper were taken from the thesis of W. D. Salmon, presented at the University of
Missouri, as partial fulfillment of the requirements for the degree of Master of Arts. The investigation
was initiated by F. B. Mumford, Dean of the College of Agriculture, and by P. F. Trowbridge, at that
time Chairman of the Department of Agricultural Chemistry. Since September 1918, E. A. Trowbridge,
Chairman of the Department of Animal Husbandry, has had general supervision of the project. This
article was prepared by A. G. Hogan, who has been in immediate charge since September, 1920. A large
number of workers have contributed to the success of the experiment.

' The original data will be reproduced in detail in a subsequent publication.

8 Armsby, H. p., and Fries, J. A. net energy values for ruminants. Pa. Agr. Exp. Sta. Bui.
142, 20 p. 1916.

Journal of Agricultural Research Vol. XXII, No. 3

Washington, D. C. Oct. 15, 1921

zv Mo. -J

(115)

ii6 Journal of Agricultural Research voi. xxn. no. 3

METHOD OF THE EXPERIMENT

RATIONS

The concentrate consisted of the following mixture: Com chop, 60
per cent; wheat bran, 30 per cent; linseed meal, 10 percent. The rough-
age fed to the 3 old steers. No. 528, 589, and 585, from the beginning of
the experiment until July 20, 191 7, was timothy. For the next 10 days
a mixture of 5 parts timothy, 3 parts alfalfa, and 2 parts oat straw was
fed. Following this the roughage consisted of a mixture of 60 per cent
alfalfa amd 40 per cent oat straw. The animals were fed twice daily and
had access to water at all times. Salt was accessible at feeding time.

PERIODS

The calculations are made for periods of 180 days, with the exception
of the first period for the 3 older steers, which was as follows: No. 528,
130 days; No. 579, 142 days; No. 585, 150 days. The warm months
of the year were selected for these periods to avoid a possible disturbing
effect of low temperatures in the winter months.

WEIGHTS

The steers were weighed each morning after feeding but before water-
ing. The weight given for the beginning of a period is the average of the
10 preceding days. The weight given at the end is an average of the last
10 days of the period

ENERGY INTAKE

The amount of dry matter consumed was calculated from the weight
and composition of the feed consumed. The net energy was computed
from this by the use of factors reported by Armsby and Fries. ^ For the
concentrates the value 83.82 therms per 100 pounds dry matter was used.
This is the factor given for Armsby's grain mixture No. 2,- which approxi-
mates the grain mixture used in this experiment. For timothy hay the
value 48.63 therms per 100 pounds dry matter was used. The factor for
the roughage mixture used in the latter part of the experiment was cal-
culated from the Armsby values, for alfalfa, 34.10 therms and for oat
straw, 26.03 therms per 100 pounds dry matter. A mixture of 60 parts
alfalfa and 40 parts oat straw would have a value of 30.87 therms per 100
pounds dry matter. The calculations of the energy value of the milk are
based on factors published by Armsby.^ There are 29.01 therms per
100 pounds whole milk (4.4 per cent) and 14.31 therms per 100 pounds
skim milk (0.2 per cent). From these values factors were computed for
the different grades of milk used.

'Armsby, H. P., and Fries, J. A. op. cit.

2 Annsby's grain mixture No. 2, 60 per cent com meal, 30 per cent crushed oats, 10 per cent O. P. linseed
meal. Our grain mixture, 60 per cent com meal, 30 per cent wheat bran, 10 per cent O. P. linseed meal.
^Armsby, Henry Prentiss, thb nutrition op farm animals, p. 719. New York, 1917.

Oct. IS, 1921 ' Influence of Plane Nutrition on Maintenance

117

CHANGES IN BODY WEIGHT

In order to obtain data concerning the maintenance requirement of
these steers, it is necessary to calculate the energy gained or lost through
changes in body weight. Our calculations are based on analyses previ-
ously made by the Department of Agricultural Chemistry, University of
Missouri.^ Control animals were selected from those on which analyses
were available, on the basis of similar weights and measurements, and
when possible of similar ages, daily gains, and daily consumption of dry-
matter. In some cases suitable control animals were not available, and
the composition of steers for those periods was estimated by interpola-
tion, with the exception of the last period for steer No. 528. In this case
a value published by Armsby^ was used. The average energy values of
a pound gain as calculated by this method for steers in the three groups
are given in Table I. For purposes of comparison the values given by
Armsby are shown in the same table.

Table I. — Energy values of a pound gain

Approximate age (months).

6.
18
36
54
66
78

Group I.

Therms.

o- 95575

1. 0918
I. 7136

2. 1993
2. 50
3.00

Group II.

Therms.

o- 95575
I- 0583
I. 1608
I. 4104

I- 5352
I. 660

Group III.

Therms.
o- 8343
•9445
I. 0548
I. 1013
I. 4790
I. 6490

Annsby's values.

Age.

Months.

2 to 3

5 to 6

II to 12

18 to 24

Energy.

Therms.

1. 170
1-374

1.680

2. 292

3. 000

Armsby's values are consistently higher, as is to be expected. Our
animals were thin and contained less than the usual amount of fat in the
gain. .; „.j'f u.-A ..■...■.:■' f.,^.. vjorf

In calculating the maintenance requirements per 1,000 pounds live
weight, Moulton's ^ formula was used. He has shown that the surface
areas of thin cattle are proportional to the ^ power of the live weight.
The results of this calculation, on the basis of dry matter consumed, are
given in Table II.

The net energy required for maintenance was also calculated by
another method,* based on the digestible organic matter of the feed.

The following factors are given for the metabolizable energy of digesti-
ble organic matter consumed: Roughage, 1.588 therms per pound; grains
and similar feeds with less than 5 per cent digestible fat, 1.769 therms per
pound. In the same publication the "Average energy expenditure by
cattle per 100 pounds of dry matter eaten" is given.

1 These have not yet been published.

* Armsby, Henry Prentiss, op. cit.

' MOULTON, C. R. THE AVAtLABILITY OF THE ENERGY OF FOOD FOR GROWTH. In Jour. Biol. Chem., V.

31, no. 2, p. 390. 1917.

* Armsby, H. P., and Fries, J. A. op. cit.

ii8

Journal of Agricultural Research voi. xxii. no. 3

Table II. — Average daily maintenance requirement as calculated from dry matter con-
sumed

Steer No.

Number of periods averaged.

Therms of net energy per i.ooo pounds,
based on H power of live weight.

Group I.

Group II.

Group III.

528

577

571

579

578

573

585

575

574

572

5. 870
5.280
5-730

4.920
3-830
4.409

•J

4. 221
4.041
4.302
3-250

•J

Average of all animals for all periods

5-523

4-485

3-830

Table III. — Energy expenditure by cattle per loo pounds dry matter consumed

Ration.

Roughage :

Timothy hay

Alfalfa hay

Oat straw

Concentrate :

Grain mixture No. 2

Energy ex-
penditure.

Therms.
35-47
53-03
46. 00

51-76

The coefficients of digestibility used in these calculations were derived
from digestion trials conducted under similar conditions at this Station.
These indicated that the digestibility of the ration varied with the rela-
tive amounts of hay and grain fed. The factors used are given in
Table IV.

Table IV. — Digestion factors for organic matter

Ratio of grain to
hay.

1. 1

2-3

I. 3

1.3.4. or 5

1.6 or 7

1. 8,9, or 10

Hay only.

Factor

.6956

.6695

•6434

.6340

. 6229

.6030

0. 5832

Inasmuch as the thermal value of a pound of organic matter from
grain differs from that of a similar weight of organic matter from rough-
age, the Armsby factors ^ previously quoted in this paper could not be
directly applied to the values obtained with the foregoing digestion
coefficients. Those factors would not provide for the widely varying
proportions of grain and hay. The following method, therefore, was

'Armsby, H. P., and Fries, J. A. op. cit.

Oct. IS. 1921 Inf,uence of Plane Nutrition on Maintenance

119

used in computing tlie energy intake on the basis of digestible organic
matter consumed. By use of the factors in Table IV, the weight in
pounds of digestible organic matter in the mixed ration was determined
for each period. This was multiplied by 1.588, the Armsby factor for
metabolizable energy in a pound of digestible organic matter from hay.
The thermal value of digestible organic matter from grain is 1.769,
however, or 0.181 therms more. Therefore, each pound of digestible
organic matter derived from grain was multiplied by 0.181, and the
product was added to the result obtained by multiplying the total
digestible organic matter by 1.588. This gave the total metabolizable
energy in both the hay and grain. The digestibility of the organic
matter of the grain was estimated by difference. This ranged closely
around 80 per cent. The factors for energy expenditure are given in
Table III.

It seemed impracticable to calculate the net energy of the milk con-
sumed on the basis of digestible organic matter, so the calculation was
based on the quantity consumed, as previously described. Since the
amount was small, however, the method of calculation would have
little effect on the final result. [i;i> 0.

The method used in correcting for changes in body weight has already
been described, and the maintenance requirement as calculated on the
basis of digestible organic matter consumed is given in Table V.

Table v.-

-Average daily maintenance requirement, as calculated front digestible organic
matter consumed

Steer No.

Number of periods averaged.

Therms of net energy per i.ooo pounds
based on 5/8 power of live weight.

Group I. Group II.

Group III.

528

577

571

579

578

573

585

575

574

572

6 . . . .

6. 261
5-412
5-174

5. 260
4. 192
4-893

4.725
4-454
4-591
3-649

■2 ...

-2 .

•2 .

Average of all animals for all
periods

5-777

4.869

4. 408

In determining the maintenance requirement on the basis of digestible
organic matter, the calculations were based on digestion coefficients ob-
tained at this Station under similar conditions. This method is probably
more accurate than that of calculation on the basis of dry matter con-
sumed, and for the animals concerned it gives a result about lo per cent
higher.

120

Journal of Agricultural Research

Vol. XXII. No. 3

In calculating average results, obtained by both methods, four periods
in which there were losses in live weight were omitted. The results for
those periods were low, and we were uncertain as to whether the result
was approximately correct or whether it was due to an incorrect assump-
tion as to the energy value of the loss in weight. Most of the dry matter
of the loss was probably fat, and if so, our calculation of its energy value
was too low and so made our calculation of the maintenance requirement
too low.

One steer, No. 585, had a navel infection during the first period,
accompanied by a very high maintenance requirement. This period also
was discarded in calculating averages.

INFLUENCE OF NUTRITIONAL PLANE

There is a close parallel between the intake of net energy and the
maintenance requirement of the animal. The record of steer 574 illus-
trates that tendency. For the first period the average daily intake of
net energy was 3.884 therms per 1,000 pounds, based on the 5/8 power
of the live weight; and the maintenance requirement was 3.818 therms.
For the second period the energy intake was increased to 5.783 therms,
and the maintenance requirement increased to 5.1 19 therms. In the
third period the energy intake was 5.253 therms, and the maintenance
requirement was 4.836 therms.

Table VI. — Dailymaintenance requirements of cattle — Net energy

RESPIRATION EXPERIMENTS

Num-
ber of

Investigator.

Condition of ani-
mal.

Therms per i.ooo pounds live weight.

ments.

Maximum.

Minimum.

Average.

Armsby and Fries ^ .

Medium

7-430
6. 780

8.871

4-723
4-921
7-319

Kellner ^

5-99^

Fat

5- 742
7.946

LIVE-WEIGHT EXPERIMENTS

Armsbv ' . .

do'. ...

Haecker * . .
Eward ^ . . .
Eckles^ ..
Shirky 2. . .
... .do. . ..
Our results .
....do. ...
....do. ...

Thin

do. . .

Medium...

do....

do3..

Thin*....
Group I . . .
Group II..
Group III.

7.044
6.039
5.676
7.850
7.079

5- OQ59
7.380
5- 724
5- 217

6. 136

4- 713
4. 662
6.450
5.841

4-953
4-915
3.809
3.276

50s
423
021
180
173
732
0245

777

5
5

4. 869
4. 408

' Armsby, Henry Prentiss, op. cit., p. 291.

2 Shirkey, S. B. extent to which growth retarded during the early life of the beef animal
CAN BE LATER REGAINED. Univ. of Mo. thesis, 1919. (Unpublished ) animal

' Corresponds to group I of this experiment.
* Corresponds to group 11 of this experiment.

Oct. 15. 1921 Influence of Plane Nutrition on Maintenance 121

In comparing the maintenance requirements of the three groups it
should be kept in mind that group I does not represent a high plane of
nutrition. The aim was to secure maximum growth with no considerable
fattening. Their maintenance requirements as computed in this paper
correspond closely to the average of 22 respiration experiments by Armsby
and Fries ^ and of 7 by Kellner,^ on cattle in medium condition.
A comparison of our results, and of those obtained by other investiga-
tions, is given in Table VI.

INFLUENCE OF AGE

The ages represented in this experiment vary from 30 days for some of
the calves at the beginning of the first period to more than 6 years at the
close of the seventh period. Apparently there was no relation between
the age and the maintenance requirement of these animals. Some of the
steers showed a gradual decrease in the maintenance cost from the begin-
ning to the end of the experiment. In such cases it was found that the
energy intake per 1,000 pounds had also decreased. On the other hand,
steers with an increasing energy intake showed an increased maintenance
requirement. Maintenance trials on young animals usually give higher
results than have been obtained with mature animals, but if age does
influence the maintenance requirement the effect is too slight to be shown
in a live- weight experiment of this kind.

SUMMARY AND DISCUSSION

There is a close relation between the amount of net energy consumed
and the maintenance requirement. Periods of high energy intake were
apparently periods of high maintenance cost, while periods of low energy
intake were accompanied by a lowered maintenance requirement.

The averages of the periods discussed show the following daily main-
tenance requirements per 1,000 pounds live weight, calculated on the
basis of digestible organic matter, and in terms of net energy: Group I,
5.777 therms; group II, 4.869 therms; and group III, 4.408 therms. If
the maintenance requirement of group I is 100 per cent, that of group II
is 84.4 per cent, and that of group III is 76.3 per cent.

The calculations on the basis of dry matter consumed indicate even
greater differences. The maintenance requirements as derived by this
method may be compared as follows: Group I, 100 per cent; group II,
81 per cent; group III, 69.3 per cent. The estimated maintenance re-
quirement of group I, as calculated by this method, is 30 per cent greater
than the total net energy intake of group III.

There is no apparent relation between the age of the animals and the
amount of energy required for maintenance.

' Armsby, Henry Prentiss, op. cit.

Turnip mosaic ^

By Max W. Gardner, Associate in Botany, and James B. Kendrick, Assistant in
Botany, Purdue University Agricultural Experiment Station

In one comer of a small field of turnips near South Bend, Ind., Octo-
ber 12, 1920, a considerable percentage of the plants were found affected
with an unmistakable mosaic disease. The symptoms were typical of
mosaic diseases in general. The leaves were stunted, misshapen, and a
lighter green with dark green blisters or puffy areas. Many of the leaves
were extremely distorted by crinkling and folding (PI. 20, A). The dis-
ease seemed to be confined to one area in the field, to some extent coin-
cident with a heavy infestation of tarnished plant bugs.

Several diseased plants were transplanted to pots in the greenhouse,
where they continued to form new leaves during the winter. The mosaic
symptoms exhibited by the new foliage formed under greenhouse con-
ditions were not quite so extreme as had been noted in the field. One
of these plants, with mottled and spindling leaves, is shown in Plate 20,
B, as it appeared in December.

Inoculation of a number of potted turnip and radish seedlings was made
by breaking off a leaf and rubbing the wound with crushed leaf tissue
from one of the mosaic plants. Out of 21 turnip seedlings inoculated
early in January, 13 developed characteristic mosaic symptoms. The
first symptoms were noted 26 days after inoculation. The turnips inocu-
lated showed some varietal difference from the plants collected in the
field in that the leaves were much less distinctly pinnatifid. Out of 46
radish seedlings, including both white and red varieties, similarly inocu-
lated, none developed mosaic symptoms.

A later series of inoculations was made January 26 by wounding the
plants with a needle and rubbing the wounded areas with a piece of cotton
soaked in the juice from mosaic leaves ground up in a mortar. Ten out
of 14 turnip plants thus inoculated developed the mosaic disease. The
first symptoms were noted 16 days after inoculation. No mosaic devel-
oped among 13 control plants similarly treated except that sterile water
was substituted for the mosaic virus. Twenty-two radish plants were
also inoculated, and none of these developed the disease. Subsequent
reinoculation of turnip plants from one of these radish plants produced
no mosaic. The mosaic disease of turnips is therefore readily transmis-
sible to turnips but not to radishes.

' Contribution from the Botanical Department of Purdue University Agricultural Experiment Station,
I,aFayette, Ind.

After this article was prepared it was learned that Eugene vS. Schultz, of the Bureau of Plant Industry,
United States Department of Agriculture, was also working on this disease.

Journal of Agricultural Research, Vol. XXII, No. 3

Washington, D. C. Oct. 15, 1921

zw Key No. Ind.- 1 1

(123)

PLATE 20

A. — Leaves from mosaic turnip plants collected October 12, tqso.
B. — Mosaic turnip plant transplanted to a pot in the greenhouse. Photographed
December 20, 1920.

(124)

Turnip Mosaic

Plate 20

Journal of Agricultural Research

Vol. XXII, No. 3

HYDROCYANIC ACID IN SUDAN GRASS'

By C. O. vSwANSON ^
Professor of Agricultural Chemistry, Kansas State Agricultural College

In a previous paper ^ it was shown that hydrocyanic acid (HCN) is
obtained from green Sudan grass by macerating, digesting in water, and
distilling into a dilute solution of sodium or potassium hydroxid. Sev-
eral experiments reported in that paper made it clear that this acid does
not exist free in Sudan grass and is obtained only if the conditions of the
determination are favorable to enzym action. It appears to be a common
belief that hydrocyanic acid is developed by freezing. This merely bursts
the green cells and thus performs the same function as maceration, with
the result that the hydrocyanic acid is rapidly lost from frosted grass.
It was also shown that while in some cases poisoning had been reported
from pasturing Sudan grass, under normal conditions no poisoning took
place either before or after the grass was frozen. It was suggested that
when frozen the hydrocyanic acid had been liberated and then evap-
orated as the grass dried.

Because of the importance of the subject it was thought worth while
to make further investigations. During the summer of 1920, material
was obtained from a i/20-acre plot of Sudan grass grown by the Depart-
ment of Agronomy of the Kansas State Agricultural College. The
Sudan grass had been planted early in June in rows about 2 feet apart.
On June 22, when the experiments were begun, the grass was about 6
inches high. These experiments were continued during the summer and

early fall.

METHOD OF DETERMINING HYDROCYANIC ACID

At present there are no satisfactory quantitative methods for esti-
mating hydrocyanic acid obtained from organic material. All are open
to some objection. After considering several, the Prussian-blue method
was adopted as best suited for the purposes of the present investigation.
Because of simplicity in manipulation it is possible to run a large number
of determinations at the same time. The amount of hydrocyanic acid
obtained from the different samples was estimated colorimetrically,
using standard solutions containing known amounts of potassium cyanid.
One objection to the colorimetric measurements was the difficulty in

' Contribution No. 92 from the Department of Chemistry, Agricultural Experiment Station of Kansas
State Agricultural College.

* Credit is due Mr. Carl M. Conrad for efficient assistance in making the determinations reported in this
paper.

' SWANSON, C. O. HYDROCYANIC ACID IN SUDAN GRASS AND ITS EFFECT ON CATTLE. Ill JoUr. Amer.

Soc. Agron., v. 13, no. i, p. 33-36. 1921.

Journal of Agricultural Research Vol. XXII, No. 3

Washington, D. C. Oct. 15, 1921

7x Key No. Kans.-J7

(125)

126 Journal of Agricultural Research voi. xxii. no. 3

obtaining a uniform blue color. Very often the precipitate was decidedly
green. It was found that by warming and letting the precipitate
stand for some time in loosely stoppered bottles a uniform blue color
could be obtained. The use of nitric or sulphuric acid instead of hydro-
chloric acid or the addition of potassium fluorid, all of which have been
suggested by other workers, did not seem to eliminate, entirely, the
green color. While the defects of the Prussian-blue method are fully
realized, it compares favorably with other methods.' In no sense are
the values reported in this paper to be regarded with the same degree of
accuracy as a protein or even a crude-fiber determination. For this
reason no conclusions should be drawn from the results unless the figures
presented are uniformly consistent or the differences large.

The calculations in this paper are based upon approximately 200 gm.
of green material. When the grass was wilted or dry the weight of sample
used was proportionately lessened. It is impracticable to secure green
samples of uniform weights of dry matter, particularly if they are
gathered during different hours of the day and throughout several weeks
and months. Then, as will be shown in what follows, the hydrocyanic
acid is localized in the plant, being present in the largest amounts in
those portions of the plant possessing the greatest vegetative activity.
For this reason leaves were separated from the stems whenever these
were present. The amount of hydrocyanic acid obtained is small in
proportion to the total weight of samples used. It was seldom more

than 0.015 per cent.

EFFECTS OF MACERATION

The first sample was collected June 22, when the grass was about 6
inches high. This was cut into pieces about % inch long and digested for
three hours in water at room temperature. Less than i mgm. hydrocy-
anic acid was obtained. Another sample, taken the next day, was cut
and thoroughly macerated by pounding in an iron mortar and was then
digested in water. This sample gave 27 mgm. hydrocyanic acid. On
June 28 a sample was secured and divided into two equal portions. One
portion was cut and macerated as described above, and the other was
cut and macerated with coarse, sharp sand. Both were digested in
water for the same length of time. The portion macerated with sand
gave 26 mgm. hydrocyanic acid, and the other gave 36 mgm. It ap-
peared from this that maceration with sand was not necessary and might
result in a loss. Subsequent experiments showed that as soon as the
grass is macerated the hydrocyanic acid is liberated and for this reason
may be lost. On August 18 a sample was divided into four portions
and, after the preliminary treatment mentioned, was digested overnight,
with the result given in Table I.

* ViEHOVER, Arno, and Johns, Carl O. on the determination of small quantities op hydrocy-
anic ACID. In Jour. Amer. Chem. Soc, v. 37, no. 3, p. 601-607. 1915.

octis. I92I Hydrocyanic Acid in Sudan Grass 127

Table I. — Effect of maceration on liberation of hydrocyanic acid

Sam-
ple
No.

Treatment.

HCN.

No cutting or maceration

Cut in feed cutter {}^ to ys inch)

Cut and macerated slightly

Cut and macerated thoroughly . . .

Mgm.

From this it appeared that if the time of digestion is sufficiently long
the amount of maceration is less important, provided, however, that the
plant tissue is cut fairly fine. The smaller amount obtained from the
grass on August 18, as compared with that obtained in June, is in accord
with a general observation made during the summer, that as the season
advanced smaller amounts were obtained from the 200-gm. portions.

TIME REQUIRED FOR DIGESTION •tM,;;-n

The time required for digestion in order to obtain the maximum amount
of hydrocyanic acid was determened. A sample collected on June 28 was
divided into three portions and similarly treated, except for the time
allowed for digestion. The results obtained are given in Table II.

Table II. — Effect of time of digestion on liberation of hydrocyanic acid

Sample
No.

Treatment.

HCN.

Digested 3 hours

Mgm.
18

Digested 6 hours

32
32

Digested 24 hours

This experiment seemed to show that digesting 3 hours was not long
enough, while 6 hours was as effective as 24. On August 28 a similar
experiment gave the results shown in Table III, the results in each case
being an average of duplicate samples.

Table III. — Effect of tim,e of digestion on liberation of hydrocyanic acid

Sample
No.

Treatment.

HCN.

Digested two days. . .
Digested three days .
Digested four days. . .
Digested seven days .

Mgm.

The portion digested seven days developed a very bad odor. Because of
these results, the usual procedure with experiments reported in this paper
was to macerate the sample and then digest at room temperature over-
night.

128

Journal of Agricultural Research voi. xxn. No. 3

LOCALIZATION OF HYDROCYANIC ACID IN THE PLANT

At three different times the grass was divided into leaves and stems.
From 200-gm. portions the number of milligrams of hydrocyanic acid
shown in Table IV were obtained.

Table 1\ . — Hydrocyanic acid in leaves and stem s of Sudan grass

Date collected.

June 30
July 7..
July 21.

stems.

Mgm.

Trace.

The immaturity of the sample collected June 30 accounts for the moder-
ate amount obtained from the stems. At a later date two tests were made
on immature heads. No hydrocyanic acid was found. In the following
tests reported in this paper leaves only were used unless otherwise stated.

INFLUENCE OF STAGE OF GROWTH

Since grass was cut almost every week throughout the summer there
were afforded several opportunities to test the comparative amounts
present in various stages of growth and development. The shorter grass
was obtained from plants which had been cut once or several times.

Table V. — Hydrocyanic acid in Sudan grass at different dates and stages of growth

Date collected.

Portion used.

Average height.

HCN.

July 22.

26.

Aug. 12
27.
30

Whole plant.

....do

Leaves

....do

Whole plant.

Leaves

I Whole plant.
Leaves
do

(Whole plant,
do
Leaves
do

fWhole plant.
\Leaves

(Whole plant,
do
Leaves
do

6 inches

8 inches

12 inches

18 inches

24 inches

30 inches

4 inches

6 inches

12 inches

Beginning to head

Partly headed

Fully headed

Partly dead

4 inches

16 inches

Headed

4 inches

10 inches

15 inches

24 inches

5 inches

20 inches

2 inches

12 inches

Ready to head. .. .
Blooming

Mgm.
27

7
24
20

9
40

32
10
12

17
20
6
II
18

Oct. 15, I93I Hydrocyanic Acid in Sudan Grass 129

The results show that more hydrocyanic acid is found in the whole
plant in the earlier stages of growth and less as the season advances.
The difference is perhaps due to the large proportion of stems in the
latter part of the season, since if leaves only are compared there is very
little difference except where they are from mature plants. This indi-
cates that most of the hydrocyanic acid is obtained from those parts of
the plants where the vegetative activity is most pronounced. This
agrees with the results obtained by Menaul and Dowell ^ at the Okla-
homa Agricultural Experiment Station. These observations support the
theory that hydrocyanic acid is an intermediate product between the
nitrates and the amino acids. ^

DISAPPEARANCE FROM MACERATED MATERIAL
As soon as the grass is macerated the hydrocyanic acid begins to pass
off. This was demonstrated several times by suspending small pieces
of sodium-picrate paper above some macerated grass in stoppered flasks.
The paper very soon assumed a brown color. The quantitative deter-
minations given in Table VI were made on samples macerated July 10
and treated as indicated.

Table VI. — Disappearance of hydrocyanic acid in macerated grass

^X!'^ Treatment. | HCN.

Digested in water two days

Placed without added water in covered mason jar for two days then
small amount of water added and distilled

Placed in flask two days so that the hydrocyanic acid could escape
only into the receiving flask, after which water was added and
distilled

4 Left in open jar for two days, digested and distilled

5 Repeat of 3 but kept in flask overnight only

Mgm.
25

26
Trace.
26

DISAPPEARANCE FROM GRASS AFTER CUTTING

In a previous paper ^ it was stated that tests made on partially wilted
grass may be worthless. In the experiments made at that time, the
amount of sulphuric acid added was not carefully enough controlled. It
will be shown in the following paragraphs that if acid is added beyond
certain limits no hj^drocyanic acid will be obtained from either green or
partially wilted grass. In each of the determinations given in Table
VII the grass was macerated after the treatment stated and then di-
gested in water overnight.

1 Menaul, Paul, and DowEi,!,, C. T. cyanogenesis in sudan grass: a modification of the franos-
CONNELI, METHOD OF DETERMINING HYDROCYANIC ACID. In Jour. Agr. Research, v. i8, no. 8, p. 447-450.
1920.

2 Ravenna, C, and Zamorani, M. ntjove ricerche sulla fxjnzione fisologica dell, acido ciani-
DRICO NEL SORGHUM VULGARE. In Atti R. Accad. Lincei, Rend. Cl. Sci. Fis., Mat. e Nat., v. 18, sem
2, no. 8, p. 283-287. 1909. Abstract in Chem. Abs., v. s, no. 6, p. 1113. 1911.

' SWANSON, C. O. HYDROCYANIC ACID IN SUDAN GRASS AND ITS EFFECT ON CATTLE In Jour. Amer.

Soc. Agron. , v. 13, no. i, p. 33-36. 1921.

I30

Journal of Agricultural Research voi. xxii. no. 3

TablR YII. — Disappearance of hydrocyanic acid from Sudan grass after cutting

Date collected.

Sample
No.

Treatment.

HCN.

June 29

July 7.

Aug. 12
Sept. I.

\Vilted in shade

Green , control sample

Wilted in sun for three hours

Dried in sun from morning till evening, outdoors over

night

Dried in shade for same length of time as 2

Dried outdoors two days and nights

Dried in the shade two days and nights

Dried in shade tliree da)'s and nights

Dried in the shade five days

Dried in the shade two days

Mgm.

28
36

15
24

32
6

While these results are not uniform, they do show conclusively that
hydrocyanic acid can be obtained from wilted grass. Because of this
result an attempt was made to determine more accurately the amount of
hydrocyanic acid that may be obtained from wilted and dried grass.

EFFECTS OF KEEPING GREEN GRASS MOIST AFTER IT IS CUT

A large sample of grass collected June 29 was placed stems down in a
large bottle so that about one-fourth was immersed in water. At the
end of different periods of time 200-gm. portions of the leaves were mac-
erated and digested in water overnight. The amount of hydrocyanic
acid obtained is given in Table VIII.

Table VIII.

— Effect of keeping grass moitt after cutting

Sample
No.

Length of treatment.

HCN.

6 hours

Mgm.
32
20

22 hours '

30 hours

48 hours

The results indicate that hydrocyanic acid slowly disappears from the
grass after it is cut, but also that the grass may be kept for a while in the
green condition without much loss of the hydrocyanic acid. Control
samples taken at this time gave 30 mgm. hydrocyanic acid.

When the grass was wholly covered with water or when the air was
excluded the results were different. In each case in the experiment re-
ported in Table IX, unless otherwise stated, the grass was macerated and
digested at the end of the treatment given.

This shows that the presence or absence of air has an intimate relation
to the evolution of hydrocyanic acid. Experiments were performed in
which the grass was kept in an atmosphere of carbon dioxid and also of

Oct. IS, 1921

Hydrocyanic Acid in Sudan Grass

131

hydrogen. No hydrocyanic acid was obtained from the grass kept in an
atmosphere of hydrogen, whereas from that kept in carbon dioxid con-
siderable amounts were obtained. Some macerated grass was also placed
in a desiccator from which the air was exhausted continuously. This
did not seem to affect the amount of hydrocyanic acid obtained, but the
experiment was not satisfactory. The effect of keeping the grass in
different atmospheres needs further study.

Table IX. — Effect of different treatments after cutting on hydrocyanic acid content

Date collected.

July 8.

July 10.

Treatment.

'Placed uncut in bottles and covered with water two days

Distillate from this water

Placed uncut in sealed mason jar with small amount of water
two da^'s

Placed uncut in sealed mason jar with small amount of chlo-
roform two days

Placed uncut in sealed mason jar for 2 days, no water

Placed uncut in bottle 2 days, covered with water

Obtained by distilling water from this

Macerated and digested 2 daj^s in water

<! Macerated and placed in bottle 2 days then water added and

distilled

[Macerated and placed in open pan 2 days

HCN.

Mgm.

12
Trace.

EFFECT OF HOT WATER

To determine this relation, enough grass was cut to make twenty-four
200-gm. portions of leaves. After the preliminary treatments as indi-
cated in Table X, one set of 12 samples was digested in cold water and
another set of 12 samples in hot water.

Table X. — Effect of adding hot water on amount of hydrocyanic acid obtained

Time of

drying in

shade.

(hours).

7-
28

Treatment before digestion.

Uncut

Cut in feed cutter ,

Macerated

Uncut

Cut in feed cutter .

Macerated

Uncut

Cut in feed cutter.

Macerated

Uncut

Cut in feed cutter.
Macerated

Time of digestion.

5 hours. . . .

do....

24 hours. . .

do....

Overnight.

do....

30 hours. . .

do....

HCN obtained after
adding —

Water at
room tem-
perature.

Mgm.

14
34

8
12

Boiling
water

Mgm.

Trace.

Trace.
2
2

65508°— 21-

132

Journal of Agricultural Research voi. xxn.No. 3

This shows that it is possible to obtain some hydrocyanic acid from the
uncut green grass if the time of digestion is sufficiently long. In every
case more was obtained when the material was cut in the feed cutter and
still more when it was macerated. Hot water placed on the green mate-
rial entirely prevented liberation. The small amount obtained from the
partially wilted grass when the hot water was added was probably in a
free condition at the time of adding the hot water. Almost as much
hydrocyanic acid was obtained from the grass that was wilted seven hours
as from the fresh grass if digested in water at room temperature suffi-
ciently long.

This experiment as well as several others show that under some
circumstances it is possible to obtain hydrocyanic acid from wilted or
dried grass both with and without digestion in either hot or cold water.
To investigate this further the following experiment was planned and
executed. Five sets of 12 samples were secured and treated as follows:
(i) Dried in the sun; (2) dried in the shade; (3) exposed in the sun,
but kept moist by frequent sprinkling with water; (4) exposed in the
shade but kept moist by sprinkling with water; (5) frozen in an ice
machine and then exposed in open pans in the shade. The duration
of these treatments was for 4, 8, 24, 31, and 48 hours, respectively.
Six of the samples from each set were macerated after the period of
the preliminary treatment, and hot water was poured on and distilled
at once. The other six were digested in cold water overnight and then
distilled. The results are shown in Table XL

Table XI. — Rate of disappearance of hydrocyanic acid from Sudan grass after it is cut

and variously handled <i

Time of treatment.

Hours
of

pre-
limi-
nary
treat-
ment.

Dried in sun,
treated with —

Exposed in
sun but kept

wet and
treated with—

Dried in shade

and
treated with —

Exposed in

shade but

kept wet and

treated with —

Frozen before

exposed in

shade and

treated with—

Hot

water.

Cold
water.

Hot
water

Cold
water

Hot
water

Cold
water.

Hot
water.

Cold
water

Hot
water

Cold
water

9 a. m first day

0
4
8

31
48

0
6
5
6

'A
Trace.

16
12
10
10
3

Trace.

0
4
I

16
14

5
7

3
Trace.

0
I
0
0

9 a. m. first day to i
p. m. first day

Trace.

8
I

Trace.

4
8
4
6
Trace.

9 a. m. first day to s
p. m. first day

9 a. m. first day to 9
a. m. second day

9 a. m. first day to s
p. m. second day ....

9 a. m. first day to 9
a. m. third day

8
4

5
8
10

Trace.

" The figures indicate milligrams of hydrocyanic acid from 200 gm. of grass and are averages of several
determinations.

The results show that no hydrocyanic acid is obtained from green
material when treated with hot water very soon after cutting and mac-
erating, but that when the grass is wilted as much as four hours in the
sun, considerable hydrocyanic acid is obtained by treating with hot
water immediately after maceration. The amount of hydrocyanic acid

Oct. IS. I92I Hydrocyanic Acid in Sudan Grass 133

obtained was not greater when the grass was wilted for a longer time.
Less hydrocyanic acid is obtained from grass that is kept moist while
in the sun than from grass that is allowed to dry rapidly. According
to Ravenna and Zamorani ^ the nitrogen passes through the following
stages in the plant: Nitrate -^ hydrocyanic acid — > amino substance
— ^ protein substance. According to this theory the cells which con-
tinue to be active use the hydrocyanic acid for the building of protein
substance, and as more nitrates from the soil are not supplied for man-
ufacture of more hydrocyanic acid, the potential amount present when
the plant is cut is soon exhausted.

When the grass was dried slowly in the shade the hydrocyanic acid
disappeared more slowly than when it was dried in the sun, and the
amount obtained from the hot-water treatment became approximately
equal to that obtained from the longer digestion in cold water. This
seems to mean that when the plant wilts the hydrocyanic acid is split
off from glucocids and held in such loose combination that it can be
set free by hot water and that practically all the hydrocyanic acid is in
such combination, since additional amounts can not be obtained by
further digestion. Splitting off begins as soon as the plant is cut.
Determinations made on grass kept moist in the shade appear to show
that after 24 hours all the hydrocyanic acid not otherwise used by the
cells is in such a condition that it is soluble in water.

In the test in which the grass was frosted the hydrocyanic acid dis-
appeared very rapidly, though the results were not very consistent.

EFFECT OF ACIDS

On June 24 a sample of grass was placed in a flask after maceration,
covered with water, and sulphuric acid was added to acid reaction.
After it was digested and distilled as usual only a trace of hydrocyanic
acid was obtained. From a sample of like material and similarly
treated, except that no acid was added, 27 mgm. were obtained. On
June 29 this experiment was repeated with the result that 8 mgm.
were obtained when acid was used and 26 mgm. when it was not used.
On June 30, i and 28 mgm. were obtained by these respective treat-
ments. These experiments clearly indicate that the presence of acid
has a very importance influence on the amount of hydrocyanic acid that
may be obtained. To test the effect of the amount of acid used, four
samples were prepared on July 14 and digested overnight in the fol-
lowing: (i) water; (2) N/o.i sulphuric acid (HjSOJ; (3) N/0.2 sul-
phuric acid; (4) N/i sulphuric acid. No hydrocyanic acid was ob-
tained from any of the treatments with sulphuric acid, whereas the
water digestion gave 30 mgm. On August 4 this experiment was
repeated, using a weaker acid solution. Digestion in water gave 10

1 Ravenna, C., and Zamorani, M. nuove ricerche sdlla funzione fisiologica dell' acido cian-
iDRico NEL SORGHUM VULGARE. In Atti R. Accad. Lincei, Rend. Cl. Sci. Fis., Mat e Nat., v i8, sem. 2,
no. 8, p. 283-287. 1909. Abstract in Chem. Abs. v. 5, no. 6, p. 1123. 1911.

134

Journal of Agrictdtural Research

Vol. XXI. No. 3

mgm. of hydrocyanic acid; N/o.oi sulphuric acid, 1 1 mgm. ; and in N/ 0.02
sulphuric acid, 4 mgm. On August 2 three samples were prepared and
digested in N/i vSulphuric acid; in N/0.2 sulphuric acid; and in N/0.05
sulphuric acid. Just before distillation, sodium hydroxid was added to
almost neutral reaction. From the N/0.05 sulphuric acid 18 mgm. of
hydrocyanic acid were obtained; a trace was obtained from the N/0.2,
and none from normal. The weakest of the acid solutions gave no
more than water alone. The smaller amounts obtained from the water
treatments at the later date is in accord with the general observation
that as the season advanced less hydrocyanic acid was present. It
was planned to determine the exact hydrogen-ion concentration at
which the hydrocyanic acid is most easily split off, but time did not
permit. It is hoped that this may be determined in the future.

It was shown in connection with the hot-water treatment that when
grass dries the hydrocyanic acid is changed into a free condition, so that
simply adding hot water and distilling will drive off the hydrocyanic acid.
To see if more would be driven off if acid was also present the following
experiment was performed. Six samples of leaves were placed in the
open in clear weather from 9 a. m. till 9 a. m. the next day. Then they
were macerated and digested in water and in different concentrations of
sulphuric acid. The results are given in Table XII.

Table XII. — Effect of acid solutions in formation of hydrocyanic acid

HiS04 added.

Nil...
NI0.2.
NI0.05

HCN ob-
tained.

Mgm.

H2S04 added.

NI0.O2
NJO.OI

Water.

HCN ob
tained.

Mgm.

Trace.
10
18

Thus, it appears that sulphuric acid is unfavorable to the liberation of
the hydrocyanic acid even in the wilted material.

To determine whether hot sulphuric acid would liberate the hydro-
cyanic acid, hot water and sulphuric acid of varying normalities were
added to green material immediately after maceration on July 16. The
results are shown in Table XIII.

Table XIII. — Effect of hot sulphuric acid on formation of hydrocyanic acid

H2SO4 added.

Nil..
NI0.5
NI0.2
NIo.i

HON ob-
tained.

Mgm.

Trace.

Trace .

H2SO4 added.

NI0.05

NI0.02

NIo.oi. .. .
Hot water

HCN ob-
tained.

Mgm.

Trace.
Trace.

Oct. 15, 1921

Hydrocyanic Acid in Sudan Grass

135

This shows that the use of hot acid is similar to that of hot water and
that acid has no power to spht off the hydrocyanic acid, at least in the
concentration used. The traces obtained in some cases were no doubt
due to liberation of hydrocyanic acid during maceration. It would,
appear, however, that hot water was less destructive than hot acid.
I/ike experiments with hydrochloric acid were performed with similar

results. ,,^.. ^.r>{' .r..>!!;.-:i r..,^

On July 2 1 eight samples were prepared and digested at room tempera-
ture in phosporic acid (Table XIV) .

Table XIV. — Effect 0/ phosphoric acid on liberation of hydrocyanic acid

H3PO4 added.

Nji..
NI0.5
NI0.2
Njo.i

HCN ob-
tained.

Mgm.

H3PO4 added.

NI0.05
N 1 0.02
Njo.oi
Water.

HCN ob-
tained.

Mgin,

The results indicate that the inhibiting power of phosphoric acid
(H3PO4) was somewhat less than that of hydrochloric (HCl) or sulphuric
acid. This would be expected since the degree of ionization of phosphoric
acid is less than that of hydrochloric or sulphuric acid. Experiments
with tartaric acid gave similar results.

EFFECT OF DIGESTING IN ALKALINE SOLUTION

On July 20, 16 samples were prepared and digested in sodium-hydroxid
(NaOH) and sodium-carbonate (Na2C03) solutions, respectively (Table
XV).

Table XV. — Effect of alkaline solution on formation of hydrocyanic acid

NaOH added.

Nji. ..
NI0.5.

NjO.2.

NIo.i.
Njo.05
N 1 0.02
Njo.oi
Water.

HCN
obtained.

Mgm.

Nas CO3 added.

Nil...
NI0.5.
NI0.2.
NIo.i .
NI0.05
NI0.02
NIo.oi
Water .

HCN
obtained.

Mgm.

Trace.

The results show the same general effect as that secured with acid
solutions.

136

Journal of Agricultural Research voi. xxii. No. 3

EFFECT OF ACID OR ALKALI ON HYDROCYANIC ACID AFTER IT IS

LIBERATED

An experiment was performed to show what effect acid or alkaline
solutions have on the hydrocyanic acid after it is liberated. The green,
macerated material was digested overnight in measured amounts of
water. Enough standardized acid or alkali was then added to give the
normality desired, and distilled. The results are given in Table XVI.

Table XVI. — Effect of acid and alkali on hydrocyanic acid after it is liberated

Solution added.

H2SO4
Hcl . . .
H3PO4
NaOH

HON obtained after treatment with solutions of-

Nlr.

Mgm.

9
16

Nlo.g.

Mgm.

N/o.i. Njo.os.

Mgm.

Mgvi.

Water.

Mg7n.

20
20

No hydrocyanic acid passed over in the first distillate from the sodium-

hydroxid solution. The mixture was acidified with sulphuric acid and

then distilled with the results given in Table XVI. While the results

obtained in this experiment are not very uniform, they do show that

hydrocyanic acid can be obtained from acid and alkaline solutions if the

hydrocyanic acid is in a free condition before the acids are added. The

experiment also appears to show that the addition of acid or alkali

resulted in diminishing the amount of hydrocyanic acid obtained. The

experiment was also tried by digesting the grass in sulphuric acid and

sodium hydroxid of the normalities N/i, N/0.2, N/o.i, and N/o.o^ and

then neutralizing before distilling. In no case was any hydrocyanic acid

obtained.

INFLUENCE OF WEATHER

From a sample taken June 23, when there had been no rain for three
weeks, 27 mgm. of hydrocyanic acid were obtained. On June 30, after
a heavy rain and a week of good growing weather, during which there
was plenty of moisture, 30 mgm. were obtained. On July 24, when
there had been a period of dry weather, the amount obtained was 7 mgm.
The next day, following a rain during the night, the amount was 16 mgm.
Several experiments indicated that the largest quantity was obtained
when the plant was in the most vigorous growing condition. This is
contrary to a common belief that stunting has some effect in increasing
hydrocyanic acid. On the contrary, the potential amount may be
lessened. Determinations were made on samples collected at sundown
and also before sunrise. The data obtained were not conclusive in deter-
mining the effect of light on the potential amount of hydrocyanic acid
present.

Oct IS. 1021 Hydrocyanic Acid in Svdan Grass 137

HYDROCYANIC ACID IN SUDAN HAY

Two samples were taken from the outside of a stack of Sudan hay
and two from the inside. No hydrocyanic acid was found.

AMOUNT OF HYDROCYANIC ACID IN OTHER SORGHUMS

On July 23 a sample of kafir was taken and separated into leaves
and stems. From the leaves were obtained 16 mgm., and from the
stems 10 mgm. of hydrocyanic acid. The kafir stems were very little
developed. Sudan grass, tested the same day, gave 8 mgm. of the acid
from the same weight of material. On July 26, just after a heavy rain,
following a period of dry weather, a sample of kafir gave 72 mgm. and a
sample of sorgo (cane) 42 mgm. of hydrocyanic acid. Sudan grass
6 inches high, tested on that date, gave 32 mgm. of hydrocyanic acid.

On August 7 a sample of second-growth sorgo (cane) was received
from LaHarpe, Kans. About one-fifth was quite dry, two-fifths were
wilted and yellow, and two-fifths were green. The sample was some-
what moldy. One portion digested in the usual manner gave 13 mgm.
hydrocyanic acid. Another portion distilled at once from hot water
gave 24 mgm., showing that the hydrocyanic acid was in free condition.
Another sample of sorgo was sent in from Seneca, Kans. This was
reported to have killed six cows. From the portion distilled from hot
water 20 mgm. were obtained and from the portion digested in the
usual way 36 mgm.

On September 2 a quantity of Red Amber kafir was collected, and
six portions were prepared, and treated with the results given in Table

XVII.

Table XVII. — Hydrocyanic acid in Red Atnber kafir

Sample

No.

Treatment.

Left in flask 15 minutes after maceration, after which hot water

was added and distilled

Digested overnight in water ^^,.^.,^,^.

Digested overnight in N/o.^ H2SO4. ...-.'.?..'..

Digested overnight in Njo.i HjSO^

Digested overnight in N/o.oi H2SO4

Digested overnight in N/o.oi NaOH

Mgm.

32
119

None.

None.
40
36

Part of this experiment was repeated by putting macerated sorgo into
boiling water at once. This gave 8 mgm. hydrocyanic acid, while that
digested overnight gave 96 mgm. Another portion was divided into five
portions. After maceration they were all digested in water overnight.
Then to these portions standardized sulphtuic acid was added so as to
make the normahties indicated. The results are given in Table XVIII.

These determinations show without a doubt that sorgo and kafir con-
tain much larger amounts of hydrocyanic acid than does Sudan grass,
and also that the conditions for obtaining it are very similar.

138 Journal of Agricultural Research voi. xxii. No. 3

Table XVIII. — Effect of different treatments on liberation of hydrocyanic acid in sorgo

Sample

No.

Treatment.

HCN.

Digested in water and distilled

Digested in water and distilled from Njo.^ H2SO4. . .
Digested in water and distilled from NIo.i H2SO4. . .
Digested in water and distilled from NI0.02 H2SO4. .

Mgni.

72
80
64
72
89

EFFECT OF HYDROCYANIC ACID FROM GREEN SORGO ON A HORSE

Ten-pound portions of green sorgo, testing the amount of hydrocyanic
acid given in Table XVIII, were fed to a horse. No effect on respiration,
pulse, or temperature could be observed by Dr. H. F. Lienhardt, of the
Veterinary Division, who made the observations. Data presented in
this paper show that such a degree of acidity as is found in the stomach
of a horse would prevent liberation of hydrocyanic acid from the green
material. Feeding wilted sorgo was not tried.

SUMMARY

(i) In this paper are presented data givmg the results of tests made
on Sudan grass for hydrocyanic acid during the summer and early fall of
1920.

(2) The maximum amount of hydrocyanic acid was obtained by mac-
erating the material and digesting in water at room temperature for about
six hours or overnight.

(3) Practically all the hydrocyanic acid was found in the leaves. In
well-developed stems none was found.

(4) More hydrocyanic acid was found in younger plants than in those
more mature. This is due mostly to stem development. If leaves only
are used the dififerences are small, except when the plants approach
maturity. More was found in the summer than in the fall.

(5) Hydrocyanic acid does not exist as free HCN in the growing plant.
It begins to be liberated as soon as the plant is macerated or undergoes
wilting.

(6) Liberation of hydrocyanic acid is intimately associated with enzym
action. If this enzym action is inhibited by addition of hot water or
acids, no hydrocyanic acid will be liberated. Hydrocyanic acid was
obtained from wilted grass when hot water was added, because during
the wilting process hydrocyanic acid was set free.

(7) Hydrocyanic acid can not be set free from the green material by
acids.

(8) The action of strong alkali is similar to that of acids.

(9) Most hydrocyanic acid is present when the plant is in a vigorous
growing condition.

(10) Sudan grass contains less hydrocyanic acid than sorgo or kafir.

NUTRIENT REQUIREMENTS OF GROWING CHICKS:
NUTRITIVE DEFICIENCIES OF CORN^

By F. E. MUSSEHL, Professor of Poultry Husbandry, J. W. Calvin, Associate Chemist,
Nebraska Agricultural Experiment Station, with the cooperation of D. L. Halber-
SLEBEN and R. M. Sandstedt

Investigators in the field of nutrition have noted that chickens behave
unlike rats and swine when limited to rations of com or wheat grains and
their products. This fact has made necessary the planning and execu-
tion of experimental work having for its object a determination of the
values and deficiencies of our common feeding stuffs when used for poultry
and egg production. The results of a series of experiments carried on at
this Station with this objective are reported in this paper.

From the experience of investigators ^ who have worked with other
species, mainly rats and swine, it has seemed that systematic inquiry
should be made into the (a) ash re-
quirements, (b) protein requirements
(quality and quantity), and (c) food
accessory requirements. Earlier in-
vestigational work with chicks by
Osborne and Mendel ^ and Hart,
Halpin, and Steenbock* indicates that
another element, (d) the physical
factor, is also of fundamental impor-
tance and must be considered in any
complete study of the nutritive values
of a particular grain or ration.

In our work lo-day-old vS ingle-Comb
White Leghorn chicks were used, spe-
cial care being taken to select for vigor,
vitality, and uniformity in each lot.
Nine chicks per lot were used for the
first series of experiments. Chicks were weighed individually every
seven days, and the growth curves selected are typical of each lot (fig.
i-ii). They show the weight of the chicks at the beginning of the
experiment and the change in weight thereafter. Records of the feed

1 Published with the approval of the Director of the Nebraska Agricultural Experiment Station.

'McCoLLUM, E. v., SiMMONDS, N. , and PiTz, W. the relation of the UNroENTiFiBD dietary fac-
tors, THE fat-soluble a, AND WATER-SOLUBLE 6, OF THE DIET TO THE GROWTH-PROMOTING PROPERTIES
OF MILK. In Jour. Biol. Chem., v. 27, no. i, p. 33-43, 6 charts (1-3, 6 in text). 1916.

'Osborne, Thomas B. , and Mendel, Lafayette B. the growth of chickens in confinement. In
Jour. Biol, chem., v. 33, no. 3, p. 433-438, pi. 4-6. 1918.

^Hart, E. B., Halpin, J. G., and Steenbock, H. use of synthetic diets in the growth of baby
CHICKS. A study of LEG WEAKNESS IN CHICKENS. In Jour. Biol. Chem., v. 43, no. 2, p. 421-442, 2 pi. 1920.

o
100

—-

» —

1125

— *

/oo

■:HiaK 1

//oa I

,3S ^2

Fig. I.— Graph showing unsatisfactory results
from feeding ration of 100 parts yellow com
and calcium carbonate grit ad libitum to
chicks of lot II. The time at which chicks
died is indicated by X.

Journal of Agricultural Research,

Washington, D. C.

(139)

Vol. XXII. No. 3
Oct. 15, 192 1
Key No. Nebr.-4

140

Journal of Agricultural Research voi. xxii. No. 3

(300

< y

300

Jj 700

\ 600

SOO

//^i

300

J-

X''

Fio. a.— Graph showing slow but continuous growth of chick in lot 211, fed ration of 95 parts yellow com

and 5 parts ash mixture

Nutrient Requirements of Growing Chicks 141

/.soo

A 200

/,/oo

/.ooo

900

/f'^

, (

i'^

ii/i

)f^(i

_,---

5<?(S'

eoo

300

2O0

fOO

700

>*^

.-^

r.K

2jj

'■^rf

^ ^

,a£^

—

I*""*'

O 7/^2/ Ze 3S -^2 4& se 63 70 77 S4 S/ S8 /OS //2 //^ /Z6

Fig. 3. — Graph showing growth of chicks in lot 213, fed ration of 80 parts yellow com, 15 parts casein,

and 5 parts ash mixture.

142

Journal of Agricultural Research voi. xxu,no.3

/,ooo

9CX)

soo

700

600

t 500

10 ^oo

300

200

/OO

if?'

rjt^

A 1

403

■y

/OO

CH/CK 2736

O 7 /4 2/ 2e 3S ^^2 ^& S6 63 70 77 S'^ S/ S3

Fig. 4. — Graph showing detrimental results of adding 5 parts butter fat to ration of 65 parts yellow com,
15 parts casein, 5 parts ash mixture, and 10 parts starch for chicks in lot 215.

Oct. IS, 1921

Nutrient Requirements of Growing Chicks

143

/ooo
soo

TOO

^00

^00
ZOO

too
o

/■

.i(^^

(?/?!

?i^

)(^t

*^^

O 7 /^ 2f 23 3,^ *Pe '^9 c5<5 G3 70 77 S^ S/ &3

Fig. 5.— Graph showing that the addition of 20 parts com gluten did not improve ration of 65 parts yellow
com, 5 parts ash mixture, and 10 parts starch for chicks of lot 208.

144

Journal of Agricultural Reserach voi. xxn. no. 3

900

aoo

700

600
\soo

\fOO

300

200

/oo

■y

aI"^

^iQ

•^

.*-'

300

^ zoo

O 7 /"f 2/ £3 3S ^2 -^9 S6 63 70 77 3^ 9/ 93

Fig. 6.— Graph showing that the addition of 5 parts butter fat (fat-soluble A) did not improve ration of s
parts yellow corn, 20 parts corn gluten, 5 parts ash mixture, and 5 parts starch for chicks of lot 209.

X-

j/r

P7f

j^..

—

-*-

rff<

27.

'^z'

Oct. IS, I93I

Nutrient Requirements of Growing Chicks

145

'POO

200

/oo

I o
%zoo

-X

TWCK

27^

consumption of each lot were also obtained. Clean wood shavings
were used for litter, and each lot was confined to a pen 2 by 8 feet
in size until the chicks were 8 weeks old, when the near normal lots
were given a yard 4 by 8 feet
in size.

Methods of feeding are known
to have considerable influence on
the efficiency of a ration; so a
standard policy was established of
dividing the ration into two parts —
the scratch or coarse feed and the
mash or fine feed. The rations
were mixed so that equal quantities
of mash and scratch feeds were
provided. The mash feed con-
tained all the supplemental ingre-
dients, such as ash, butter fat, and
purified casein.

The casein used in the rations was
purified by extracting repeatedly
with distilled water slightly acid-
ified with acetic acid . After extrac-
tion the casein was drained and
dried in an air oven at 70° to 100° C. and then ground. The butter fat
was purified by melting at 40° in a water bath and was then centrifuged
to remove ash, casein, and other material. The ash mixture^ used in
our rations was composed of the following ingredients, parts by weight:

Bone ash .jV^/:'^: 't';^;! . V'^'v'. .'. 50

Calcium carbonate

Sodium chlorid

Dipotassium phosphate

Calcium lactate

Magnesium sulphate

Sulphur

Iron sulphate

^i9

Fig. 7. — Graph showing that the addition of is parts
soybean meal did not improve ration of 80 parts
yellow corn and 5 parts ash mixture for chicks of
lot 210. The time at which chicks died is indicated
by A'.

JXi.i . U.

"■^^rA.

14
IS
10

5
3
2

The results of our inquiries may briefly be summarized as follows:
(i) Yellow com (maize) is deficient in several of the essential qualities
necessary for the complete nutrition of growing chicks. A deficiency in
the ash content of the yellow com kernel is no doubt responsible for the
early failure of baby chicks when restricted to a ration of corn alone.
Supplementing the corn kernel with 5 per cent of a complete ash mix-
ture improved the ration so as to enable very slow but persistent growth.

1 Philips, A. G., Carr, R. H., and Kennard, D. C. meat scraps versus soy-bean proteins as a
StJPPLEMENT TO CORN FOR GROWING CHICKS. In Jour Agf. Research, v. i8, no. 7, p. 391-398, i fig. , pi. 50.
1920

146

Journal of Agricultural Research

Vol. XXII, No. 3

JOO

% °
S 100

JOO

•^

frH

CHICK

Vr\3

— X

C»/Ch
Z770

"■"^x

CHICK

zeas

— X

£>i9y.s

Fig. 8.— Graph show-
ing bad results from
lack of roughage in
ration of 65 parts
yellow com, 15 parts
gelatin, 5 parts but-
ter fat, 5 parts ash
mixture, and 10
parts starch for
chicks of lot 217.
The time at which
chick died is indi-
cated by X.

(2) Yellow corn is deficient in quality and quantity of protein required
for normal growth of chicks. The addition of more com protein by
including corn gluten in the ration did not markedly
improve the efficiency of the ration. Compare growth
curves, lots 208 and 211.

(3) The addition of 15 per cent purified casein to a
basal ration of yellow corn and ash did improve the
ration decidedly. Compare growth curves, lots 211 and
213. The amino acid deficiencies of the corn proteins
are no doubt supplemented by the amino acid contribu-
tions of the casein.

(4) Supplementing the basal yellow com ration with
certain other proteins, ^^g albumen, and gelatin, lowered
rather than raised the efficiency of the ration. The poor
results with rations 217 and 218 were probably due to
a distinctly sticky physical quality which prevented
normal nutrition.

(5) The fat-soluble food accessory does not appear to
be a limiting factor in a yellow com diet for baby
chicks. The addition of butter
fat to a yellow corn, casein,

and ash ration (lot 215) did not improve but
rather lowered the efficiency of the ration. A
slight change in the physical condition of the
ration may explain this lowered efficiency,
though it is more probable that the butter fat
addition temporarily stimulated growth so that
the supply of some other essential accessory
was exhausted earlier than would have been
the case had the butter fat been omitted. It
is apparent at least that the failure of chicks
on ration 215 was not due to fat-soluble A
starvation.

(6) Green feeds make certain very valuable
contributions to a ration for growdng chicks.
The addition of wheat greens to a yellow com,
casein, and ash ration effected a decided im-
provement in the efficiency of the ration. An
excess of the wheat greens was offered, and
subsequent observations indicate that about 5
per cent (dry matter basis) of this kind of green
food are consumed when offered regularly in excess. The helpful influ-
ence of the wheat greens may have been due to, first, an improvement
of the physical condition of the ration; second, a food accessory con-

zoo

/oo

o
200

/oo

I o
^200

/oo

o
/oo

_„--'

5^'

^A//'^

Thi^

$73?

Thi^

Z73\

/«? 2/

Fig. 9.— Graph showing that be-
cause proper physical quality
was lacking the addition of 15
parts egg albumen did not im-
prove ration of 65 parts yellow
com, 5 parts butter fat, s parts
ash mixture, and 10 parts starch
for chicks of lot 218. The time
at which chicks died is indi-
cated by X.

Nutrient Requirements of Growing Chicks

/.■300

/.OOO

900

eoo

<f) 700

1 —

ndi

, 6

^LQ^

soo

400

200

/OO

eoo

600

.soo

Vi 300

400

<:'..

♦;-^

'f.i

^ff

-3

. o

^$'i.

W'^

ii
^

€^

'0.

l'^

i* d

zoo

/OO

~ O 7 /4 2/ 23 33 42 49 S6 63 TO 77 34 S/ S3 /OS //2 //S /ZS

Fig. io. — Graph showing that the addition of excess of wheat greens improved ration of 8o parts yellow
com, 15 parts casein, and s parts ash mixture for chicks of lot 227.

65508°— 21 3

148

Journal of Agricultural Research voi. xxu,no.3

/./oo

/,ooo

900

800

700

(ft 600

400

300

200

/CO

(

0 7 M 2/ Z8 3S f2 -^9 S6 63 70 77 S'^ 3/ 38 /OS //2

Fig. II.— Graph showing normal growth produced by ration of 65 parts wheat, 15 parts casein, s parts
butter fat, 5 parts ash mixture, 10 parts starch, and excess of wheat greens for chicks of lot 205.
Birds were kept on this ration until they produced eggs, at age of about 200 days.

Octis, I92I Nutrient Requirements of Growing Chicks 149

tribution; third, an increased food consumption, due to a stimulatory
effect on the appetite. A series of experiments to shed further hght
on the specific contributions which green feeds make to the ration is in
progress at this time.

(7) It is possible to raise to normal maturity chicks confined to a small
pen. Drummond ^ reports great difficulty in rearing chicks in confine-
ment, and other investigators have noted some of the problems, especially
leg weakness.' Our lot 205 grew to normal maturity, some of the pullets
producing eggs when about 200 days old, though never having more
range than was provided in a yard 4 by 8 feet in size. Ration 205, though
not synthetic, is of interest because of its comparative simplicity.

1 Drummond, Jack Cecil, observations upon the growth of young chickens under laboratory
CONDITIONS. In Biochem. Jour., v. lo, no. i, p. 77-88, i pi. 1916.

2 Hart, E- B., Hai,pin, J. G., and Steenbock, H. op. ai.

AECIAL STAGE OF THE ORANGE LEAFRUST OF
WHEAT, PUCCINIA TRITICINA ERIKS.^

By H. S. Jackson, Chief in Botany, and E. B. Mains, Associate Botanist, Purdue
University Agricultural Experiment Station, and Agents, Office of Cereal Investiga'
tions. Bureau of Plant Industry, United States Department of Agriculture ^

This paper presents, in part, the results of a study of the leafrusts
of wheat, rye, barley, com, and related grasses which was begun in 191 8.
One of the important phases of this investigation is the determination of
the aecial relationships of the various races or species included in the
collective species, Puccinia Clematidis (DC.) Lagerh. (P. Agropyri Ellis
and Ev.), and other closely related forms. While a number of the rusts
of this group which occur on wild grasses have been connected with
aecia, their host limitations and interrelations are not well understood.
This study is especially important in the case of the leafrust of wheat,
P. triticina Eriks. So long as the aecial stage of this species was un-
known, little progress could be made in developing our knowledge with
reference to its origin, development, spread, and relation to other rusts.
The results of the investigation of the aecial relationship of this rust
are presented in the following pages.

HISTORICAL REVIEW

Three rusts are known to attack wheat : the black or stemrust, Puccinia
graminis Pers. ; the stripe or yellow rust, P. glumarum (Schmidt) Eriks.
and Henn. ; and the orange or leafrust, P. triticina. Of these the stem-
rust is the only one for which the aecial stage has been determined.
This rust was shown by De Bary to have its aecial stage on Berberis
vulgaris L., and this relationship has since been demonstrated repeatedly
by a number of workers in various parts of the world. The discovery
of the place of Aecidium Berberidis Pers. in the life cycle of P. graminis
caused De Bary (4, p. 207-211)^ to turn his attention to the study of
other grass rusts having incomplete life cycles. This resulted in the
discovery that P. rubigo-vera (DC.) Wint. (P. straminis Fckl.) on rye
was connected with aecia on Anchusa officinalis and Anchusa arvensis.
Sowings made with teliospores from rye resulted in the production of

' Published with the approval of the Director as a contribution from the Department of Botany, Purdue
University Agricultural Experiment Station. Cooperative investigation between the Purdue University
Agricultural Experiment Station and the Office of Cereal Investigations, Bureau of Plant Industry, United
States Department of Agriculture.

' The writers wish to acknowledge their indebtedness to various pathologists throughout the country
for aid in obtaining material for the cultural studies upon which this paper is based, and to Mr. Forest
Fuller, temporary culture assistant during the spring of 1919, and Mr. Emile Mardfin and Miss Florence
M. Smith, Scientific Assistants, Ofifice of Cereal Investigations, Bureau of Plant Industry, for assistance
in carrying out the cultural investigations.

' Reference is made by number (italic) to "Literature cited," p. 170-171.

Journal of Agricultural Research, Vol. XXII, No. 3

Washington, D. C Oct. 15, 1921

zz Key No. G-347

1^2 Journal of Agricultural Research v^oi. xxii. No. 3

aecia on Anchusa, and when sowings were made with aeciospores from
Anchusa, uredinia on rye were developed. Sowings of basidiospores from
rye upon Berheris vulgaris Hoi., Rhamnus Frangida, Rhamus cathartica,
Ranunculus acris, Ranunculus bulbosus, Taraxacum officinalis, and Urtica
dioica were without result. Nielsen {20, p. 37) 10 years later reported
obtaining infection with aeciospores from Anchusa officinalis on both rye
and wheat. Plowright {21, p. 168) states that in the fall of 1885 he
obtained aecia upon Anchusa arvensis by placing wheat straw rusted
with P. rubigo-vcra near that host.

At the time this work was carried out the name Puccinia rubigo-vera
was used for the leafrusts of wheat, rye, and barley, as well as for similar
grass rusts having globoid urediniospores and long covered telia.
Eriksson and Henning (11, p. 197-203, 2^7-2^9) separated this species
into two — Puccinia glumarum, the stripe rust, and Puccinia dispersa
Eriks., the brown rust. Under the latter they included the rust of wheat
as well as that of rye. The rust of wheat, however, was considered as a
forma specialis, Tritici, of Puccinia dispersa. As the leafrust of rye had
been shown by De Bary (4) to be connected with aecia on Anchusa,
Eriksson (jo, p. 254-2^7) sought for the same connection for the leafrust
of wheat. His sowings of basidiospores from wheat upon Anchusa
officinalis and A. arvensis, however, produced no infection, as was also
the case when aeciospores from Anchusa were sown on wheat. No results
were obtained when basidiospores were sown on Nonnea rosea, Myosotis
arvensis, M. alpestris, Symphytum asperrimum and Pulmonaria officinalis,
species of Boraginaceae related to Anchusa upon which unconnected
aecia were known to occur. As a result of these cultures, Eriksson {10,
p. 270) concluded that the orange leafrust of wheat was a distinct species
and gave it the name, Puccinia trificina.

Klebahn (17, p. 85-86; 18, p. 246) made rather extensive cultures in
an endeavor to discover the aecial host of Puccinia triticina. Besides
sowing aeciospores of Aecidium Anchusae Eriks. and Henn. on wheat he
made sowings of basidiospores on Anchusa arvensis and Anchusa officinalis
without result. Sowings of basidiospores also were made without success
upon Triticum vulgare, Ranunculus acer, Ranunaihis asiaticiis, Ranun-
culus auricomus. Ranunculus bulbosus, Ranunculus Ficaria, Ranunculus
flammula, Rammculus lanuginosus , Ranunculus repens, Anemone ranun-
culoidcs, Aconitum Lycoctonum, Aconitum Napellus, Berberis vulgaris,
Nasturtium sp., Barbaraea vulgaris, Melandryum albutn, Coronaria fl.os-
cuculi, Agrostetnma Githago, Rhamnus cathartica, Lythrum Salicaria,
Ribes Grossularia, Aegopodium Podagraria, Pastinaca sativa, Valeriana
dioica, Knautia arvensis, Tussilago Farfara, Taraxacum, officinale, Cen-
taurea Cyanus, Achillea Ptarmica, Campamila rotundifolia, Ligustrum
vulgare, Phillyrea sp., Echium vulgare, Lithospermum purpureo-coeruleum,
Myosotis sp., Symphytum officinale, Glechoma hederacea, Prunella vulgaris,

Oct. IS, 1921 Aecial Stage of the Orange Leaf rust of Wheat 153

Rumex acetosa, and Urtica dioica. These results substantiate those
obtained by Eriksson and indicate that the positive results reported by
Nielsen {20) and Plowright {21) were probably due to a mixture of rusts
or of hosts.

The failure to obtain infection on Boraginaceous hosts has influenced
other workers to turn their attention to other families in a search for the
aecial hosts. Arthur (j, v. 9, p. 304), largely as a result of morphological
studies, reached the conclusion that Puccinia triticina was best con-
sidered a race of P. Agropyri, and upon this basis Arthur and Fromme
(j. P' 333~337) have placed it in the collective species Dicaeoma Clerna-
tidis (DC.) Arth. Several races of this collective species had been shown
by workers in Europe and America to go to species of Clematis. Arthur
thought that the aecial host might be either Clematis ftamm.ula or C.
vitalha as these were the only common species of Clematis found in the
wheat-growing regions of southern Europe, northern Africa, and western
Asia, a region which at that time was considered as the probable home
of the original wild wheat. His culture with wintered telia of the leaf-
rust of wheat on C. flammula, however, was unsuccessful.

According to Butler (d, p. 75) Cunningham and Prain (9) considered
that there was considerable ground for believing that an Aecidium on
Launaea asplenijolia, one of the Cichoriaceae, was the aecial stage of
Puccinia triticina, as it was found throughout the greater part of the
wheat-growing area of India. Butler, however, sowed aeciospores from
this host upon wheat without obtaining infection.

These unsuccessful attempts to demonstrate an aecial stage for Puc-
cinia triticina have resulted in the development of the idea that the
aecial stage of this rust has been lost and that it is able to maintain itself
without one. In this connection a number of important facts have
been established and a number of interesting hypotheses proposed. It
has been shown by BoUey (5, p. 13-14), Hitchcock and Carleton (15, p.
1—2), Carleton {8, p. 21-22), and others that in certain regions, P. tri-
ticina is able to overwinter by means of its uredinal mycelium and that
no aecial host is necessary for the maintenance of this species. This
does not appear, however, to be true for all regions where P. triticina is
abundant {6, p. 11). A number of suggestions have been made to ex-
plain the 5'early appearance of the rust in regions where the uredini-
ospores or uredinial mycelium does not overwinter. It was considered
possible that spores may be carried from other regions by the wind.
The mycoplasm theory of seed transmissal has also been put forward as
a possible explanation. Whatever may be the merits of these hypothe-
ses, they have resulted in recent years in directing attention away from
a search for the aecial host of this species.

154 Journal of Agricultural Research voi. xxii, no. 3

BASIS OF CULTURAL INVESTIGATIONS

A study of Puccinia trUicina in comparison with other grass rusts with
long covered teha shows that it can not be readily separated morpholog-
ically from the leafrust of rye. The separation of this form as a species
was made by Eriksson {10) because he obtained only slight infection on
rye with urediniospores and was not able to obtain infection on Anchusa
with basidiospores and because the teliospores germinated in the spring,
while those of the rye rust germinated in the fall. The close morphological
similarity, however, furnished considerable grounds for the assumption
that the aecial host of leafrust of wheat was likely to be some species of
Boraginaceae other than Anchusa, especially as another rust of this type,
Puccinia hromina Eriks., has since been found to have its aecia on the
Boraginaceous hosts Symphytum officinale and Pulmonaria montana, with
very weak development of aecia on Anchusa {ig, p. 182-202). Unfin-
ished investigations now being conducted in this laboratory strongly
indicate that in America certain grass rusts having aecia on Boraginaceous
hosts are very similar to the leafrust of wheat and rye. For these reasons
it was considered desirable to test as many Boraginaceous hosts as were
available, as possible aecial hosts for the leafrust of wheat.

There is, however, still another group of grass rusts very similar to the
orange leafrust of wheat to which Arthur (j, v. 9, p. 304) has called atten-
tion. This group has aecia upon various Ranunculaceous hosts and in-
cludes forms which have been separated from time to time, according to
their aecial connection, together with slight morphological variation, into
a number of species, including Puccinia persistens Plowr., P. perplexans
Plowr., P. Agropyri, and P. alternans Arth, The writers felt from the
beginning that the greatest possibility of success in the search for the
aecial stage was to study thoroughly the genera of this family on which
aecia were known to occur.

The idea that Puccinia tritichia has lost its ability to develop an aecial
. stage through long propagation by urediniospores, while admittedly pos-
sible, was not considered to be fully substantiated.

CULTURES MADE IN 1919

With these considerations in mind rather extensive sowings were made
in the spring of 19 19 upon a considerable number of species of the fami-
lies Ranunculaceae and Boraginaceae and the closely related family
Hydrophyllaceae. For this purpose, 20 collections of telia of Puccinia
tritici'na were obtained from various sections of the country during the
summer and fall of 1918 and placed outdoors to winter. Early in March
these began to germinate. Ten of the 20 collections gave good germina-
tion and were sown upon various species of the above-named families and
upon Ornithogalum umbellatum L., Impaiiens sp., and Catnassia esculenta
(Ker.) Robins. (Quamasia hyacinthina). The results obtained are given
in Table I.

Oct. IS. 1921 Aecial Stage of the Orange Leaf rust of Wheat

155

Table l.—Data obtained in igig from sowing teliospores of Puccinia triticina, from 10
different localities, on various host plants, mostly of the families Ranunculaceae and
Boraginaceae o-

Host inoculated.

No. ii8
(Okla.).

No. 218
(Ala.).

No. 418
(Tenn.).

No. 618
(Ga.).

No. 718
(Ga.).

No. 818
(S.C).

No. 918
(Ind.).

No.

3518

(Wis.).

No.

3818

(Wis.).

No.

4Si8

(Wis.).

■~

Anemone japonica Sieb. &

Aquilegia canadensis L

■■■-'■'

Aquilegia glandulosa Fisch. .

Aquilegia vulgaris 1,

■:':;::;:::::

Cimicifuga racemosa (L.)
Nutt

Clematis virginiana 1,

■■'-"■

Delphinium "Belladonna."
(Hort.)..

" "—

Mertensia virginica (L-)
Link

—

Ornithogalum umbellatum.
L

—

Camassia esculenta (Ker.)

b "—
b "—

Ranunculus repens L

Miihl

—

" — No infection.
" Pycnia produced.
^ Two sowings were made,
the other.

Pycnia were produced from only one sowing, no result being obtained from

The telial collections used in the cultures were all obtained from Triti-
cum aestivum (T. vulgare) in the following localities :

118, from Stillwater, Okla., collected by J. D. Moore.
218, from Flint, Ala., collected by McClellan.
418, from Tennessee, collected by W. T. Evans.
618, from Carrolton, Ga., collected by R. O. Bums.
718, from Carrolton, Ga., collected by R. O. Burns.

1:^6 Journal of Agricultural Research , ,l Voi. xxii, No. 3

818, from Anderson, S. C, collected by R. O. Burns.

918, from La Fayette, Ind., collected by E. H. Toole.

3518, from Menah, Wis., collected by E. H. Toole.

3818, from Wisconsin, collected by E. H. Toole.

4518, from Superior, Wisconsin, collected by E. H. Toole.
Negative results were obtained on all but two species of the hosts used.
The collection from La Fayette, Ind. (No. 918), gave infection upon
Thalictrum angustifolium and T. aquilegijolium, producing, however, only
pycnia. It was impossible to carry this study further in 1919, as the
above results were not obtained until late in the spring.

The failure of aecia to develop from the two successful infections could
be explained on either of two hypotheses. The conditions in the green-
house may have been unfavorable, or the species of Thalictrum used may
have been resistant. In either case, however, these results were inter-
preted as indicating that the aecial host of the leafrust of wheat was some
species of ThaHctrum. There was considerable basis for this assumption.
All of the culture studies being carried on in this laboratory with the
related rusts, occurring on wild grasses, and having aecia on members of
the family Ranunculaceae, have indicated that while a given race may
develop aecia on several species in one host genus with varying degrees
of virulence it will not go to species of more than one genus. The rusts
of this group show a very high degree of specialization. The two species
of Thalictrum on which infection was obtained were foreign species,
while the North American species, Thalictrum dioicum and T. polygarnum,
were not infected. On this account it was thought that the susceptible
aecial hosts for the leafrust of wheat probably were foreign species of
Thalictrum. As the leafrust of wheat presumably is an introduced form,
as explained in the following pages, this would be expected, and on that
basis the species of Thalictrum should be western Asiatic or eastern
European, corresponding to the region in which wheat is believed to have

originated.

CULTURES MADE IN 1920

In preparation for cultural studies for the spring of 1920 an effort was
made during the summer and fall of 19 19 to obtain as many species of
Thalictrum as possible. It was impossible to obtain material from for-
eign botanical gardens in time to be of use, and the best that could be
done was to secure such species of Thalictrum as were carried by nursery-
men in this country, together with such native species as could be obtained
through collectors in various parts of the United States. As a result 14
species were brought together. An appeal was also made to the plant
pathologists in the various agricultural experiment stations tliroughout
the country for aid in securing telial material of the leafrust of wheat.
A very gratifying response to this appeal was made, and in this way 80
collections of telia were obtained and placed out to overwinter. Of
these, 51 collections germinated in the spring of 1920 and were sown.
The number of collections was so great that it was not possible to sow

Oct. IS, 1921 Aecial Stage of the Orange Leaf rust of Wheat

157

them on as large a number of species as was done in 19 19, and attention
was devoted mainly to sowing upon species of Thalictrum. Of the 51
collections used 9 were sown upon as many Thalictrum species as possi-
ble in order to determine the relative susceptibility of these species.
The results are given in Table II.

Tabl:^ II. — Data obtained in ig20 from sowing teliospores of Piiccinia triticina from
nine different localities on -various species of ThalictrumO'

Host inoculated.

Labor-
atory
No.

No.
5619
(Ga.).

No.

6019
(N. C ).

No.

7219
(N.C).

No.

7819

(Tenn.).

No.

8019

(Mich.).

No.

8719

(Miss.).

No.
11619
(Pa.).

No.

12519
(Idaho).

No.
15119

(Nev.)

Thalictrum angustifo-

56
16
53
17

114

61
0

I
I

0
0

-(2)

0
I

0
— 0

-(2)

61
0

0
0

I
bi

Thalictrum aquilegifo-

i>i

Thalictrum aquilegifo-

Thalictrum dasycarpum
Fisch. and LaU

Thalictrum De lavayi

I
I

-(3)

Thalictrum diocium X,

Thalictrum flavum I/. . . .
Thalictrum minus 1,

I
0

- (2)

Thalictrum, minus adi-

Thalictrum occidentale
Gray

Thalictrum polycarpum
S. Wats

Thalictrum polygamum
Muhl

- (2)
I

-(2)
I —

a — Noinfection, ■ . v, u'.vr^'! •,>'.t>.-.i, T

0 Pycnia only produced.

1 Aecia following pycnia.

A numeral in parenthesis following the sign indicates the number of times the results were obtained.
^ Although aecia were produced, the infection was weak.

The following is a list of the sources of the telial material used in the
cultures :

5619, from Athens, Ga., collected by C. A. Ludwig.

6019, from W. Raleigh, N. C, collected by Ludwig and Wolf.

7219, from Hickory, N. C, collected by C. A. Ludwig.

7819, from Tennessee, collected by C. A. Ludwig.

8019, from Coldwater, Mich., collected by B. W. Mains.

8719, from Canton, Miss., collected by C. A. Ludwig.

11619, from State College, Pa., collected by J. T. Adams.

12519, from Moscow, Idaho, collected by C. W. Hungerford.

15 1 19, from Reno, Nev., collected by G. R. Hoemer.
In addition to the sowings indicated in Table II, culture 8719 was
sown upon Aquilegia glandulosa F'isch., A. olympica Boiss., Clematis
heraclaefoHa DC, C. paniculaia Thunb., C. recta L., and Ranunculus acris
L-, all without infection.

An examination of Table II shows that 12 out of the 14 species of
Thalictrum were infected, Thalictrum occidentale and T. aquilegifolium
apparantly being immune. The species on which infection occurred
showed varying degrees of susceptibility. Thalictrum dasycarpum and

158 Journal of Agricultural Research voi. xxii. no. 3

T. polygamum gave mostly negative results or the occasional production
of pycnia. T. angustifolium, T. aquilegijolium (5), T. minus, T. minus
adiantifolium, and T. polycarpum showed occasionally a weak develop-
ment of aecia, but usually only pycnia developed or no infection occurred.
T. dioicum, in one case, showed a moderate development of aecia; in
all other cases only pycnia developed, or no infection resulted. T.
Delavayi and T. sp. (98) (PI. 21, A, B) showed fairly vigorous infection,
accompanied in most cases by more or less hypertrophy and usually by
well-developed aecia. T. flavum (PI. 21, C) and T. sp. (55) (Pi. 21,
D) showed a very vigorous infection accompanied usually by pronounced
hypertrophy of the infected leaf and petiole tissue and practically always
with the production of well-developed aecia.

An attempt has been made to check the determination of the species
of Thalictrum used in these studies, but this has been difficult because
a number of them have produced neither flowers nor fruit, and the leaf
characters in this genus are in most cases extremely variable. Specimens
of most of the species have been sent to Mr. S. F. Blake, of the Bureau
of Plant Industry, Washington, D. C, who has kindly compared them
with specimens in the United States National Herbarium and has given
his opinion as to the identity of our material. The following list gives
the species used above, their sources, and native distribution as accurately
as they could be determined. The accession number of this laboratory
follows the name of each species. ,

Thalictrum angustifolium L. (6) . Source : Seed from Brooklyn Botanic
Garden. Distribution: Central Europe and Asia Minor.

Thalictrum aquilegifolium L. (5 and 66) . Source : Bobbink and Atkins
Nursery Co. Distribution: Europe, Middle and Northern Asia. (No.
66 was purchased for T. paniculatum.)

Thalictrum dasycarpum Fisch. and Lall. (65). Source: Department of
Botany, Michigan Agricultural College. Distribution: Northern and
central United States and southern Canada.

Thalictrum Delavayi Franchet (56). Source: Farr Nursery Co. Dis-
tribution: Western China.

Thalictrum dioicum L. (16). Source: LaFayette, Ind. Distribution:
Eastern United States.

Tlialictrum flavum L. (53). Source: Farr Nursery Co. Distribution:
Europe, Western Asia, and Asia Minor.

Thalictrum minus L. (17). Source: An American nursery. Distribu-
tion: Europe, Asia, and eastern and southern Africa.

Thalictrum m,inus adiantifolium (63). Source: Seed from Brooklyn
Botanic Garden. Distribution: See T. minus.

Thalictrum occidentale Gray (115). Source: Corvallis, Oreg. Distribu-
tion: Mountains, California to British Columbia.

Thalictrum, polycarpum S. Wats. (114). Source: Berkeley, Calif. Dis-
tribution: California.

Oct. IS, 1921 Aecial Stage of the Orange Leaf rust of Wheat

159

Thalictrum polygamum Muhl. 19. Source: Ithaca, N. Y. Distribution:
Eastern United States.

Thalictrum sp. (98). Source: Palisade Nursery Co. Distribution:
Exotic.

Thalictrum sp. (55). Source: Farr Nursery Co. Distribution: Exotic.

From the data presented above it is evident that a number of species
of Thalictrum are susceptible hosts for Puccinia triticina. As far as the
host determinations are at all certain, the evidence would indicate that
the most susceptible hosts are from western Asia and eastern Europe,
and doubtless in this region other species will be found of as great or
greater susceptibility.

The remaining collections of telia showing good germination were
sown on one or more of the susceptible species of Thalictrum in order
to determine how uniformly Puccinia triticina from the United States
would go to Thalictrum. Table III gives the results of these cultures.

Table III. — Data obtained in ig20 from sowing teliospores of Puccinia triticina, from
many different localities, on four especially susceptible species of Thalictrum o-

Nmnber and source of telia.

5119 Pa

11719 Pa.. . .
9819 W. Va.
6319 N. C. .
6419 N. C. .
7319 N. C. .
5819S. C. ...
5019 Ga. . . .
4119 Ala. . . .
4519 Ala. . . .
4419 Tenn.^.
17419 Tenn.
6519 Ky . . . .
4819 Ind. . . .
5319 Ind....
10119 Ind. . .
10419 111 . . .
12419 Minn. .
3619 Iowa. .
9219 Mo. . . .
9319 Mo . . . .

9519 Mo

9619 Mo . . . .

3819 La

6719 Tex. .. .
7019 Tex.c. .
3219 Ariz. . .
61 19 Calif. .
122 19 Wash.
165 19 Wash.
199 19 Wash.
I39i9 0reg. .

T. sp. (ss).

T Delavayi
<S6).

T. flavum
(Si).

T. sp. (98)

I-(2)

o — No infection.

0 Pycnia produced.

1 Aecia following pycnia.

A numeral (in parenthesis) following the sign indicates the number of times the results were obtained.
*> Sown also on T. angustifolium, producing pycnia, and on T. aquilegifolium without results.
e Sown also on T. dasycarpum without results.

i6o Journal of Agricultural Research voi. xxii.no. 3

Source of telial material used in cultures :

51 19, from York, Pa., collected by F. D. Kern.

1 1 719, from Bradford Count}^ Pa., collected by E- T. Nixon.

9819, from Morgantown, W. Va., collected by N. J. Giddings.

6319, from Statesville, N. C, collected by C. A. Ludwig.

6419, from Statesville, N. C, collected by C. A. Ludwig.

7319, from Biltmore, N. C, collected by C. A. Ludwig.

5819, from Clemson College, S. C, collected by C. A. Ludwig.

5019, from Tifton, Ga., collected by C. A. Ludwig.

41 19, from Bay Minette, Ala., collected by C. A. Ludwig.

4519, from Auburn, Ala., collected by C. A. Lud\vig.

4419, from Union City, Tenn., collected by Carl Kurtzweil.

1 7419, from Johnson City, Tenn., collected by C. A. Ludwig.

6519, from Lexington, Ky., collected by R. S. Kirby.

4819, from Mount Vernon, Ind., collected by E- B. Mains.

5319, from Washington County, Ind., collected by H. S. Jackson.

10119, from La Eayette, Ind., collected by E. B. Mains.

10419, from Bloomington, 111., collected by Koehler and Toole.

1 24 1 9, from Wasioja, Minn., collected by G. W. Martin.

36^9, from Ames, Iowa, collected by I. E. Melhus.

9319, from Columbia, Mo., collected by W. E. Maneval.

9419, from Columbia, Mo., collected by W. E. Maneval.

9519, from Columbia, Mo., collected by W. E. Maneval.

9619, from Columbia, Mo., collected by W. E. Maneval.

3819, from Baton Rouge, La., collected by Thiel and Lud\\4g.

6719, from Dallas, Tex., collected by W. H. Ballamy.

7019, from San Antonio, Tex., collected by R. S. Kirby.

3219, from Yuma, Ariz., collected by L. Y. Leonard.

61 19, from Chico, Calif., collected by R. M. Kelia.

122 19, from Dayton, Wash., collected by J. W. Hotson.

165 19, from Colton, Wash., collected by J. W. Hotson.

19919, from Puyallup, Wash., collected by G. R. Hoemer.

13919, from Oregon, collected by G. R. Hoemer.
The data in this table, taken with those in Table II, show that Puccinia
triticina from Pennsylvania, West Virginia, North Carolina, South Caro-
lina, Georgia, Alabama, Mississippi, Tennessee, Kentucky, Indiana,
Michigan, Illinois, Minnesota, Iowa, Missouri, Louisiana, Texas, Arizona,
California, Washington, Oregon, Idaho, and Nevada gave positive results
when sown upon Thalictrum.

The following collections showed some germination but produced no
infection when sown on Thalictrum :

11819, from Hopkinsville, Ky., collected by Carl Kurtzweil.

9419, from Columbia, Mo., collected by W. E. Maneval.

3919, from Fayetteville, Ark., collected by H. R. Rosen.

5419, from Memphis, Tenn., collected by A. F. Thiel,

Oct IS, 1921 Aecial Stooge of the Orange Leaf rust of Wheat 161

7519, from Southampton, N. Y., collected by H. S. Jackson.
8319, from Rocky Ford, Colo., collected by J. G. Leach.
10019, from Buffalo, Minn., collected by G. W. Martin.
105 19, from Plainview, Nebr., collected by H. W. Thurston.
11019, from Vermillion, Minn., collected by G. W. Martin.
1 12 19, from Newark, Del., collected by T. F. Manns.
Besides the above, the following collections were wintered, but no
germinating teliospores were found, and in consequence they were not

sown.

1419, from Santa Rosa, Calif., collected by H. S. Jackson.

3319, from Sonora, Mexico, near Yuma, Ariz., collected by L. Y.
Leonard.

3419, from St. Louis, Mo., collected by E- B. Mains.

3719, from Jackson, Tenn., collected by Kurtzweil and Thiel. .5

4019, from Corvallis, Oreg., collected by G. R. Hoemer. ,i

5519, from St. Paul, Minn., collected by A. F. Thiel. .^ |

6619, from Hiawatha, Kans., collected by W. H. Ballamy.o\^ |

6819, from Marshall, Mo., collected by R. S. Kirby. ) n

6919, from Guthrie, Okla., collected by R. S. Kirby.

71 19, from Wellington, Mich., collected by G. H. Coons.

7919, from Nashville, Tenn., collected by C. A. Ludwig.

91 19, from Madison, Wis., collected by E. B. Mains.

108 1 9, from Manhattan, Kans., collected by L. E. Melchers.

Ill 19, from Toledo, Iowa, collected by I. E- Melhus.

11919, from Fort Collins, Colo., collected by J. G. Leach.

12019, from East Lansing, Mich., collected by Acelia M. Leach.

12 1 19, from East Lansing, Mich., collected by Acelia M. Leach.

12319, from Pullman, Wash., collected by F. D. Heald.

1 38 1 9, from Fort Collins, Colo., collected by J. G. Leach.

21019, from Moscow, Idaho, collected by G. R. Hoemer.

25519, from Murfreesboro, Tenn., collected by Carl Kurtzweil.

25719, from Clarksville, Tenn., collected by Carl Kurtzweil.
The accompanying map (fig. i) shows the source of collections used
in the work together with the results obtained with them at La Fayette,
Ind. This map shows that material from the States of Pennsylvania,
West Virginia, Indiana, Illinois, North Carolina, South Carolina, Georgia,
Alabama, Mississippi, Louisiana, and Texas gave germination uniformly
and infected Thalictrum in all cases. A region represented by the States
of Colorado, Oklahoma, Arkansas, Kansas, Nebraska, Minnesota, Iowa,
and Missouri, with one arm running through Wisconsin into Michigan
and another through Tennessee into Kentucky, gave material which
usually did not germinate or, if germination was obtained, produced
infection on Thalictrum in only a few cases. Whether this situation
indicates the presence of another strain of the leafrust having different
characteristics as regards its viability and power to infect Thalictrum,
or whether it means that the season or climate was of such a nature that

l62

Journal of Agricultural Research voi. xxii. No. 3

teliospores of a low vitality were produced, remains for future investiga-
tion to decide. Telial material from the Pacific coast, while not viable
in a number of cases, produced infection on Thalictrum in all cases where
germination was obtained.

The aecia produced from the following telial collections were sown
back upon wheat:

4519, from Auburn, Ala.

4819, from Mount Vernon, Ind.

5019, from Tifton, Ga.

5619, from Athens, Ga.

5819, from Clemson College, S. C.

6019, from W. Raleigh, N. C.

6319, from Statesville, N. C.

/u_ p/ \ ■ — j —

I T^ L 1 L

V \ / /^i-

0 i
.+

500~^

?pr+^

1.
+0

— <

•
\

\ \ /— ~— ^/ *

0°

vXT r

I XI A/ 0 A/OT SOi^A/.

,->

\ai4

Flo. I, — Map showing results obtained at LaFayettc, Ind., with leafrust material collected in different

parts of the United States.

7219, from Hickory, N. C.

7819, from Tennessee.

8019, from Coldwater, Mich.

8719, from Canton, Miss.

9619, from Columbia, Mo.

9819, from Morgantown, W. Va.
10419, from Bloomington, 111.
11619, from State College, Pa.
1 24 1 9, from Wasioja, Minn.
125 1 9, from Moscow, Idaho.
151 19, from Reno, Nev.
165 19, from Colton, Wash.
1 7419, from Johnson City, Tenn.

Oct. 15, 1921

Aecial Stage of the Orange Leaf rust of Wheat

163

These were each sown upon the variety of wheat known as Dawson
Golden ChaflF, and in all cases positive infection was obtained followed by
the development of uredinia which were typical of Puccinia triticina.

Sowings of aeciospores also were made upon a number of grasses.
Aecia which were produced from telia obtained from Hickory, N. C,
Canton, Miss., and Moscow, Idaho, were used and Table IV gives the
results.

Table IV. — Results obtained in ig20 from sowing the aeciospores of Puccinia triticina
produced from telia obtained in three different areas, on wheat and related grasses

Host inoculated.

Arrhenatherum elatius (L.) Mert & Koch. . .

Agropyron caninum (L.) Beauv

Agropyon cristatum Beauv

Agropyron desertorum Schult

Agropyron in£rme (Schribn. & Sm.) Rydb .

Agropyron repens (L.) Beauv

Agropyron tenerum Vasey

Elymus atistralis Schribn. & Ball

Elynius canadensis L

Elymus glaucus Buckl

Elym^us triticoides Buckl

Elymus virginicus L

Hordeum caespitosum Schribn

Hordeum gu^soneanum Pari

Hordeum jubatum L

Hordeum pusillum Nutt

Hordeum inurinum I,

Hordeum vulgare L

Hystrix Hystrix (L.) Millsp

Notholcus lanatus (L.) Nash

Secale cereale L

Sitanion Hystrix (Nutt.) J. G. Sm.

Triticum aegilops Beauv

Triticum aestivum L

Number and source of aecia.

No. 7219
(N.C.).

t Many.

No. 8719
(Miss.).

t One ure-
dinium.

t Many.

No. 12519
(Idaho).

t Few.
t Many,

— No infection.

t Uredinia produced.

Except for the one uredinium produced on Secale cereale, Triticum
aestivum and T. aegilops were the only species infected

DESCRIPTION OF AECIA

The following description has been drawn from the aecia obtained
in the cultures discussed above.

Pycnia amphigenous, mostly epiphyllous, numerous, crowded upon
more or less swollen reddish brown to yellowish areas 2 to 15 mm. in
diameter, conspicuous, subepidermal, honey-yellow, globoid or flattened
globoid, 80 to 145 /i broad by 80 to 130 fx high; ostiolar filaments 95 to
190 jj, long, agglutinated to form a prominent, broad column.
65508°— 21 4

164 Journal of Agricultural Research voi. xxii. no. 3

Aecia hypophyllous, crowded in more or less swollen, gall-like, reddish
brown or yellowish areas 2 to 15 mm. in diameter, cupulate or short
cylindric, 0.2 to 0.6 mm. in diameter, up to 0.5 mm. high; peridium white
or yellowish, the margin erose or somewhat lacerate, recurved; peridial
cells oblong or somewhat rhomboidal in longitudinal radial section, 14 to
19 by 18 to 29 IX, abutted or slightly overlapping, the outer wall 6 to 7 /x
thick, transversely striate, the inner wall thinner 2 to 3 yLt, very coarsely
verrucose; aeciospores angularly globoid or ellipsoid, 16 to 20 by 16 to
26 /x; wall colorless, thin, i /i or less, very closely and finely verrucose.

The pycnia and aecia usually were produced in definite galls or swellings.
These galls apparently were formed by the excessive enlargement of the
cells of the infected areas, especially those of the mesophyll. When
infection took place in the young, rapidly developing tissue of the petiole,
galls developed (PI. 21, D) which were 10 or 15 times as large as the
normal petiole. A very noticeable odor, resembling that of the hyacinth,
was often detected as the pycnia reached maturity.

GENERAL DISCUSSION OF RESULTS

The discovery that species of Thalictrum are the aecial hosts for
Puccinia triticina goes to support Arthur's contention {i, v. 9, p. 304) that
the leafrust of wheat is closely related to grass rusts of the type of Puccinia
Agropyri, having aecia on species of the family Ranunculaceae. A num-
ber of cultures have been made with rusts of this type, connecting them
with various species of Thalictrum. Plowright, in England {21, p. j8i),
connected aecia on Thalictrum flavum with a rust on Agropyron repens.
To this rust he gave the name Puccinia persistens Plowr. He considered
Aecidium Ranunculacearum 7 Thalictri flavi DC, and Aecidium Thalictri
fiavi (DC.) Winter as synonyms, and describes the aecia as occurring
on thickened spots with aeciospores subglobose 17 to 20 by 20 to 30 /i.
Fischer (12, p. 37-63), in Switzerland, cultured a rust from Poa nemoralis
var. firmula on Thalictrmn minus, T. aquilegijolium and T. foetidum.
On account of the morphological similarity, he concluded that his material
belonged to Puccinia persistens, although he made no cultures on either
Agropyron repens or T. flavum. He describes the aecia (ij, p. 347-349)
as having peridial cells with the outer wall 4.5 to 13.5 ^ thick and the inner
2 to 6 fjL and aeciospores 10 to 21 /x broad and up to 28 /i long. An exam-
ination of Sydow's Uredineen No. 725, issued as Puccinia persistens on T.
aquilegifolium, shows the following measurements : Peridial cells, 18 to 23
by 21 to 26 /x; outer wall, 7 to g fx; inner, 3 to 5 /x; aeciospores, 16 to 19
by 19 to 26 n.

Juel (j<5, p. 411), in Sweden, made cultures connecting aecia on Thalic-
trum alpinum with a rust on Agrostis borealis and Anthoxanthum odor-
atum. To this rust he gave the name Puccinia borealis Juel, and con-
sidered Aecidium thalictri Grev. as a synonym. His description follows:
Pycnia not present; aecia not causing hypertrophy of host tissue; aecio-

Oct i^. 1921 Aecial Stage of the Orange Leaf rust of Wheat 165

spores about 13 ju in diameter. The Sydows {23, p. 718-719) give the
measurement of the aeciospores as 13 to 18 /x in diameter or 13 to 16 by
18 to 20 M and note that no swelHngs are produced on the leaves of the host.
An examination of Eriksson's Fungi Parasitici Scandinavici 432a, col-
lected by Juel in Norway, gives the following measurements: Peridial
cells, 16 to 19 by 19 to 29 /x; the outer wall, 10 /x; the inner, 3 to 4 ju;
aeciospores 14 to 16 by 16 to 21 m-

Rostrup (22, p. 269-273), in Denmark, obtained infection with aecio-
spores from Thalictrum minus on Elymus arenarius and considered the
rust to be Puccinia Elymi Westendorp. The writers have seen neither
description nor material of these aecia.

The Sydows (23, p. 827) mention that Lindroth in Finland connected
an aecidium on Thalictrum m/ijus with a rust on Agropyron caninum.
No description or material of this connection is available for study.

In North America a number of connections have been established by
the cultures of Arthur and of Fraser. Arthur (/, v, i, p. 248-249) reports
culturing a rust found associated with aecia on Thalictrum sparsifiorum
from Bromus Porteri to T. dioicum. To this he gave the name Puccinia
alternans. He describes the aecia as having peridial cells 2 1 to 29 /x long
with the outer wall 9 to 12 /x thick and the inner 5 to 7 /x and with aecio-
spores 15 to 20 by 17 to 24 IX. A number of other species of Thalictrum
are given as hosts.

Arthur (j, v. 2, p. 226) also reports obtaining infection from telia on
Agropyron resulting in aecia on Thalictrum alpinum but not on T. dioicum.
This material he considered as belonging in Puccinia obliterata Arth.,
which he had previously shown as having aecia on Aquilegia. A study
of the material obtained by this culture shows little or no hypertrophy
of the host tissue. The peridial cells measure 16 to 21 by 24 to 32 /x,
having the outer wall 7 to 9 ^u thick and the inner 3 to 5 /x. The aecio-
spores measure 14 to 18 by 18 to 23 jx.

Still another connection was obtained by Arthur (i, v. 8, p. 132-133)
when he cultured a rust on Festuca Thurberi to Thalictrum dioicum,
producing aecia. To this he later (2, p. 113) gave the name Puccinia
Cockerelliana Bethel. He gives the peridial cells as 16 to 23 by 27 to
36 /x with the outer wall 6 to 8 /x and the inner 2 to 3 a« and aeciospores 18
to 24 by 20 to 29 IX with a wall 1.5 to 2.5 ix thick. The natural host for the
aecia is given as T. Fendleri.

Fraser {14, p. 131-133) reports sowing aeciospores from Thalictrum
dasycarpwn on Elymus canadensis, E. virginicus, Agropyron tenerum,
A. Richardsonii, Hordeum jubatum, Triticum vulgare, and Bromus
ciliatus, obtaining infection on E. canadensis, E. virginicus, H. juba-
tum, and B. ciliatus. When, however, the rust obtained upon B. cili-
atus was sown on E. virginicus, A. tenerum., A. Smithii, A. repens, and
H. jubatum no infection was obtained on these species. From these
results Fraser concludes that two strains of Puccinia Agropyri KHis and E.,

1 66 Journal of Agricultural Research voi. xxn. no. 3

were present in the aecial material on Thalictrum which he used for the
culture. A study of the aecia used in these cultures shows the dimensions
of the peridial cells to be 15 to 19 by 23 to 29 n with the outer wall 7 to
10 /x and the inner 3 fx thick and the aeciospores 14 to 19 by 19 to 23 p. in
diameter.

A comparison of the foregoing description of the aecia of Puccinia
triticina with the measurements given for the various grass rust aecia on
Thalictrum shows surprisingly little variation. The aecia of P. Cockerel-
liana show the greatest difference, having larger peridial cells and some-
what larger aeciospores with much thicker walls than the aecia of P. triti-
cina. Slightly smaller aeciospores occur in P. horealis and P. obliterata,
and the aecial infection causes little or no hypertrophy of the host.
The remaining aecia differ mainly in slightly thicker walls of the peridial
cells.

It is evident that Puccinia triticina is closely related to P. persistens.
Whether the name Aecidium Tlialictri-flavi (DC.) Wint. should apply to
the aecial stage of the former is a question which can not be answered
with the available information. De Candolle (7, p. giy) described
A. Ranunculacearum for aecia occurring on the family Ranunculaceae
and as a variety of this gives Thalictri-flavi without further description.
Winter (24, p. 269) raises this variety to specific rank and gives a
description which, however, could apply to the aecia of either rust.
As both P. triticina and P. persist-ens are common rusts throughout
Europe, there is no way of determining definitely to what aecia the name
was applied beyond the fact that they were on Thalictrum flavum. As
it has been shown that at least some aecia on that host in England belong
to P. persistens the name A. Thalictri-flavi should be retained for the
present as a synonym of that species, at least until aecia can be found
in Europe upon T. flavum which will produce the leafrust of wheat.

Upon their grass hosts these rusts present a somewhat greater varia-
tion. They all have uredinia with globoid or ellipsoid urediniospores
with a varying number of scattered pores, usually more than six, and
with few or no paraphyses. The telia are long, covered by the epidermis,
usually with more or less stroma present, and the teliospores are cylindric,
more or less flattened at the apex, and with a very short pedicel. Puc-
cinia Cockerelliana differs most noticeably from P. triticina in that the
teliospores are much longer and the telia do not remain entirely covered
by the epidermis at maturity. P. Elymi differs especially in the thicker
and darker walls of the urediniospore and in the longer teliospores which
are often many-celled. P. alternans, P. borealis, P. obliterata, and P.
persistens differ but little, mostly in the tinting of the urediniospore wall
and a slight variation in pore number.

Although the morphological differences between Puccinia triticina and
the related rusts discussed above are not great, their biologic specializa-

Oct. IS, 1921 Aecial Stage of the Orange Leaf rust of Wheat 167

tion to their hosts is very pronounced. This appears to hold true for the
aecial as well as the grass hosts. P. Cockerelliana and P. alternans go to
Thalictrum dioicum, and P. Elymi to T. minus, as aecial hosts, neither of
which is a favorable host for P. triticina. The rust of T. dasycarpum,
used by Fraser {14) in his cultures, is on a host which was not infected
by P. triticina. P. horealis and P. ohliterata on T. alpinum offer no com-
parison, as P. triticina was not sown on that host. P. persistens, as
cultured by Fischer on T. minus and T. aquilegifolium, is upon species of
Thalictrum unfavorable for P. triticina, while P. persistens as originally
cultured by Plowright upon T. flavum is on the most congenial host for
the leafrust of wheat. It is very probable that Plowright and Fischer
were working with two distinct biologic strains. Although T. flavum
appears to be a favorable host for both P. persistens and P. triticina, and
these two rusts are very similar in their morphology, the inability of the
latter to infect Agropyron repens shows that it is biologically distinct
from the former. A study is being made of the relationship of P. triticina
to grass hosts other than wheat. From the data now at hand, it would
appear that, in addition to the grasses listed in Table IV, species of
Bromus, Festuca, Agrostis, Poa, and Anthoxanthum are immune from
the leafrust of wheat. These results indicate that, as far as its telial
host is concerned, P. triticina also is biologically distinct from other grass
rusts having aecia on Thalictrum.

A similar situation exists in the relationship of Puccinia triticina to
rusts having aecia upon species of other genera of the Ranunculaceae.
Slight morphological differences, such as urediniospore size, wall color,
and pore number, exist among the different races producing aecia upon
species of such genera as Actaea, Anemone, Clematis, Delphinium, etc.
A similar, or perhaps greater, biologic specialization is also to be found
among these races. The importance of these morphological characters
and biological differences which occur among the members of this group
can not be fully determined at present on account of our comparatively
limited knowledge of but few races. Any final interpretation must
await further study of a greater number of such races. On the basis of
our present knowledge, the disposition of P. triticina must depend largely
upon the species concept held. In Europe there is a tendency among
certain students of the rusts to consider as species those rusts showing
distinct biologic specialization regardless of the absence of morphological
difference. In this country, on the other hand, the general tendency is
to include in a single species all closely related forms having but little
difference in their morphology. Forms limited to a definite host, or
hosts, are considered as races of such species. On the former basis, P.
triticina would be considered a distinct species comparable to P. Elymi,
P. Agropyri, P. persistens, etc., while with the latter concept it would be
united with all or part of these, each being considered a race of a

168 Journal of Agricultural Research Voi. xxii. no. 3

collective species to be designated, according to the limitations of
the species concept held and the system of nomenclatm-e used, as
P. Agropyri B. and E. (j, v. 9, p. 304), P. Clematidis (DC.) Lagerh., or
Dicaeoma Clematidis (DC.) Arth. (j, p. 333-337).

The close biological specialization of Puccinia triticina to wheat is of
considerable significance with respect to the bearing it has upon the
possible origin of this rust and of wheat itself. Since wheat is an intro-
duced plant, it is logical to assume that a rust showing such close biolog-
ical specialization to it is also introduced and of foreign origin.

It is generally recognized among students of the rusts that a high de-
gree of host specialization must have been acquired in certain groups of
species at a very early stage in the evolutionary history of this group of
fungi. It is also recognized that the host is the most important factor
in the evolution of highly specialized pai-asitic fungi. As the higher
plants have gradually developed during geological times, their rust
parasites have developed with them. It therefore appears reasonable to
assume that Puccinia triticina, which shows such a high degree of spe-
cialization to wheat at the present time, had its origin as a distinct strain
comparatively early in the development of the group of grasses from
which our cultivated wheats have originated. The original distribution
of the rust presumably would coincide with the distribution of the
ancestral wheats.

A study of the relative susceptibility of various species of Thalictrum
to infection by this rust is of interest in this connection. The four most
susceptible species of Thalictrum encountered in this investigation are
all of foreign origin. The most susceptible of our native North American
species, Thalictrum dioicum, does not compare in susceptibility with
these four foreign species— T. flavum, T. Delavayi, T. sp. 55, and T. sp.
98 — but is comparable to the resistant foreign species such as T. minus.
That these foreign susceptible species of Thalictrum are also to be con-
sidered as indicating a foreign origin of the rust would appear to follow if
the nature of aecial infection is considered. Heteroecious rusts in most
cases infect their aecial hosts only for a comparatively short period of
tune while the teliospores are germinating in the spring. The infection
produced, not being able to propagate itself upon such hosts, causes little
or no damage, and they are in most cases soon able to outgrow it. On
this account it is hardly to be expected that a natural selection of resist-
ant strains of aecial hosts takes place in nature comparable to that which
occurs where the host is killed or prevented from producing seed. Should
this occur in heteroecious rusts which are not able to survive adverse
conditions in winter or summer by means of urediniospores, such a selec-
tion would be fatal to the rust itself. For this reason the susceptibility
of the aecial hosts of P. triticina may be taken as indicative of its origin.
It is true that susceptibility of a host species does not necessarily indi-
cate that such a species was a native host of the rust nor does resistance

Oct. IS, i92t Aecial Stage of the Orange Leaf rust of Wheat 169

of some one species denote that the rust is not to be found in the habitat
of such a resistant species, for susceptibility or resistance is not dependent
upon the presence or absence of the rust but may develop with the species
in any region. It is regarded as significant, however, that of the species
of Thalictrum tested the most susceptible are exotic. This fact, taken
with the foreign origin of wheat itself, is confirmative of the foreign
origin of the rust.

The native habitats of two of these species of Thalictrum are known
with some degree of certainty. Thalictrum flavurn is found throughout
Europe, western Asia, and Asia Minor. T. Delavayi is given by the Index
Kewensis ^ as occurring in western China, probably indicating a distri-
bution in the little-known mountainous regions of Tibet and Chinese
Turkestan. These two species, taken together, would therefore indicate
as the most probable original distribution a region in which the two
Thalictrum species may border or overlap, such as that of southwestern
Asia. Such an origin would indicate a like origin for wheat itself,
which, we believe, would agree with the latest theories advanced as to
the original home of wheat.

Concerning the occurrence and distribution of the aecia of Puccinia
triticina but little can be said with the data at hand. It is also probable
that the aecial stage occurs, and probably assumes greater importance,
in other regions than it may in either Europe or North America, where the
rust is known to overwinter in its uredinial stage. Thus in such countries
as India, where Butler has shown there is no oversummering of the rust,
the Thalictrum species of the foothills may be of importance in starting
the rust the next season. The question of the role which the aecia of the
leafrust of wheat plays in its life history and distribution must, however,
be left for future research to solve. Whether native species of Thalictrum
serve as aecial hosts in North America and, if so, whether they serve as
important factors in the development of the leafrust of wheat and
whether there is more than one race of the leafrust, as indicated by the
results obtained from the Great Plains area, or whether these results
were due to other causes, such as climatic or seasonal effects weakening
the vitality of the teliospores, are all questions on which further investi-
gation is planned. Other species of Thalictrum from foreign botanic
gardens also v/ill be studied in regard to their susceptibility to the orange

leafrust of wheat.

SUMMARY

(i) The aecial stage of Puccinia triticina has been produced in green-
house cultures upon several species of Thalictrum.

(2) The various species of Thalictrum show varying degrees of sus-
ceptibility to the rust. Thalictrum occidentale was apparently immune.
Upon T. dasycarpum and T. polygamutn an occasional devlopment of

> INDEX KEWENSIS PLANTARUM PHANEROGAMARUM. SUPPI.EMENTUM PRIMUM . . . CONFECERUNT
THBOPHIlrUS DURAND ET B. DAYDON JACKSON. p. 42^. BniXCllis I9OI-06.

lyo Journal of Agricultural Research voi. xxn. no. 3

pycnia took place. When T. angustijoliuvi, T. aqtiilegifolium, T. dioicum,
T. minus, T. minus adiantijolium, and T. polycarpum were inoculated
usually only pycnia resulted, with an occasional weak development of
aecia, while in other cases no infection occurred. Two undetermined
species of Thalictrum, as well as T. Delavayi and T. flainmi, when inocu-
lated, showed a vigorous development of aecia, increasing in suscepti-
bility in the order named.

(3) Puccinia triticina is apparently limited to species of the genus
Thahctrum, no infection being obtained upon species of Aconitum,
Actaea, Anemone, Aquilegia, Cimicifuga, Clematis, Delphinium, Echium,
Ilepatica, Hydrophyllum, Impatiens, Mertensia, Myosotis, Ornithogalum,
Phacelia, Camassia, Ranunculus, or Trollius.

(4) On account of the morphology and host relationships, Puccinia
triticina is considered to be very closely related to P. persistens, P.
borealis, P. alternans, P. ohliterata, P. Elymi, and P. Agropyri, but is
separable from these rusts by its sharp biologic limitation to wheat.

(5) Puccinia triticina is considered to be of foreign origin, because
wheat, for which it shows close specialization, is an introduced host, and
because the most susceptible species of Thalictrum which serve as aecial
hosts also are exotic.

LITERATURE CITED
(i) Arthur, Joseph Charles.

1909-17. CtliTURES OF UREDINEAE IN 1908, I909, 1915, 1916 AND 1917. In

Mycologia v. i, no. 6, p. 225-256; v. 2, no. 5, p. 213-240; v. 8, no. 3,
p. 125-141; V. 9, no. 5, p. 294-312.
(2)

1919. NEW SPECIES OF UREDINEAE — XI. In Bui. Torrey Bot. Club, v. 46,

no. 4, p. 107-125.

(3) and Fromme, Fred. Denton.

1920. DICAEOMA ON POACEAE. In North American Flora, v. 7, pt. 4-5, p.

269-341.

(4) Bary, Anton de.

1866. NEUE UNTERSUCHUNGEN ubER urEdinEEn. In Monatsber. K. Preuss.
Akad. Wiss. Berlin, 1866, p. 205-215, i pi.

(5) BoLLEY, H. L.

1889. WHEAT rust. Ind. Agr. Exp. Sta. Bui. 26, 19 p., 9 fig.

(6) Butler, E. J., and Hayman, J. M.

1906. INDIAN wheat rusts. In Mem. Dept. Agr. India, Bot. Ser., v. i, no. 2,
p. 1-52, I fig., 5 pi. (1-4 col.).

(7) CandollE, a. p. de.

1815. FLORE FRANfAisE. • • t. 5 (v. 6). Patis.

(8) Carleton, Mark Alfred.

1899. CEREAL RUSTS OF THE UNITED STATES. A PHYSIOLOGICAL INVESTIGA-
TION. U. S. Dept. Agr. Div. Veg. Physiol, and Path. Bui. 16, 74 p.,
I fig., 4 col. pi. Bibliography, p. 70-73.

(9) Cunningham, D. D., and Prain, D.

1896. A note on INDIAN WHEAT-RUSTS. In Rec. Bot. Survey India, v. i,
no. 7, p. 99-124.

Oct. IS, 19" Aecial Stage of the Orange Leaf rust of Wheat 171

(10) Eriksson, Jakob.

1899. NOuvELLES ^TudES sur LA rouillE brunE dES c^r^alES. In Ann.

Sci. Nat. Bot. ser. 8, t. 9, no. 2/4, p. 241-288, pi. 11-13 (col.). Lit-
tdrature citee, p. 286-287.

(11) and Henning, Ernst.

i8g4. DIE hauptrESULTATE EinER nEUEN unTERSUCHUNG ubeR die GETREI-
DEROSTE. In Ztschr. Pflanzenkrank., Bd. 4, p. 66-73, 140-142, 197-
203, 257-262.

(12) Fischer, Eduard.

1898. ENTWICKLUNGSGESCHICHTLICHE UNTERSUCHUNGEN UBER ROSTPIUZE. . .

X, 120 p., 16 fig., 2 pi. (Beitrage zur Kryptogamenflora der Schweiz,
Bd. I, Heft. I.)

(13)

1904. DIE UREDINEEN DER SCHWEIZ. xciv, 590 p., 342 fig. Bern. Literatur-
verzeichniss, p. 558-576. (Beitrage zur Kryptogamenflora der vSchweiz
Bd. 2, Heft. 2.)

(14) Eraser, W. P.

1919. CULTURES OF hETEroecious RUSTS IN 1918. In Mycologia, v. 11, no. 3,
p. 129-133.

(15) Hitchcock, A. S., and Carleton, M. A.

1894. SECOND REPORT ON RUSTS OP GRAIN. Kans. Agr. Exp. Sta. Bui. 46, 9 p.

(16) JuEL, H. O.

1894. MYKOLOGISCHE BEITRAGE I. ZUR KENNTNISS EINIGER UREDINEEN AUS

DEN gebirgsgEgendEn skandinaviens. In Ofvers. K. Svenska
Vetensk. Akad. Forhandl., Arg. 51, no. 8, p. 409-418.

(17) Klebahn, Heinrich.

1900. beitrage zur kenntnis der getreideroste. II. In Ztschr. Pflanzen-

krank., Bd. 10, Heft. 2, p. 70-96, I fig.

(18)

1904. DIE wirtswechselnden rostpilze. . . 447 p. Berlin. Literatur, p.
ix-xxxvii.

(19) MtJLLER, Fritz.

1901. BEITRAGE ZUR KENNTNIS DER GRASROSTC. In Bot. Centbl., Beihefte.
Bd. 10, Heft 4/5, p. 181-212, I fig..

(20) Nielsen, p.

1877. bemaerkninger om nogle rustarter, navnlig om en genetisk

FORBINDELSE MELLEM AECIDtUM TUSSILAGINIS PERS. OG PUCCINIA POARUM

N. SP. In Bot. Tidsskr., Raekke 3, Bd. 2, p. 26-42, 3 fig.

(21) Plowright, Charles B.

1889. A monograph of the BRITISH UREDINEAE AND USTILAGINEAE . . . vii,

347 p., 13 fig., 8 pi. London. Authors quoted, p. 309-315.

(22) ROSTRUP, E.

1898. ET NYT VAERTSKIFTE HOS UREDINACEERNE OG KONIDIER HOS THECAPHORA

CONVOLVULI. In Overs. K. Danske Vidensk. Selsk. Fordhandl., 1898,
No. 5, p. 269-276.

(23) Sydow, p., and Sydow, H.

1904. MONOGRAPHiA uredinearum . . . V. I. Lipsiae.

(24) Winter, Georg.

1884. die PILZE DEUTSCHLANDS, OESTERREICHS UND der SCHWEIZ. ABT. I.
SCHIZOMYCETEN, SACCHAROMYCETEN UND BASIDIOMYCETEN. In Raben-

horst, L. Kryptogamen-Flora von Deutschland, Oesterreich und der
Schweiz. Aufl. 2. Bd. i, Abt. i. Leipzig.

PLATE 21

A.— Infection produced upon Thalictrum sp. (98) inoculated with Puccinia triticina
from Colton, Wash. (16519).

B. — Infection produced upon Thalictrum sp. (98) inoculated with Puccinia triticina
from Canton, Miss. (8719).

' C. — Infection produced upon Thalictrum flavjim (53) inoculated with Puccinia iriii-
ana from Hickory, N. C. (7219).

D. — Infection produced upon Thalictruin sp. (55) inoculated with Puccinia triticina
from Hickory, N. C. (7219).

(172)

Aecial Stage of the Orange Leafrust of Wheat

PLATE 21

Journal of Agricultural Research

Vol. XXII, No. 3

A Transmissible mosaic disease oe Chinese

CABBAGE, MUSTARD, AND TURNIP

By E. S. ScHUivTz

Pathologist, Office of Cotton, Truck, and Forage Crop Disease Investigations, Bureati of
Plant Industry, United States Department of Agriculture

In the fall of 19 19, while the writer was selecting different kinds of
plants for inoculation experiments with mosaic of Irish potatoes (Sola-
num tuberosum Linn.), Dr. W. A. Orton called his attention to mottling
in plants of Chinese cabbage (Brassica pekinensis (Lour.) Gagn.), mus-
tard {B. japonica Coss.), and turnip {B. rapa, Linn.). The mottling
resembled that of mosaic plants of other species, such as potato and
tobacco. Diseased and healthy individuals were found in the same plot;
the former appeared in groups in some parts of tlie field, suggesting an
infectious character of this malady. Evidence bearing upon the nature
of this disease, its symptoms, and means of transmission is presented in

this paper.

SYMPTOMS

Mosaic of Chinese cabbage, mustard, and turnip produces a distinct
mottling of the leaves, very similar to that of mosaic diseases of the
Solanaceae. This mottling is produced by the appearance of irregular
light green and dark green areas on the leaves (PI. B ; 22, D, E ; 24, A, B).
These light green areas usually adjoin the veins, from which they may
extend so as to include a considerable area of the leaf surface between
the veins. Another very common macroscopic symptom of this disease
is the characteristic ruffling and distorting of the leaf surface (PI. 24,
A, B). On the raised areas the dark green patches appear. The leaf
margins frequently are much more irregular than in healthy plants,
causing some of the leaves to appear somewhat unsymmetrical. In addi-
tion to these common abnormalities on the leaves the entire plant may
be dwarfed, and the flower stalk and number of blossoms may be con-
siderably reduced (PI. 22, B; 23, B).

OCCURRENCE IN THE FIEED

Since mosaic individuals appeared among Chinese cabbage, mustard,
and turnip plants growing in adjoining plots, interspecific susceptibility
was suggested. Furthermore, it was found that a large percentage of
the plants were infested with aphids, Myzus persicae Sulz.,^ one of the

1 Identified by Dr. A. C. Baker, Entomologist, Bureau of Entomology, United States Department of
Agriculture.

Journal of Agricultural Research, Vol. XXII, No. 3

Washington, D. C. Oct. 15, 1921

aaa Key No. G-248

(173)

174

Journal of Agricultural Research voi. xxn. N0.3

casual agents in the transmission of mosaic and leafroll of Irish potato.^
In view of these field observations experiments on this disease were
conducted in the greenhouse at Washington, D. C, during the winters of
1919-20 and 1920-21.

TRANSMLSvSION WITH PLANT JUICE

Chinese cabbage, mustard, and turnip plants showing mosaic mottling
were taken from the field and planted in pots in the greenhouse. Only
a small percentage of these mature and mosaic plants survived trans-
planting, so that the supply of mosaic material for inoculations was thus
considerably reduced, and therefore only a small number of healthy
plants were inoculated at one time.

Inoculations with juice were made by rubbing the leaves between the
fingers so that considerable sections of the leaflets were crushed, apparently
permitting the applied juice to be absorbed by such areas of the leaf as
still remained free or partly free from mutilation. Such operations were
performed chiefly upon the youngest leaves, the first applications being
made when the plants had developed about five leaves. In Table I the
results of these inoculations are presented.

Table I. — Inoculations with juice from mosaic plants

Variety and species
inoculated.

Time of inocu-
lation.

Source of juice.

Southern

turnip.

Do. .

Prize

Mustard .
Do.

Do.
Do.

Pe-tsai or Chinese
cabbage.

Dec. 4, 1919 .

Dec. 6, 1919 .

...do

/Mar. 8, 192 1

\Mar. 21, 192 1

fMar. 9, 192 1

\Mar. 21, 1921

Jan. 15,1921

Dec. 4, 1919

....do

Dec. 6, 1919

Mosaic Southern

Prize tiu-nip.
Healthy turnip. . . .
Mosaic Green Moun-
tain potato.
do

}....do

[Mosaic mustard. . . .

Mosaic pe-tsai or

Chinese cabbage.

Healthy

Mosaic Green Moun-
tain potato.

Number

of plants

inoculated.

Number
of plants
mosaic."

0
0

5
4

0
0

Per cent-
age mosaic.

63
60

<» Date of last observation April 2, 1920 and 1921.

The data indicated in Table I disclose the fact that mosaic mottling
was obtained only when juice from a mosaic plant was introduced into

I ScHULTz, E. S., FoLSOM, Donald, Hildebrandt, F. Merrill, and Hawkins, Lon A. investigations
ON THE MOSAIC DISEASE OF THE IRISH POTATO. In Jour. AgT. Research, v. 17, no. 6, p. 247-274, pi. A-B
(col.), 25-30. 1919. Literature cited, p. 272-273.

ScHULTz, E. S., and Folsom, Donald, transmission of the mosaic disease of irish potatoes. In
Jour Agr. Research, v. 19, no. 7, p. 3x5-338, pi. 49-56. 1920.

ScHULTZ, E. S., and Folsom, Donald, leafroll, net-necrosis, and spindling-sprout of the irish
POTATO. In Jour. Agr. Research, v. 21, no. i, p. 47-So, pi. 1-13 1921. Literature cited, p. 7S-80.

Oct. IS, I92I Transmissible Mosaic Disease of Cabbage 175

a plant of the same or a closely related species.^ No mosaic mottling
appeared on any of the cruciferous plants inoculated with juice from
mosaic potato. With a more adequate supply ofcrucifer mosaic mate-
rial and repeated applications it is probable that every plant treated
would have developed mosaic mottling, such as has frequently been
obtained with mosaic potato juice inoculations on the Irish potato.^

The first mosaic mottling was observed from 20 to 30 days after inoc-
ulation, which also corresponds very closely with the incubation period
for mosaic of Irish potato. The results in Table I also disclose successful
inoculations on plants in different species of Brassica. Further evidence
on this interspecific infection is presented in Table II on transmission by

means of aphids.

TRANSMISSION WITH APHIDS

Since aphids were found on every mosaic plant examined in the field
and on account of the fact that these insects have been found to transmit
mosaic of tobacco,^ spinach blight/ and mosaic of potato,'' experiments
were carried on with these insects. Aphids belonging to Myzus persicae
Sulz. were used in this investigation. These insects were originally
collected from the morning-glory and transferred to healthy turnip and
mustard plants on which they were cultured while confined under cages
until needed for inoculation. Neither the morning-glory nor the turnip
or mustard plants on which these insects fed before being transferred
to mosaic Chinese cabbage and turnip developed mosaic mottling.
This indicates that the morning-glory apparently was free from mosaic,
at least from the type which could infect the crucifers used in this
experiment.

When the healthy plants for inoculation had developed from five to
eight leaves, aphids were transferred from the cultures to mosaic plants,
where they were allowed to feed for a few days before they were intro-
duced to the healthy plants. All inoculated plants also were confined
in cages so as to prevent dispersal from one species to another. After
the aphids had fed from 7 to 14 days on the inoculated plants they were
killed by tobacco fumigation in a fumigation chamber. These plants
were now allowed to grow without cages in a greenhouse where fumiga-
tion was practiced at regular intervals for the control of aphids. Since
mosaic mottling developed from 12 to 30 days after these insects were
killed by fumigation, mosaic mottling can not be attributed simply to
the mechanical injury produced by the aphids. This fact is further

J Gardner, Max W., and Kendrick, James B. turnip mosaic. In Jour. Agr. Research, v. 22, no. 3,
p. 123-124, I pi. 1921.

- ScHXJLTz, E. S., Foi^oM, Donald, Hudebrandt, F. Merrill, and Hawkins, I,on A. op. ot.

' Allard, H. A. THE MOSAIC disease OF TOBACCO. U. S. Dept. Agf. Bul. 40, 33 p. , 7 pi. 1914.

* McCuNTOCK, J. A., and Smith, Loren B. true nature of spinach-blight and relation of insects
TO ITS transmission. In Jour. Agr. Research, v. 14, no. i, p. 1-60, pi. A (col), i-ii. 1918.

6 Schultz, E. S., Folsom, Donald, Hildebrandt, F. Merrill, and Hawkins, Lon. A. op. ciT.

176

Journal of Agricultural Research voi. xxn. no. 3

confirmed by the control plants which remained free from mosaic mottling
after aphids taken from healthy plants had fed upon them. The results
secured from inoculation with aphids are presented in Table II.

Table II. — Transmission of mosaic of m,ustard, pe-tsai, and turnip by means of aphids

Variety inoculated.

Date of inocu-
lation.

Ap-
proxi-
mate
num-
ber of
aphids
trans-
ferred.

Source of aphids.

Num-
ber of
plants
inocu-
lated.

Date of first
symptoms.

Num-
ber of
plants
mo-
saic. «

Per-
centage
mo-
saic.

Southern Prize tur-
nip.

Jan. 12,1920

Jan. 26, 1920
Jan. 12,1920
Jan. 22,1920

Feb. 24, 1920

Mar. 5, 1920
Mar. 10,1920
Jan. 12,1920
Jan. 19, 1920
do

5°

SO
5°
12

100

100
50

100

so
50
25

2S
12

2S
25
SO

Mosaic Southern

Prize turnip.
do

3
3
3

3
I
I
3

I
3
I

S
3
3

I
5
S

Feb. 3 , 1920
Feb. 20, 1920

3
0
3

2
I
I
3
0

I
3
0

100

Healthy turnip. . .
Mosaic Southern

Prize turnip.
Mosaic mustard. . .

Purple Top turnip..

Seven Top or South-
Prize turnip.
Do

Feb. 14, 1921

Mar. 20, 1930

Mar. 31,1920
Apr. 2,1920
Feb. 11,1920
Feb. 19, 1920

100
100

Healthy mustard .
Mosaic mustard . . .
Healthy mustard. .
Mosaic mustard . . .
Mosaic Southern

Prize turnip.
do

Jan. 26, 1920
. . .do

Mar. 12,1921
Jan. 29,1921

Feb. 19, 1921
Feb. 8, 1921
Jan. 15, 1921

Jan. 29, 1921
Feb. 19,1921
Mar. 2,1921

Apr. 2,1921
Feb. 20, 192 1

Mar. 25,1921

Mosaic Chinese

cabbage.
do

Feb. 14,1921

Feb. 20,1921
Mar. 38,1921

Mosaic Green
Mountain potato.

« Date of last observation, Apr. 2, 1920 and 1921.

From the data indicated in Table II it is evident that aphids transmit
mosiac of the crucifers in question between different species as well as
among plants of the same species, as was suggested in Table I on juice
inoculations by means of rubbing. It will also be noted that the period
in which the first mosaic mottling appeared corresponds very closely to
that obtaining with mosiac diseases of other plants. As with the plants
inoculated by rubbing, the plants inoculated by means of aphids devel-
oped the mosaic symptoms only on the younger leaves. Mosaic symp-
toms on the inoculated plants were like those which were observed on
mosaic lots taken from the field.

Since turnips from mosaic plants taken from the field continued to
produce mosaic foliage it is apparent that such plants become a source
of infection if planted near susceptible varieties. Mustard seed from
mosaic mustard plants apparently develop healthy seedlings. This was
observed in loo seedlings, which were grown from seed from mosaic
mustard in the fall of 1920; in this test every seedling was free from
mosaic mottling.

oct.is. I92I Transmissible Mosaic Disease of Cabbage 177

SUMMARY

From these preliminary observations and experiments it appears that
the crucifers here mentioned may be added to the Hst of plants suscepti-
ble to mosaic, a disease whose cause has not been discovered but which
can be transmitted from mosaic to healthy plants by direct transfer of
juice as well as by means of aphids which apparently are very effective
natural agents in the dissemination of this disease.

PLATE B

I. — Leaf from healthy turnip, control to mosaic turnip in figure 2.
2. — Leaf from mosaic turnip, mosaic induced by aphids transferred from mosaic
turnip plant.

3. — Leaf from healthy mustard, control to mosaic mustard in figure 4.

4. — Leaf from mosaic mustard, mosaic produced by aphids from mosaic mustard.

(178)

A Transmissible Mosaic Disease of Chinese Cabbage

Plate B

Journal of Agricultural Research

Vol. XXII, No. 3

Transmissible Mosaic Disease of Cabbage

Plate 22

Journal of Agricultural Researcli

Vol. XXII, No. 3

PLATE 2 2

A. — Healthy turnip plant, control to B. Aphids from healthy turnip were allowed
to feed on this plant. Planted the same time as B.

B. — Mosaic on turnip plant, variety Seven Top or Southern. Mosaic mottling
appeared 26 days after the introduction of aphids from a mosaic Southern turnip
plant.

C. — Leaf from A, healthy.

D, E. — Two mosaic leaves from B. Mosaic mottling and ruflSing apparent on the
diseased leaves.

65508°— 21 5

PLATE 23

A. — Healthy mtistard plant, control to B. Planted the same time as B.
B.^ — Mosaic on mustard plant produced by transferring aphids from mosaic mustard.
Distinct mosaic mottling was noted 28 daj^s after introduction of aphids.

Transmissible IVlosaic Disease of Cabbage

Plate 23

Journal of Agricultural Research

Vol. XXII, No. 3

Transmissible Mosaic Disease cf Cabbage

Plate 24

Journal of Agricultural Research

Vol. XXII, No. 3

PLATE 24

Leaves from plants shown in Plate 23, A, B.

A, B. — Mosaic leaves showing mottling and ruffling.

C— Healthy leaf.

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Vol. XXII OCTOBER 22, 1921 No. ^

JOURNAL OP

AGRICULTURAL
RESEARCH

coN'rE>N'rs

Page

Flora of Corn Meal --------179

CHARLES THOM and EDWIN LeFEVRE

( Contribution from Bureau of Chemistry )

Hopkins Host-Selection Principle as Related to Certain
Cerambycid Beetles ------- 189

F. C. CRAIGHEAD

( Contribution from Bureau of Entomology)

Notes on the Organic Acids of Pyrus coronaria, Rhus
glabra, and Acer saccharum - - - - - - 221

CHARLES E. SANDO and H. H. BARTLETT

( Contribution from Bureau of Plant Industry and University of Michigan )

Fertility in Shropshire Sheep - - - - - -231

ELMER ROBERTS

( Contribution from Illinois Agricultural Experiment Station )

PUBUSHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE,

WITH THE COOPERATION OF THE ASSOCIATION OF

LAND-GRANT COLLEGES

^W:A.SHlNaTON, D. c.

EDITORIAL COMMITTEE OF THE

UNITED STATES DEPARTMENT OF AGRICULTURE AND

THE ASSOCIATION OF LAND-GRANT COLLEGES

FOR THE DEPARTMENT

KARL F. KELLERMAN, Chairman

Physiologist and Associate Chief, Bureau
of Plant Industry

EDWIN W. ALIyEN

Chief, Office of Experiment Stations

CHARLES L. MARLATT

Entomologist and Assistant Chief, Bureau
of Entomology

FOR THE ASSOCIATIOII

J. G. UPMAN

Dean, State College of Agriculfurei and
Director, New Jersey Agricultural Experi-
ment Station, Rutgers College

W. A. RILEY

Entomologist and Chief, Division of Ento-
mology and Economic Zoology, Agricul-
tural Experiment Station of the University
of Minnesota

R. L. WATTS

Dean, School of Agriculture; and Diredor:
Agricultural Experiment Station; Tht
Pennsylvania State College

All correspondence regarding articles from the Department of Agriculture should be
addressed to Karl F. Kellennan, Journal of Agricultural Research, Washington, D. C.

All correspondence regarding articles from State Experiment Stations should be
addressed to J. G. Lipman, New Jersey Agricultxiral Experiment Station, New
Brunswick, N. J.

joim OF AGrtrmL re

Vol. XXII Washington, D. C, October 22, 1921 xM'^j^^ 4

FLORA OF CORN MEAL '^ >".:-.*.

By Charles Thom, Mycologist in Charge, and Edwin LeFevre, Scientific Assistant,
Microbiological Laboratory, Bureau of Chemistry, United States Department of Agri-
culture

INTRODUCTION

Corn meal as it comes from the mill carries the mycelia of certain fungi
which infect unground grain. In addition, numerous species of molds
and bacteria, present in spore form as contaminations upon the surfaces
of sound kernels or as saprophytes in partially spoiled grains, are recov-
erable by routine cultural examination of the finished meal. Many
experiments, extending over several years and including the work of vari-
ous members of the Microbiological Laboratory, show that certain groups
of organisms are practically always abundant in such cultures. Other
species are usually present, but in smaller numbers, and many forms are
obtained occasionally as accidental contaminations. In undertaking to
study this complex flora, it may be possible to determine by routine cul-
ture the species represented and something of their relative abundance in
the sample, but the list so obtained gives little information as to the
relative importance of the individual species as causes of spoilage in the
product.

The culture media commonly used in such routine examination of food-
stuffs present conditions for the growth of microorganisms which differ
greatly from those found in corn meal. The nutrients used in preparing
such media are selected because they are readily assimilable to most
organisms. These nutrients appear in solution or in jelly-like masses
which contain high percentages of moisture. Corn meal, on the other
hand, presents a range of composition, according to Winton and his
associates {8),^ approximately as follows: Moisture, 10 to 18 per cent,
but under usual commercial practices ranging from 12 to 15 per cent;
protein, 5 to 10 per cent; fat, i to 5 per cent, according to the method of
milling; nitrogen-free extract, including starch and sugar, 68 to 78 per
cent. Of the nitrogen-free extract, sugars constitute perhaps 3 per
cent, and gums and dextrin, some of which are readily fermentable,,
perhaps an equal quantity. In dealing with this product as a substratum
for organisms, the percentage of water found is an important limiting

I Reference is made by number (italic) to " lyiterature cited," p. i88.

Journal of Agricultural Research, Vol. XXII, No. 4

Washington, D. C. Oct. 22, 1921

aab Key No. E-16

(179)

i8o Journal of Agricultural Research voi. xxii, no. 4

factor. Obviously this product, 'even at its maximum moisture content,
presents a marked contrast to laboratory media as usually prepared.
Nevertheless, corn meal has been so often found an unstable product that
it is commonly milled only for consumption within a few weeks or by
methods intended to eliminate the most readily fermentable portions of
the grain.

Under ordinary conditions of handling, spoilage in this product ap-
pears in one of the following forms: Souring, rancidity, mustiness, the
formation of clumps or balls, extensive concretions which may involve
the solidification of an entire bag, or the formation of a hard, cylindrical
outer mass with the center loose and mealy. Heating occurs only in
the wettest samples. Much com meal, if held beyond a very short
period, develops a musty, moldy, or sour odor and shows occasional
balls or masses of meal held together by mold, which bring about losses
in palatability and market quality in the product. Such changes as
rancidity and the formation of extensive concretions into moldy masses
are so obviously due to high moisture content and involve such losses
that they have been almost eliminated from commercial practice.
When losses occur the meal is found to carry more than a critical mois-
ture percentage. This may be due either to milling com which is in-
sufficiently dried or to the storage of the meal under conditions which
will maintain a moisture content above the danger point. For the
samples used in all series reported here this figure was approximately

13 per cent (2).

CULTURAL EXAMINATION

In routine cultural examination reported here, plain agar was used
for bacterial counts, wort agar for mold counts, and dextrose-litmus
shake agar to determine acid, gas, and anaerobic growth. The presence
of particular organisms was determined by tlie use of special methods
on special media. Experimentation covered a range wide enough to
justify the restriction of routine cultures to the media already noted.

After comparative study of many series of cultures. Table I is intro-
duced as giving a group of cultural results fairly typical for commercial
meal in sound, merchantable condition. The nine samples reported
were purchased in different retail stores of Washington, D. C, during
October and November, 1920. Four of them were yellow and fairly
coarsely ground. The white meals were softer or more finely ground.
All were bolted. All showed by microscopic examination traces of
both bran and germ, although these portions were scanty in certain
samples. The history of the samples was not obtained.

These samples were sound in appearance and odor. There was no
evidence of the multiplication of microorganisms. Among the bacterial
colonies micrococci, members of the mesentericus and of the colon-
aerogenes groups were characteristically present. Special tests in cab-

Oct. 22, I92I

Flora of Corn. Meal

I8i

bage juice showed, in four of the nine samples, the presence of lacto-
bacilH with the morphology and cultural characters of the organism of
pickle and sauerkraut fermentation. N>) bacterial colonies were obtained
in plain agar from two of the samples. A duplicate of sample 9 proved
equally negative. Mold colonies were obtained in all samples. These
represented in varying proportions Aspergillus repens De Bary, A. niger
Van Tieghem, A. fiavus hink, Fusarium, various mucors, and unidentified
colonies.

Table I. — Results of cultural examination of commercial corn meals

Sample No.

Bacteria

per gram

on plain

agar.

Molds per

gram on

wort agar.

Bacteria
per gram

on
dextrose-
litmus
agar.

Acid
colonies.

10, 000

TO. 000

16, 000

Per cent.
50

5, 000 I, 000

t;=;, 000 T3. 000

•^

42, 000

13, 000

10, 000

8, 000

60, 000

70, 000

5,000

10, 000
400, 000

20, 000
3,000

11, 000
5,000

50
60

10, 000

4, 000
3,000

A more extensive series of studies was conducted in cooperation with
the Plant Chemical Laboratory of this bureau. The general results
of this experiment are described elsewhere (2). In brief, during the
spring of 1920, a series of bags of meal were prepared for this storage
experiment from com bought by the mill in the regular course of business.
This grain, while sold as No. 2, was obviously wet and barely passable
as a fair product. Infected and even badly decomposed ears were not
uncommon among the ears of corn received in bulk. Although the lots
of meal included were milled at water contents varying from 12.7 to
16.18 per cent, the conditions of storage were such that no spoilage
determinable by the senses took place. Cultures were made from the
meal as freshly ground in April, then, beginning May 5, once each week
until July. In all these cultures no evidence of multiplication of either
mold or bacteria was found. It was, therefore, possible to follow the
relative numbers of viable organisms in the various groups from the
time of grinding through the four months of storage.

In the freshly milled samples the average count of colonies of bacteria
upon plain agar was about 1,000,000 per gram of meal, with variations
from 600,000 to 1,600,000. Upon wort agar the count of mold colonies
averaged about 100,000 per gram of meal, with variations in different
samples from 70,000 to 160,000. Of the bacterial colonies observed
about 60 per cent were acid producers.

1 82 Journal oj {Agricultural Research voi. xxn, no. 4

For comparison a special series of samples were prepared by adding
5 per cent of meal made from com markedly rotted with Diplodia and
Fusarium. In the freshly grouted meal of this series the bacterial count
upon plain agar was about 2,600,000. The count of mold colonies upon
wort agar was about 1 10,000. About 70 per cent of the bacterial colonies
were acid producers.

After storage for approximately one month (May 20 and 21) samples
from a particular lot of five bags of the regular meal showed an average
count of 108,000 bacterial colonies and 15,000 molds. Samples from the
same bags on June 30 showed an average count of 12,600 bacterial
colonies, and 7,600 mold colonies. Without placing emphasis upon
exact figures, these cultural results are fairly typical of the mass of
figures obtained from cultures made weekly from representative samples
involving the whole series of 88 bags of meal. These figures are readily
comparable with those obtained from commercial samples (Table I).
Discrepancies which occur may perhaps be accounted for by the fact
that samples 3, 4, and 5 were evidently the product of local mills, sold
fairly quickly after milling, while samples 2, 7, and 9 were clearly the
product of special processes and handled under conditions involving
much slower distribution.

In this lot of meal, therefore, the conspicuous change due to storage
was the drop in the number of viable organisms to about i per cent of
the original number of bacteria and perhaps 10 per cent of the original
number of molds. The larger part of this decrease occurred during the
first six weeks, with a slow reduction throughout the succeeding periods.

In connection with the study of these figures, data obtained by Thom
and Stiles (unpublished) in examining Winton's {8) samples ^ in 19 14
were restudied and compared with the results here considered. Win-
ton's com meal varied in initial moisture content from 19.27 to 10.79
per cent. In those lots of meal (A, B, and C) carrying moisture mark-
edly above 13 per cent, the evidence of multiplication of molds and
bacteria was clearly discernible. Musty odors and balls of meal held
together by mold were present in every sample. In cultures, the count
of colonies of molds and bacteria reached 13 million in the wettest lot.
Of these several million were Aspergillus flavus. The predominant or-
ganisms were molds rather than bacteria, but there was fairly clear
evidence of some bacterial multiplication at the higher water percentages.

In the roller-gi'ound samples of lots D, E, F, which did not spoil and
whose water percentage was near to or less than 13, the total counts
found by Stiles approached very nearly those already given in this paper.
These examinations began too late in the storage period to show that
part of the bacterial flora which dies off rapidly. The stored samples
still showed some acid organisms, but micrococci and aerobic spore

' Samples of the meal studied were examined bacteriologically by G. W. Stiles, formerly of the Bureau
of Chemistry, and for mold activity by Charles Thom, then in the Bureau of Animal Industry {8, p. zs)-

Oct. 22, 1921 Flora of Corn Meal 1 83

formers of the mesentericus group formed the majority of the bacteria
obtained.

In the lots with moistm^e content decreasing toward 13 per cent there
was progressive reduction in the number of active species of molds.
Bxtensive experimentation showed clearly that Aspergillus repens was
the agent which formed the balls of meal loosely held together with
mold hyphae, which characterized meal containing barely enough
water to start spoilage. In another series of experiments A. flavus
began to be active only in samples containing about 16 per cent of water.
Yeasts, mucors, and Penicillia were reported by Stiles only in the sample
carrying about 19 per cent of water.

During the examination of the preliminary samples in the 1920 ex-
periment, an effort was made to identify the groups or actual species
represented. As a matter of routine, inoculations were made from each
flask prepared for diluting plates (consisting of 5 gm. of the meal to
45 cc. of sterile water) into the following media: Plain milk, gelatin,
and litmus lactose broth. Smears were also made on Endo's agar in
each instance. In every case there was prompt coagulation of the milk,
with extrusion of whey, but no digestion of curd. Pink rings formed
near the surface. Gelatin was liquefied in every instance, and acid and
gas formed in all broth tubes. Growth in Endo's media indicated the
presence of Bacterium aerogenes Escherich. Further cultural studies
showed that Bad. aerogenes was the predominant bacterial species pres-
ent in all these samples. This predominance was maintained throughout
the series of examinations made. Microscopical examinations of smears
made in each case, however, showed the presence of spore-bearing bac-
teria, especially the mesentericus group, and micrococci of various kinds.
Dextrose agar tubes often contained colonies growing deep in the media,
indicating the presence of anaerobic bacteria. Yeasts were found in all
samples, their growth being largely of the mycoderma type. The plates
showed many mold colonies. Various mucors, species of Fusarium,
Aspergillus flavus, A. niger, and occasional green Penicillia were ob-
served. The species of molds present on the plates varied from period
to period and with the sample. Molds were always more numerous on
plates made from meal to which Fusarium and Diplodia had been added,
but growth on these plates did not show dominance of these particular
forms.

Evidence of the effect of bolting upon the abundance of organisms
was furnished in the 1920 experiment by the examination of samples
of two series of five bags each, representing a single lot of meal, one-half
of which was bolted and the other half unbolted. The bolting to which
these samples were subjected removed a considerable part of the bran
but little of the germ from the meal. After one month of storage, the
bolted meal showed an average of 34,000 bacterial colonies and 20,000
mold colonies. The unbolted samples showed 108,000 bacterial colonies

184 Journal of Agricultural Research voi. xxii. no. 4

and 15,000 molds. This observation was confirmed by a restudy of
Stiles's unpublished examination of Winton's (8) samples. Of every
lot of com handled, part was ground in a stone mill without sifting or
bolting and part was carefully " degerminated " and "roller" ground.
In the bolting process all of the bran was taken out, and many of the
samples consisted almost completely of homy endosperm. In that part
of this series made up of meals in which no multiplication of micro-
organisms occurred, bolting consistently reduced the cultural count of
microorganisms below that of the stone-ground meal. Frequently the
number found in the bolted meal was less than one-tenth of that in the
stone-ground meal.

By removing the bran, bolting takes away the largest area of contami-
nation with saprophytic organisms. The tip of the kernel and the
germinal area carry the majority of the infections found in com. Study
of many samples of com over a period of years shows that invasion of the
germinal area by molds is not uncommon in com which has not been
fully matured or has not been promptly and thoroughly dried. Sam-
ples have frequently shown the invasion of the germ in every kernel by
Aspergillus repens. Recently samples representing a bulk shipment
have shown nearly every grain to contain one or the other of two species
of Penicillium. Meal therefore may be so milled and sifted or bolted
as to remove the larger part of all contaminations, as well as those mold
infections which do not involve general disintegration. The cleaning
process before milling removes the grains thoroughly rotted by Fusarium
and Diplodia. Corn has still been seen going into the rolls of a mill
in which the low grade of the stock could not have been concealed if it
had passed through a stone mill without being bolted. The product,
however, was going into human food without showing tangible evidence
of the low quality indicated by the unground grain. In other words,
the fractional milling of low-grade grain makes possible such separation
as turns the infected portions of the grain into oil stock or cattle feed
and the solid or homy portions which are less obviously damaged into
meal.

The literature of maize deterioration is reviewed by Alsberg and Black
up to 19 13 (7). The activity of Fusarium and Diplodia as causes of
rotting in ear com was discussed by Burrill and Ban-ett (5) and that of
Diplodia alone by Heald, Wilcox, and Pool (4).

More recently McHargue (6) has studied the activities of certain fungi
and their relation to commercial conditions in the handling of the product.
Excessive moisture in the grain is regarded as the limiting factor in most
cases of such spoilage. The factor of temperature must not be over-
looked. The moisture content limit may be materially increased during
the winter without evidence of the activity of microorganisms. The
agents of spoilage in all the cases under review were primarily molds.
The results already given in this paper harmonize in general with those

Oct. 22, I92I Flora of Corn Meal 185

of McHargue. It has been possible, however, to go farther and indicate
more clearly the groups of organisms regularly present and to record
the conditions under which certain of them become active factors in
spoilage.

Routine mass or dilution cultures show that certain molds are recov-
erable from practically all samples of meal. Among these are Rhizopus
nigricans Ehrenberg and some of the mucors which frequently overgrow
plate cultures within two days of incubation, although they probably
are present only in spore form in the meal. Syncephalastrum, belonging
to the same group, is not uncommon. Aspergillus flavus and A. niger
are only occasionally visible factors in the infection of the unground
grains, but they always appear as rapidly growing colonies in the mass
or dilution cultures made. The brown masses of A. tamari Kita are
commonly found with A. flavus. A. fumigatus Fres. and A. terreus Thorn.
are frequently present but are quickly overgrown by the more active
species already mentioned. A. repens, though practically always pres-
ent, can be found only by careful search in the presence of these rapidly
growing forms.

Several strains of Penicillium are found in meal cultures. Peniciilia
of the group with submerged orange mycelia and of the Citromyces group
are probably most common. Penicillium expansum Link is reported by
McHargue. P. oxalicum. Thom and Currie is found in many samples of
meal, but rarely in miscellaneous cultural work. Strains related to
P. luteum Zukal and P. purpurogenum O. Stoll are frequently present but
usually indicate soil contamination rather than active growth in the corn
or meal. One sample of corn rotted by a member of this series has been
examined, but the conditions shown clearly indicated that the product had
contained high percentages of moisture at the time the rotting occurred.

Colonies of Fusarium develop from almost every sample of meal.
Infections of this group are so abundant that conidia or grains of meal
containing living hyphae are rarely absent. Cladosporium and Alter-
naria are frequently found but represent spore contamination rather than
infection. The other organisms observed in culture from time to time
appear to represent excessive contaminations with spores due to unfavor-
able conditions in the handling of the product, or, in certain species, to
actual infection of the grain locally by the mold.

The bacteria found in the fresh samples here considered were pre-
dominantly Bacterium aerogenes. Certain other organisms have been
regularly obtained in culture. When the necessary moisture is present,
souring is so characteristic of the product that Round and Gore (7) found
the addition of 3 per cent of fresh meal an adequate starter to insure the
dominance of lactic acid fermentation in potato silage. Lacto-bacilli
were present in four of the nine lots reported in Table I. According to
unpublished records in the Microbiological Laboratory, Round found
organisms of this group abundant also in fresh meal, but occasionally

1 86 Journal of Agricultural Research voi. xxn. no. 4

absent in old meal or meal made from old and thoroughly dried corn.
Micrococci are constantly encountered in culture but have not been
typed. Aerobic spore formers of the mesentericus group are always
present, and in spore form they constitute the larger part of the living
bacteria in some meals after long storage.

This was clearly demonstrated by a series of experiments upon the
possibility of producing a sterile meal with steam, dry heat, or both
(unpublished cultural results of Ruth B. Edmondson). The spores of
this group survived more heating than could be applied under practical
working conditions to the product. Aside, however, from meal so wet
as to be unmarketable, these experiments show no evidence of bacterial
activity. One sample of apparently sound yellow meal showed the
presence of Bacillus niger Migula in such extensive numbers that masses
of meal placed upon culture media were promptly overgrown and with
the agar turned bluish black with this species. The meal was contributed
by Dr. S. S. Adams, of Washington, D. C, who reported the feces of a
child apparently well to have been blue when fed this meal.

When, however, com or meal is bottled and incubated at laboratory
temperature (20° to 30° C), those species capable of developing under
the conditions presented show active growth. In the authors' series
such growth was not detected by physical appearance in products carry-
ing less than 13 per cent of moisture. Certain stone-ground samples of
Winton's series (8) showed some evidence of mold activity below that
figure. Measurable changes in quality certainly occur in such meals
during storage. Some experimental results have suggested the possibility
that these changes in such meal are due to the distribution of infected
material throughout the mass by the grinding of infected corn. This
conflicts with the current trade belief that the natural enzyms of the
germinal area are the chief causes of such deterioration, but reflects the
findings of HofTer (5) and his coworkers that even selected seed com
may be extensively infected. Examinations of commercial samples in
the Microbiological Laboratory have shown extensive development of
molds within the grain itself in com of other than the higher grades.

In samples carrying 14 to 15 per cent of water the formation of balls
and concretions in the meal begins to be evident. The principal agent
in their formation appears to be Aspergillus repens, although many diffi-
culties are encountered in fixing a minimum moisture percentage for
the activity of this species. Changes involving the development of
mold mycelium in the meal begin within the limit of 13 to 15 per cent
of moisture. Incubation at 20° to 30° C. merely accelerates changes
which would progress more slowly in colder places. Moist chamber
experiments with meal inside this range of water content show the
presence of active mycelia of more than a single species, but principally
Aspergillus repens. When the percentage of moisture reaches 16, sev-
eral species are clearly able to grow. Special studies with Aspergillus

Oct. 22, I92I Flora of Corn Meal 187

flavus show that very little development of this species occurs below 16
per cent, but that from 16 per cent upward development of this species
rapidly increases and the number of forms capable of growing rapidly
rises. Among the characteristic saprophytic molds observed under
these conditions, in about the order of their abundance under the con-
ditions, are Aspergillus repens, Aspergillus flavus, Actinomyces sp.,
Penicillium sp. and Citromyces sp., Fusarium sp., Aspergillus candidus,
Aspergillus ochraceous Wilhelm, Aspergillus iamari, and Aspergillus niger.

Bacterial activity appears to be a concomitant of the disintegration
due to mold action in such rotting processes as this. As indicated by
Bailey and Thom (2, Table I), active disintegration by molds is
accompanied by an increase in the water percentage of the sample.
Bacteria follow rather than initiate the process in the samples studied,
thus becoming a small factor in the merchantable product.

Throughout this investigation a close correspondence has been observed

between the flora of deterioration in unground com and the flora of the

milled product.

SUMMARY

In seeking possible causes for the well-recognized instability of com
meal, cultures show considerable numbers of molds and bacteria to be
generally present. Among these the following species of molds were
characteristic of many series of cultures: Fusarium sp., Aspergillus
repens, A . flavus, A . tamari, A . niger, Citromyces (or Penicillium section
Citromyces) sp., Penicillium oxalicum, P. luteum varieties, Mucor sp.,
Rhizopus nigricans, and Syncephalastrum sp., together with various
yeasts and yeast-like fungi. Among bacterial groups, the colon- aerogenes
group and lacto-bacilli were most abundant in fresh meal. Aerobic
spore formers and micrococci were always present and persisted in the
stored product.

Within the range of composition found in merchantable meals, no
bacterial activity was detected. Only one grade of unbolted meal
showed signs of mold development below 13 per cent of moistiu-e. Above
13 per cent moisture, Aspergillus repens begins to be an active agent
of spoilage somewhere between 13 and 15 per cent of moisture, varying
with the form of milling practiced. Several other species of molds are
active in meal containing 16 per cent moisture; and numerous forms,
including some bacteria, develop when 18 to 20 per cent of moisture is
found.

Many samples of corn are found to carry extensive infections with
Fusarium, Diplodia, Aspergillus repens, or Penicillium, especially in the
germinal area and in the tip of the kernel. These sections of the kernel
are removed in varying degrees by different milling systems. The
bolted meals examined show a corresponding reduction in count of
viable organisms as shown by culture.

1 88 Journal of Agricultural Research voi. xxu, no. 4

LITERATURE CITED
(i) Alsberg, Carl L., and Black, Otis F.

19 13. CONTRIBUTIONS TO THE STUDY OF MAIZE DETERIORATION. BIOCHEMICAL
AND TOXICOLOGICAL INVESTIGATIONS OF PENICILLIUM PUBERULUM ANI>

PENiciLLiUM STOLONiFERUM. U. S. Dept. Agr. Bur. Plant Indus.
Bui. 270, 48 p., I pi. Bibliographical footnotes.

(2) Bailey, L. H., and Thom, C.

1920. SOME observations of corn meal in storage. In Operative Miller,
V. 25, no. 12, p. 368-371, chart A-D.

(3) BuRRiLL, Thomas J., and Barrett, James T.

1909. ear rots op corn. 111. Agr. Exp. Sta. Bui. 133, p. 63-109 incl. pi.
i-ii, I col. pi.

(4) Heald, F. D., Wilcox, E. M., and Pool, Venus W.

1909. THE life-history AND PARASITISM OF DIPLODIA ZEAE (SCHW.) LEV. Itl

Nebr. Agr. Exp. Sta. 22nd Rept. [1908], p. 1-19 incl. 10 pi. Biblio-
graphy, p. 7.

(5) HoFFER, George N., and Holbert, J. R.

1918. SELECTION OF DISEASE-FREE SEED CORN. Ind. Agt. Exp. Sta. Bui. 224,
16 p., 20 fig.

(6) McHargue, J. S.

1920. THE cause OF DETERIORATION AND SPOILING OF CORN AND CORN MEAL.

In Jour. Indus, and Engin. Chem., v. 12, no. 3, p. 257-262.

(7) Round, L. A., and Gore, H. C.

1916. A PRELIMINARY REPORT UPON THE MAKING OF POTATO SILAGE FOR CATTLE

FOOD. In Proc. 3rd Ann. Meeting, Potato Assoc. America, p. 75-79.

(8) WiNTON, A. L., Burnet, W. C, and Bornmann, J. H.

I915. COMPOSITION OF CORN (mAIZE) MEAL MANUFACTURED BY DIFFERENT PRO-
CESSES AND THE INFLUENCE OF COMPOSITION ON THE KEEPING QUALI-
TIES. U. S. Dept. Agr. Bui. 215, 31 p.

HOPKINS HOST-SELECTION PRINCIPLE AS RELATED
TO CERTAIN CERAMBYCID BEETLES

By F. C. Craighead

Specialist in Forest Entomology, Bureau of Entomology, United States Department of

Agriculture

INTRODUCTION

In connection with the reported dying of lodgepole pine (Pinus con-
torta Loud.) over extensive areas in northeastern Oregon caused by the
mountain pine beetle {Dendroctonus moniicolae Hopk.) and the threat-
ened invasion by this beetle of the adjacent areas of yellow pine {Pinus
ponderosa Laws.), detailed investigations were made by the Bureau of
Entomology under the direction of Dr. A. D. Hopkins. Manuscript
reports of these investigations, submitted in the summer of 19 lo, showed
that the infestation by the beetle in the lodgepole pine was so extensive
that there was no hope of controlling it, but that the comparatively small
amount of infestation in the valuable stands of yellow pine was such as
to warrant the undertaking of control, provided the beetle did not
migrate from the lodgepole pine to the yellow pine.

In a letter from Dr. A. D. Hopkins under date of July 30, 19 10, relating
to a manuscript report of Mr. H. E. Burke, the following statement
occurs which appears to be the first written reference to the host-selection
principle :

The more I consider the various features of the problem, the more I am convinced
that it is entirely practicable to protect the yellow pine, even if we leave all but the
immediately adjacent lodgepole pine to take care of itself. This is based on my
belief that the majority of the broods of the beetles which have been breeding in the
lodgepole will continue to confine their attack to that species, and gradually diminish
with the reduced supply and their increased struggle to adapt themselves to the
yellow pine. I may be wrong in this, but it is a matter worthy of careful considera-
tion. Remember, that in all these years, there has been no marked or general migra-
tion of beetles from lodgepole to the yellow pine. Therefore, it appears that the
broods which are most dangerous to the yellow pine are those which have been breed-
ing in it, and that these are the broods we will have to deal with mainly in our efforts
to protect the best bodies of yellow pine.

The control operations that were carried on during the following year,
191 1, were confined mainly to the yellow pine area. In manuscript
reports by Messrs. W. D. Edmonston and George Hofer on a special
examination of the yellow pine and lodgepole pine areas in the summer
and fall of 1913, it is stated by Edmonston:

In 1912 the examination of the areas on which insect control work was carried on
during April, May, and June, 191 1, showed an average reduction of the infestation
on the entire area, 76,430 acres, of close to 85 per cent.

Journal of Agricultural Research Vol. XXII, No. 4

Washington, D. C. Oct. 22, 1921

aac (1S9) Key No. K-ioa

190 Journal of Agricultural Research voi.xxn.No. 4

Examinations made this season, 1913, show a still greater reduction in the infesta-
tion; in fact, the infestation is so light tliat it is actually less on the treated areas
than it is throughout any other area on this Forest.

and —

There was no reoccupation of the treated areas by broods from the lodgepole infested
trees at higher elevations.

and by Hofer —

As we reached the summit near the North Powder Peaks we attained an altitude of
8,000 feet; the elevation at the Sheep Ranch is about 4,000 feet. From the summit
of this divide for a distance of 10 miles north, 10 miles east, and 16 miles west we
noted heavy infestation, both old deadings and the new work also, in both the lodge-
pole pine and white bark pine, especially on both slopes of Antone Creek.

No new infestation was found on the treated areas on Anthony Creek, Camp area.

This seemed to furnish substantial evidence that the principle would
hold.

The principle as defined by Dr. Hopkins ^ is that an insect —

species which breeds in two or more hosts will prefer to continue to breed in the host
to which it has become adapted.

In order to secure further evidence relating to this principle, the writer,
after consulting with Dr. Hopkins, began a series of experiments in 19 14
with insects which infest two or more species of wood. The wood-boring
Cerambycidae, or long-homed beetles, offered material which was very
well adapted to the conduct of such experiments. Many species were
easily available which exhibit great diversity in their selection of hosts
in nature, as illustrated by those breeding exclusively in a single species
of plant and those apparently attacking almost any wood. This varia-
tion in host habits at once brought up the following questions : Will those
species confined to a single host live in any other, and do the individuals
coming from a certain plant of those species breeding in a variety of hosts
select the same species of plant on which to oviposit ? Again, if such is
the case, how do these host strains originate in nature?

As these experiments progressed new problems came up demanding a
broadening of the experiments from- year to year until, during the season
of 1918, over 100 individual experiments were in progress. Fourteen
species of insects and 21 species of plants were used, combining to form
45 host strains. It was thought desirable to conduct experiments on
more species rather than more intensive experiments on a few species.
It will be noted that certain experiments were not carried as far as others,
due to the fact that time was not available or due to the absence of the
writer at the critical time. At present several points remain to be con-
clusively settled, and investigation of these will be continued another
year or so. Nevertheless it is believed that sufiicient data have been
accumulated to show definitely the extent to which the influence of the
host applies to these insects.

1 Hopkins, A. D. economic investigations of the scox,ytid bark and timber beetles of north
AMERICA. In U. S. Dept. Agr. Program of Work, 1917, p. 353. 1916.

Oct. 22. I92I Hopkins Host-Selection Principle 191

HISTORICAL

Very few references to the adaptation of insects to their host plants
or the variation in their selection of host plants can be found. The
most important paper dealing with the subject is that by Pictet.^ This
author shows by several examples, (Ocneria) Porthelria dispar for
one, that caterpillars of the second and third generations may be made
to change their preferred food plants and that the adults reared from
them exhibit changes in size and coloration. This paper is reviewed,
and supplemented with reports of corresponding observations, by
Schroder,^ who in a previous article ^ showed that even nidification (in
Gracilaria stigmatella F.) and habits of feeding, combined with
changes in reproduction (in the beetle Phratora vitellinoe L.). can be
changed and that these acquired characters are transmitted spontane-
ously from the third generation.

In 1907 and 1908 Paul Marchal ^ succeeded in transferring numerous
specimens of Lecanium corni Bouche from the peach (Amygdalus
persica Linn.) to the black locust {Rohinia pseudacacia Linn.).
Eggs hatched and larvae developed on the new host plant, spreading out
over the leaves in large numbers, and in the fall migrating from the leaves
to the wood for hibernation. In the summer of 1908 the insects com-
pleted their development and had then the large size, deep coloration,
and characteristic appearance of the insect described by Douglas as L.
robiniarum, the attacks of which on the black locust had been severe
in several European localities. This indicated that L. robiniarum was
only a race of L. corni, resulting from individuals that had become
transferred in some manner from the peach to the introduced American
black locust. Dr. Marchal found great difficulty in reestablishing on
the peach individuals of L. corni produced on the black locust.

There are other records showing the acquired adaptation of certain
species to new host plants, similar to those here cited. The practical
application of such phenomena, however, has, so far as can be ascer-
tained, first been recognized by Dr. A. D. Hopkins (referred to on p. 189
of this article) and presented by him in concrete form.

In a paper prepared by M. Joseph Capus ^ on a nematode disease of
peas in the Gironde and read by Paul Marchal at the session of July 10,
19 1 8, of the French Academy of Agriculture there is a record of injury to
peas by a fungus (Fusarium vasinfectum var. pisi van Hall, considered as
the conidial form of Necosmopora vasinfecta E. F. Smith, accompanied by

1 PicTET, Arnold, influence de l'alimentation et de l'humidite sur la variation des papillons.
In Mem. Soc. Phys. et Hist. Nat. Genfeve, v. 35, fasc. i, p. 45-127, pi. 2-5. 1905.

2 Schroder, Chr. die literatur uber die FARBtmo der insekten des jahres 1905. In Ztschr.
Wiss. Insektenbiol., Bd. 3, p. 162-164. i907-

3 i'BER experimentall erzielte instinktvariationen. In Verhandl. Deut. Zool. GeseU.,

Jahresversamml. 13, p. 158-166. 1903.

* Marchal, Paul, le lecanium du rohinia. Compt. Rend. Soc. Biol. [Paris], t. 6s, p. 2-5. 1908.

^ Capus, Joseph, and Marchal, Paul, sur la maladie vermiculaire des pois dans la gironde. In
Compt. Rend. Acad. Agr. France, t. 4, no. 25, p. 712-716. 1918.

192 Journal of Agricultural Research voi. xxii, no. 4

the nematode Heterodera schachti Schmidt). After pointing out the
interdependence and relation of the two, M. Capus says:

One might ask himself why this species, everywhere known for its injury to beets,
does not establish itself on this plant in the Gironde and appears so abundantly
on peas.

Following M. Capus's explanations of this phenomenon. Dr. Marchal
observed :

Among the very interesting facts pointed out by M. Capus in his note I wish to
call attention to the following: That injury to beets by Heterodera in the Gironde is
not constant, is rare. We should recall in this connection the observations, already
old but interesting, of the Dutch naturalist Ritzema-Bos. He has shown that when
nematodes multiply in course of years without interruption on the same host, biologic
races are formed adapted to this host which later pass to other vegetation with greatest
difficulty, even when these are of those preferred by the species. It must be, there-
fore, that, by virtue of the conditions of pea culture in the Gironde, a race of Heterodera
schachti was formed especially adapted to peas and to the attack of which beets are
resistant up to a certain point. There is no doubt that it will adapt itself to beets
cultivated for a number of years in succession in the same soil infested with H. schachti.

The experiments conducted at the Gipsy Moth Laboratory^ show that
of the many plants tested a decided variation was found in regard to
the susceptibility to attack by this insect. The plants were divided into
four groups: I, favored species; II, favored food species after early
stages; III, species on which a small proportion may develop; IV, species
that are unfavored food. These results show that, although this insect
has a wide variety of hosts on which it is capable of feeding, certain ones
are selected in preference to others in the natural forests. As far as
known, no observations have been reported showing whether or not
several years' feeding on any particular host produces a strain which
selects that in preference to others.

In a recent paper Dr. C. T. Brues^ writes as follows (on p. 328-329):

It has been claimed that the food habits may be modified experimentally, in that
caterpillars reared on a strange plant (where they could be induced to select it) give rise
to moths whose progeny more readily accept the new plant. It is very difficult to
accept such evidence, at least as having any general application, without very clear
and incontrovertible proof. If such transformations can occur so easily and become
hereditary so quickly they should have entirely destroyed the coherent habits now
existent, during the enormous period which has elapsed, for example, since the
violet-feeding Argynnids were differentiated, since the holarctic and nearctic Vanes-
sids have been separated, or while the world-wide Aristolochia-feeding Papilios were
attaining their present distribution. That such a change has actually occurred in
the caseof other groups seems equally evident, althoixgh, as has been shown, we can
more easily believe that they may have arisen through mutations in maternal instinct
not incompatible with larval tastes and then only in extremely rare cases and con-
fined to certain groups.

METHODS OF CONDUCTING EXPERIMENTS

In connection with the experiments by the writer several types of
cages, the particular type determined by the amount of material handled

> MOSHER, F. H. FOOD PLANTS OF THE GIPSY MOTH IN AMERICA. U. S. Dept. Agr. Bui. 250, 39 p., 6 pi.

2 Brues, Charles T. the selection of food-plants by insects, with speoal reference to lepi-
DOPTEROUS LARVAE. /» Amer. Nat., V. 54, no. 633, p. 3i2-33"2. 1920.

Oct. 22, 1921 Hopkins Host-Selection Principle 1 93

and the exact conditions required, have been used in confining the colonies
of beetles. It is essential to duplicate as closely as possible the conditions
in which the insects are found in nature.

For the larger logs and for experiments in which a large amount of
material was used, an open wire insectary was constructed. This
insectary is 40 feet long by 10 feet wide by 7 feet high. The foundation
is of concrete, the side walls and top of i8-mesh galvanized wire screening,
and over all a removable lattice- work roof was placed. This roof was
adjusted to simulate shade conditions in the woods. It was removed
in winter and replaced in summer. The floors were made of ashes to
give good drainage. Cross partitions divided the insectary into seven
compartments of different sizes. One room was entirely boarded in
and roofed over. It was used for seasoning wood. Another was lined
with cheesecloth, which was used for holding different cuts of wood under
natural conditions until desired for use. In the other compartments
were placed logs containing various species of insects. Where no danger
is present of any infestation from the original host wood into cuts of
different wood, it was possible to place several beetle species in the same
compartment and continue their breeding in the same host from year to
year. In this way forms such as Callidium in pine {Pinus spp.) , Neodytus
capraea Say in ash {Fraxinus spp.), and Cyllene pictus Drury in hickory
{Hicoria spp.) were placed together.

The smaller insects, especially those in twigs and branches, were
confined in glass museum cylinders of various sizes. The tops were kept
in place so that a very constant degree of humidity could be maintained.
This cage was found to give best results for the development of the larvae
and, as no sand was needed, the adults were easily found in the cages.
These jars were kept under a roof all the year in another insectary.

Each of these insects has a particular preference for a certain condition
or seasonal cut of wood. Also in some species the adults require food
before ovipositing, consisting of green bark from twigs, leaves, or fungus
s'pores. In the latter case the pustule of the chestnut blight {Endothea
parasitica (Murr.) P. J. and H. W. Anderson) was used. Again, some
require much moisture, others rather dry surroundings. The determi-
nation of these factors sometimes delayed the successful continuance of
a species for a year or more. When a new colony was collected from
nature it was ascertained as nearly as possible when the tree died and the
condition of the wood, also what degree of humidity was desirable. For
instance, those insects naturally feeding in dead branches of a standing
tree required drier conditions than those attacking branches fallen to the
ground.

In order to meet these conditions, wood of the various species used was
cut every month or every other month of the year and stored under
different conditions. Part was placed in the dry shed for dry seasoning,
part hung or stood up in the open-air cage for normal air seasoning, and

194 Journal of Agricultural Research voi. xxii, no. 4

part laid on the ground in the wire cages for wet seasoning. The con-
dition of the wood on which the insects were first found ehminated the
use of certain of these periodic cuts and conditions of seasoning. How-
ever, the first-year adults were usually caged with the choice of many of
these cuts and the one infested most heavily was considered as the
optimum condition and used afterwards for continuation of the succes-
sive broods. The optimum cut could only be determined when sufficient
material was given for the number of insects present, as an unfavorable
cut may be attacked when the adults are confined on it without sufficient
optimum material.

Wood used a month or two after being cut is spoken of as green or
freshly cut material.

In many cases wood from several individual trees was used to avoid
any possibility of offering an undesirable individual.

To illustrate the variation in optimum conditions of wood, several
examples are given: Callidium antennatum Newm. requires wood dry-
seasoned over winter; Neoclytus capraea, wood cut during the late winter
with the inner bark still sappy ; Liopus alpha Say in hickory, twigs cut
in the early fall, air-seasoned for a while and then left on the ground
over winter so that the inner bark sours somewhat. (This condition is
brought about by the girdling habit of Oncideres cingulata Say.)

Several terms which may need explanation are used in reference to
the species of host wood: Primary host, or original host, refers to the
wood in which the insect is found in nature and first caged in these experi-
ments ; as secondary host is understood wood in which a colony has been
successfully produced in the experiment, but it may or may not be
recorded as a host in nature; an unfavorable host is one not recorded
from nature and in which attempts to produce a colony have not been
entirely successful.

All experiments conducted are here given, although a few have been
unsuccessful or have given no results. Occasionally failure to continue
a colony is recorded. In all cases an explanation can not be given. It
may be because of an improper cut of wood or of a peculiarity of the
individual host. In one case partial failure was due to a nematode
parasite causing sterility of the females; in another, the parent insects
were entangled in spider webs and killed before ovipositing.

Reference is made to larval transfers from one host to another. This
is accomplished by making a smooth cell through the bark of the new
host, partially filling it with frass from the larval mines of the original
host, then placing the larva in this cell and finally tightly fixing a piece
of bark over the cell. Such transfers do not injure the larva or affect
its development. Many cases of transfer to the same host resulted in
the survival of every larva.

These experiments were conducted at tlie Eastern Field Station, East
Falls Church, Va., and all flight dates of the adults and times of cutting
of the wood refer to this locality unless otherwise stated.

Oct. 22, I92I Hopkms Host- Selection Principle 195

OUTLINE OF EXPERIMENTS ON EACH SPECIES
XYI^OTRECHUS COI,ONUS. EXPERIMENT I

Xylotrechus colonus Fab. is found in nature in a wide variety of hosts.
In fact, it feeds in nearly all hardwood deciduous trees of the eastern
and central United States. It shows little or no preference for any
exact condition of the wood, except that it will not attack perfectly
seasoned material. The larvae can be found in dying standing trees or
in logs felled in any month of the year provided they still contain a
certain amount of moisture.

The first flight of the year occurs in the last week of May or first week
of June, reaching the maximum in about two weeks. A few adults
emerge sporadically throughout the summer. From eggs deposited in
June a few adults usually emerge in September, but the main brood
remains as larvae until the next spring. These fall adults have never
oviposited under confinement.

The larvae feed entirely beneath the bark, or in the bark if it is thick.
The pupal cell is made in the outer sapwood or in the bark.

The wood of all species for this experiment was cut on April 15 unless
otherwise stated. The colony was started by felling a red oak tree in
March, 19 14. The wood was attacked during that June, caged soon
afterwards, and the colony has since been maintained in red oak. From
the original oak form colonies were secured in hickory (Hicoria), chestnut
{Castanea dentata {M.QXsh..) Borkh.), locust {Robinia pseudacacia Linn.),
red maple {Acer rubrum Linn.), and ash (Fraxinus sp.), in the following
manner :

QuERCUS. Experiment I. — During May, 1915, hickory logs were
placed in the cage with oak intended to carry on the colony. Many
adults were present, somewhat over 100, and the hickory as well as the
oak was subsequently found infested.

In June, 19 16, in the same cage stocked with oak for continuing the
colony, chestnut and hickory wood was placed. There was again an
overabundance of adults and all woods were infested.

In June, 191 7, oak was placed in this cage to continue the colony
and also ash, chestnut, locust, hickory, and red maple logs, all cut in
February except the hickory, which was cut in April, 191 7; extra pieces
of chestnut and maple, cut in November, 1916, and September, 1916,
respectively, were also placed in the cage. There was an abundance of
adults. In July these logs were examined and it was found that the oak
was heavily infested; the chestnut and hickory were lightly infested;
the ash, maple, and locust had no infestation. This same year, 191 7,
adults were isolated on ash (I^), maple (I*), and locust (I^) with results
as described in later paragraphs.
65583°— 21 2

196 Journal of Agricultural Reserach voi.xxn. no. 4

In June, 19 18, two pairs of adults from oak were caged on a collection
of oak, hickory, ash, maple, and chestnut logs, all cut April 15, and of
about equal size. Examination in July showed that the oak was heavily
infested (over 50 larvae present), the chestnut contained 10 larvae, the
hickory 7 larvae, and the ash and the maple none.

At the same time a similar cage was prepared, and six pairs of adults
were placed in it to test the influence of a greater number of beetles
on the selection of hosts. The results showed the same relative propor-
tion of infestation except that ash also was attacked. The maple was
not infested.

In June, 1919, this experiment was repeated with the same conditions
except that the hickory sticks were accidentally omitted. They were
examined in July and the infestation was as follows : The woods in the cage
of two pairs of adults contained 28 larvae in oak, 22 in chestnut, and none
in ash and maple; that of six pairs contained over 50 in oak, 19 in chest-
nut, and none in ash or maple.

HicoRiA. Experiment I ^ — May, 1915, hickory logs were placed (as
before described) in the oak cage with the wood intended to carry on
the colony. They were infested and in subsequent years kept isolated
and continued as the hickory form.

In June, 191 6, oak was placed in this cage, together with the hickory
to continue the colony, and was subsequently found heavily infested.

In June, 19 17, together with the hickory for reinfestation, chestnut
and locust were placed in the cage. An examination in July showed
that the chestnut contained a few larvae and the locust none.

In June, 1918, two pairs of adults were isolated in a cage containing
oak, hickory, ash, chestnut, and maple, all cut April 15, and of equal
size. The results showed that the hickory was heavily infested by over
50 larvae, the oak contained 7 larvae, the chestnut i larva, and the maple
and ash none.

In June, 19 19, selection tests and selection quantity tests were carried
out with this strain. The quantities and cuts of wood were the same
as before, except that oak was accidentally omitted. In one cage two
pairs of adults were isolated, the resulting infestation being hickory
18 larvae, chestnut 12 larvae, maple and ash none. In another cage six
pairs of adults were isolated, the resulting infestation being hickory over
50, chestnut 40, and maple and ash none,

Castanea. Experiment I ^. — In May, 1916, chestnut logs were placed
(as before described) in the oak cage with the wood intended to carry on
the colony. The wood was heavily infested, and these individuals have
since been confined to chestnut.

In May, 191 7, together with the chestnut, hickory was placed in this
cage. The hickory was lightly infested.

In June, 19 18, two pairs of adults were isolated in a cage containing
oak, hickory, ash, chestnut, and maple, all cut April 15 and of equal

Oct. 22, 1921 Hopkins Host-Selection Principle 197

size. An examination in July showed that the oak and chestnut were
equally well infested, the hickory contained one larva, and the maple
and ash none.

RoBiNiA, Experiment I ^. — In June, 19 1 7, eight adults from oak were
isolated on black locust cut in February, 191 7. The adults laid eggs, but
all died later.

In June, 19 18, the experiment was repeated with 15 adults and wood
cut April 15. Many young larvae entered the bark, but by August 15
nearly all had died, and none lived to transform the next spring. The
experiment was not repeated in 19 19.

Acer rubrum. Experiment I*. — In June, 191 7, eight adults from
oak were isolated on a piece of red maple cut February i, 19 17. A few
larvae lived and three adults (two males and one female) emerged in 19 18.
They were isolated in a cage containing oak, chestnut, hickory, ash, and
maple, all cut April 15, but no infestation occurred in any wood. During
August, 19 18, twenty larvae were transferred from oak to maple to con-
tinue the species in this host. A few adults emerged in 1919, and were
recaged on maple to develop a larger colony, which will be continued
several years before testing the selection again.

Fraxinus. Experiment I ^. — In June, 191 7, eight adults from oak
were isolated on a piece of ash cut January i, 19 17. A fair infestation
occurred, but the larvae developed slowly, and in May and June, 1918,
only seven adults (both sexes represented) emerged, while many larvae
were still in the wood. These adults were transferred to a cage contain-
ing oak, chestnut, hickory, ash, and maple, all cut April 15, but no infesta-
tion occurred in any wood. Larvae were again transferred to ash, and a
few adults emerged in 19 19. These were recaged on ash, and several
adults emerged in June, 1920, but failed to develop any larvae in the new
wood.

I, I\ I^, P. — During June, 1920, adults emerged from the oak, hickory,
chestnut, and maple; strains and adults from all were recaged on the
same wood and produced new colonies. No selection tests were made,
and these strains will be continued for several years in the same wood
before similar experiments are again attempted.

CONCLUSIONS

The original oak strain of Xylotrechus colonus shows a decided prefer-
ence for a few woods, notably oak, chestnut, and hickory. Two years'
trial failed to produce larvae capable of completing their development in
locust, while the ash and maple colonies were maintained with difficulty.
In nature these woods (ash, maple, and locust) have been found contain-
ing thrifty colonies of this species.

Originally the oak strain showed little preference as between oak, hick-
ory, and chestnut; yet, after several years, strains were developed in each
wood that showed a growing preference for the given wood.

198 Journal of Agricultural Research voi. xxn. no. 4

The number of insects present under identical conditions influences
their selection of hosts. When few are present they concentrate on
original or favored hosts; when more than can successfully oviposit on
original hosts are present, less favored hosts are taken.

CYIvI,ENE PICTUS, HICKORY HOST STRAIN. EXPERIMENT II

The larvae of Cyllene pictus feed almost exclusively in hickory. A few
specimens have been taken in wild grapevine (Vitis sp.), mulberry {Moriis
rubra Linn.), osage orange (Toxylon pomiferum Raf.), and hackberry
{Celtis occidentalis Linn.), but such instances are rare and of very local
occurrence. In one locality near Harrisburg, Pa., all except one of these
unusual food plants have been recorded. This borer is found generally
distributed east of the Mississippi River. The optimum condition of
wood is that cut during the winter, preferably in January, and left lying
on the ground. November cuts are sometimes attacked, but no wood is
suitable unless the inner bark is still sappy. Sticks cut at the time of
emergence are too green for attack.

The first emergence occurs about the middle of April and continues
for three weeks. By September the larvae are full grown and have con-
structed their pupal cells in the wood. They soon pupate, and in this
stage they overwinter. The larvae feed about equally beneath the bark
and in the wood.

These experiments were started in April, 19 15, when adults were
found ovipositing on a hickory log cut during the winter at Falls Church,
Va. The strain has since been continued in January and February cuts of
this wood, and other host strains have been attempted with varying suc-
cess in grape {Vitis sp.), locust (Robinia pseudacacia) , ash {Fraxinus sp.),
and mulberry {Morus rubra). Experiments were conducted as follows:

ViTis. Experiment II ^ — In April, 1917, a piece of grape, cut in
January, was placed in the same cage with much hickory used for the
continuation of the hickory form. This grape was not infested.

June 26, 19 1 7, sixteen larvae, about half grown, were transferred from
hickory to grape cut in January. Nearly all these larvae lived, and the
following spring 12 adults emerged. They were isolated in a cage con-
taining several pieces of grape and one of hickory, both cut in February.
Examination in June showed the grape to be heavily infested while the
hickory contained no larvae.

In April, 191 8, a large number of adults emerged from the grape.
Two pairs were isolated in a cage containing one piece of grape 2 inches
in diameter and 2 feet long, and one piece of hickory of the same size,
both cut in January. Examination in July showed the grape to be very
heavily infested, whereas the hickory contained only a few larvae.

Robinia. Experiment II ^ — April 21, 191 7, three females and two
males from hickory were caged on a piece of locust cut a month pre-
viously. The females laid all their eggs on the locust and the young

Oct. 22, 1921 Hopkins Host-Selection Principle 199

larvae bored through the bark, but by the middle of June all had died.
June 15, twelve larvae 5 mm. long were transferred to locust and these
all died by July 11, when three more, over half grown, were transferred.
These lived to construct pupal cells and pupated, but all the pupae died
during the winter.

In April, 1918, five adults (three females and two males) from hickory
were caged on locust cut during January, 19 18. The females laid all
their eggs, but only a few larvae lived. These constructed pupal cells
and pupated beneath the bark. In locust the larval mines are not
normal, lying in almost all cases immediately beneath the bark instead
of extending deep into the wood. About half the larvae made pupal cells
in the outer sapwood while the others pupated beneath the bark instead
of, as normally, deep in the wood.

April 20, 19 19, a total of six adults had emerged and two pairs were
caged on pieces of locust and hickory cut in January, 19 19. An exami-
nation July 16 showed no infestation in either.

Fraxinus. Experiment II ^. — April 24, 191 7, three females and two
males were isolated on ash cut during January. The females laid all
their eggs and the young larvae bored through the bark, but all died
before June 15. At this time fourteen larvae 5 to 7 mm. long were trans-
ferred to the same ash, and all died within a month. July 15, five more,
over half grown, were transferred. They mined extensively beneath the
bark, but all died before the end of September without pupating.

July 24, 19 18, fifteen larvae, one-half to three-fourths matured, were
transferred to ash cut in January.

April 21, 19 19, a total of eight adults had emerged. One pair was
caged on the January cut of ash, and two pairs were caged on January
cuts of ash and hickory.

July 16 the wood was examined, but in no case was it infested.

MoRUS. Experiment II*. — April 29, 1918, three females and two
males from hickory were caged on mulberry cut in January. The females
laid eggs, and a very heavy infestation was secured. They developed
normally and suffered little more than the normal rate of mortality
experienced in hickory.

April 21, 19 19, a total of 17 adults had emerged; two pairs were trans-
ferred to a cage containing two pieces of mulberry and one piece of
hickory cut during January, 19 19. In another cage containing the same
quantity of wood four pairs of adults were transferred. In neither case
was the quantity of mulberry sufficient to permit the development of
all the larvae. Each piece was 2 inches in diameter and 14 inches long.

July 16 the cages were examined, and that containing two pairs of
adults was infested as follows: Hickory 6 larvae, mulberry over 30;
that containing four pahs, hickory 13 larvae, mulberry very heavily in-
fested, over 40.

200 Journal of Agricultural Research voi. xxn.No.4

Seasoned Hicoria. Experiment II*. — ^Attempts were made in
April, 191 7, to develop a colony adapted to seasoned wood by caging two
females and one male on wood cut in October and dry seasoned. Eggs
were laid and larvse entered the bark but developed very slowly, never
entering the wood. Only three lived to pupate, and these made their
pupal cells between the bark and wood. All three pupae died during
the winter.

In May, 1918, the experiment was repeated with three females and
three males and wood cut in November, 191 7. In the fall of 19 18 a
number of larvse lived and pupated, but all were below normal size.
Only a few adults emerged in April, 191 9, and these were below normal
size.

1 1^. — Dr. A. D. Hopkins, in 19 16, recorded a dying hickory tree heavily
infested by Cyllene picius with no evidence of primary injury from other
causes.

This suggested that a strain capable of attacking living trees might
be produced, and attempts were made to secure a colony in such a tree.
A small hickory 3 inches in diameter was selected and entirely stripped
of leaves August 11, 1916. April 30, 191 7, it was again defoliated, and
80 adults were caged on it. The adults laid eggs and the young larvae
entered the bark, causing sap to flow from the wounds. However, all
died after growing to 3 mm. in length.

In April, 19 18, the tree was again defoliated, and 156 adults were caged
on it. The same results were observed.

During both years the tree put out healthy foliage after artificial defo-
liation, but it died in August, 19 17. In no case did the Cyllene larvae hve
to mine more than % inch beneath the bark.

QuERCUs. Experiment II^ — In transferring adults during the
spring of 1918 to new hickory wood to continue a large colony, a piece of
oak was unintentionally left in the cage. This cage contained six large
hickory logs 4 to 6 inches in diameter and 5 feet long. The oak log
was 3 inches in diameter and 4 feet long.

During September, 1918, work of Cyllene was noticed on this piece of
oak, and in the spring of 1919 it was separately caged. Five adults
emerged in April — all very small, much below normal size.

Two females and one male were transferred to a cage containing only
oak; one pair to a cage of oak and hickory. These cages were examined
July 16. Neither wood of the selection test was infested, but the oak
wood on which two pairs were caged contained a few very small larvae.

11^^ — ^To test the influence of host selection on the condition of host.
In April, 19 18, two males and two females from hickory were isolated in
a cage containing a piece of grape and a piece of hickory of equal size —
the grape of optimum cut, January, 19 18, the hickory less favorable,
November, 191 7. Examination in July showed the grape to be heavily
infested while the hickory contained very few larvae.

Oct 22, 1921 Hopkins Host-Selection Principle 201

11^^. — To test the influence of an overabundance of adults and scarcity
of the primary host on the host selection. Three pairs of adults from
hickory were caged on a small piece of grape and a small piece of hickory
(each 2 inches in diameter and i inch long), each cut during January,
19 1 8. Examination in July showed both woods to be infested, the grape
containing a few more larvae than the hickory.

In 1920 no adults emerged.

II, 11^. — In 1920 only two strains were continued, those in hickory and
those in mulberry. No attempt was made to reestablish the others that
failed.

CONCLUSIONS

This species, although most commonly found in hickory, will readily
adapt itself to several other plants, notably mulberry and grape, both of
which are recorded as natural hosts.

In some unfavorable hosts, or in an optimum host hi an unfavorable
condition, the larvae may become established, but the mortality is high
and the progeny seem to be sterile.

After one year's feeding in a new host the larvae may select that host in
preference to others.

The selection of a host is influenced by the number of adults present
and the quantity of the primary host, in that adults will prefer a sec-
ondary host to overinfesting the original host.

The selection of a host is influenced by the condition of the host, a
favorable condition of secondary host being preferred to an unfavor-
able condition of the original host.

The optimum condition of any host capable of properly supporting
growth of the larvae is of very restricted limits.

CYI,I,ENE PICTUS, GRAPK HOST STRAIN. EXPERIMENT III

This is the same species as previously discussed, having the same
biological habits except that this host strain in grape (Vitis) was taken
in nature at Hummelstown, Pa., in January, 1916, by J. N. Knull.
Since then the colony has been continued at East Falls Church, Va., in
grape cut in January or February. Other host strains have been pro-
duced and experiments conducted as follows :

HicoRiA. Experiment III ^ — When these adults emerged from
grape (May, 19 16), three females and two males were isolated in a cage
containing 10 pieces of grape and i piece of hickory cut in February, 19 16.
No eggs were laid on the hickory. April 17, 191 7, two pairs of adults
were isolated on hickory cut in January, 19 17. Eggs were laid and the
larvae developed but not so rapidly as in the grape. June 15 they were
under normal size. By September only two larvae were alive. One of
these pupated but died during the winter.

202 Journal of Agricultural Research voi. xxn, N0.4

Many adults emerged from the grape in 19 18 and five pairs were caged
on three small pieces of grape i}4 inches in diameter and 2 feet long and
one piece of hickory about the same size, both cut in Januar}^ 1917- In
July they were examined, and both hickory and grape were heavily in-
fested.

Quantity selection. Experiment III^. — To again test out the
effects of host selection when an insufficient amount of wood is given than
that required for the number of adults present, in April, 19 18, two pairs
from grape were caged on grape and hickory cut in January, 191 8. The
piece of grape was 2 inches in diameter and i foot long, the hickory 2
inches in diameter and 2 feet long. An examination in July showed both
grape and hickory infested.

This experiment was repeated in April, 1919, using two pieces of grape
and one piece of hickory, all of optimum cut and equal size. In one case

1 pair of adults was isolated, in another case 3 pairs were used. The wood
on which i pair was caged contained 5 larvae in hickory and 3 larvae in
grape ; that on which three pairs were caged contained 4 larvae in hickory
and over 25 in grape.

The grape colony was not continued in 1920.

CONCLUSIONS

This host variety from nature had acquired a decided preference for
grape.

The selection of a host is influenced by the quantity of wood present for
a given number of adults, in that the adults will select a new host in
preference to overinfesting the original host.

The tendency in this species in nature to confine itself to a certain host,
either hickory or grape, is not as marked as in some other species.

CYIvLENE PICTUS, HICKORY STRAIN II X GRAPE STRAIN III

In order to determine whether crossing of these two host strains would
influence the progeny in the selection of the host, males and females were
isolated from their pupal cells in the spring of 191 7. April 17 three
females from hickory and two males from grape were isolated in a cage
containing hickory. Two females from grape and two males from hickory
were isolated on grape. In neither case did mating occur as readily as
when both sexes from the same host were paired. The sexes often ap-
proached each other and moved away before finally copulating.

Good infestations were secured in both cases. In April, 1918, one pair
from hickory was caged on equal amounts of grape and hickory cut in
January, 19 18. Only the hickory was infested. Two females and two
males from grape were isolated on the same amount of grape and hickory
cut in January, 19 1 8. Both woods were infested. These pieces were all

2 inches in diameter and 1 8 inches long.

Oct. 22, 1921 Hopkins Host-Selection Principle 203

CONCIvUSIONS

This crossing of the two host forms had no influence on the selection of
hosts.

The amount of wood and number of adults present influence the host
selection, as shown when one female and two females were given the same
amount of wood.

CYI,I,SNE CRINICORNIS. BXPERIMENT XI,I

Cyllene crinicornis Che v., found in the southwestern United States, is
known to feed only in mesquite (Prosopis juliflora (Swartz) de C.) and
occasionally on an allied legume, paloverde {Parkinsonia microphylla
Torr.). In general its biology is similar to that of C. pictus, and it pre-
fers the same conditions of wood. Adults begin emerging in the natu-
ral range during late February, and part of this generation emerges in
September.

Mesquite infested with these larvae was sent to Falls Church, Va., by
T. E. Snyder from San Antonio, Tex., April 27, 191 7. Adults emerged
at Falls Church the following May and were caged on mesquite cut in
March, 1918. A good infestation was secured and has since been con-
tinued on mesquite.

RoBiNiA. Experiment XLr. — In May, 19 18, two pairs were isolated
on locust cut February, 19 18. May 31 the females were dead, and the
abdomens were dissected and found to contain eggs. Probably no eggs
were laid, and in July no evidence of larval work could be found.

July 24, 19 1 8, seven nearly matured larvae were transferred from the
mesquite to locust cut January, 1918. During May, 1919, five adults
emerged. One pair was caged on locust cut in January, 1919, and one
pair on both locust and mesquite. The mesquite was cut October, 19 18.

July 16, 1919, these cages were examined, and the selection test showed
that mesquite was not infested, whereas the locust contained several
small larvae. The cage containing only locust was lightly infested.
None of these larvae transformed in 1920.

CONCLUSIONS

The locust was such an unfavorable host that the adults would not
oviposit on it, but larvae may live and transform for one or two genera-
tions when forced to take it.

CALUDIUM ANTENNATUM. EXPERIMENT IV

Some confusion exists as to the taxonomy of the blue species of Calli-
dium allied to Callidium antennatum. A number of species have been
described of questionable validity. Two species have been experi-
mented with — C. antennatum and C janthinum Lee. These two adults
are easily separable, and their habits are also quite distinct. The

204 Journal of Agricultural Research voi. xxn, no. 4

former, so far as the Forest Insect records are concerned, feeds only in
pine (Pinus) and spruce (Picea), and for this discussion will be confined
to the form occurring in the northeastern United States. It shows a
decided preference for a certain condition of the host, requiring wood
that has been cut in the early fall or winter and is well seasoned. When
the inner bark is still sappy the insects will not normally make their
attack.

Adults first appear about the middle of April, and the flight period
continues about a month. One year is required to complete the life
cycle. The larvae feed beneath the bark until half grown, then enter
the wood to construct a long pupal excavation, at the end of which the
pupal cell is chambered off.

These experiments were started in December, 19 16, when infested
Virginia pine was caged. In 19 16 and the following years the colony
was continued in seasoned pine, and a form was also developed in spruce
and freshly cut or green pine. Unsuccessful attempts were made to
produce a juniper (Juniperus) strain. This insect has been reported as
feeding in juniper (Juniperus) and maple (Acer).

Juniperus. Experiment IV^ — In April, 19 16, juniper was placed
in the cage together with pine; in addition, about 20 adults were isolated
on a stick of juniper (both woods were cut in January, 19 16). In neither
case was the juniper attacked, and the females isolated on juniper failed
to oviposit.

The same test was repeated in 191 7 with juniper cut in October, and
the same results were obtained. During June and July, 20 larvae, from
small to over half grown, were transferred to juniper. All finally died,
some living a month. None increased in size before death.

Picea. Experiment IV^. — April 12, 1916, a piece of seasoned
spruce was placed in the cage, together with the pine, for reinfestation.
An examination in June showed only one larva in the stick, and this was
far below the normal size of those in pine. By July 11 this larva had
died. It is possible that more eggs were laid on the spruce but the larvae
died earlier.

June 29, 1916, nine larvae about half grown were transferred to the
same spruce wood. July 11, 191 6, three larvae were alive and 17 more
were transferred. From these larval transfers 10 adults were secured
in April, 191 7. They were caged on four small pieces of seasoned spruce
and a piece of seasoned pine placed in the cage for one week, both cut in
October, 19 16. Examination in July showed the spruce well infested,
but only 4 larvae were found in the piece of pine.

In 19 1 8 the same experiment was repeated, four females and three
males from spruce being caged on four sticks of spruce and one of pine
cut in September, 191 7. An examination in July showed that the spruce
contained many larvae, but none were found in the pine.

Oct. 22, 1931 Hopkins Host-Selection Principle 205

In 19 1 9 this was again repeated, but the results showed that pine was
infested while the spruce contained no larvae. Both woods were of No-
vember cut, but it is not likely that this would have such a decided in-
fluence. However, six adults were isolated on spruce to continue the
colony, and this wood was very lightly infested. No explanation can be
offered as to the reason for this discrepancy from former results unless the
spruce wood was in an unfavorable condition.

During the same season another cage was prepared of pine cut during
November, 1918, and green spruce cut in April, 1919. The pine was
heavily infested ; the spruce contained no larvse.

Experiment IV^. To test effects of condition of wood on selection. —
April 23, 1920, one pair of adults from spruce was caged on a favorable
cut of pine (November) and freshly cut spruce (April) . When the woods
were examined on July 5, 1920, the pine contained many larvae while the
spruce contained none. At the same time a pair was caged on November
pine and November spruce, both optimum cuts. Several days later
the female was found dead in the cage and had laid no eggs.

Acer. Experiment IV^. — ^As maple has been reported as a host of this
species, attempts were made in 19 16 to start a colony in this wood. On
July 6, six half- grown larvae were transferred to a seasoned piece of
wood, but by July 21 all but one had died and it was smaller than when
transferred. This one died soon after. None of the larvae fed on the
maple.

Green pine. Experiment IV^. — In 1916 some of the wood used to
carry this colony along was cut March i, and consequently little seasoned.
It was very unfavorable for the ovipositing of the adults, but some eggs
were laid on the pieces. The larvse developed slowly and at the time
of pupation were below normal size. The adults secured in 1 9 1 7 averaged
about one-half normal size. They were caged again on wood cut in
March, 191 7. Adults were secured in 1918 and again caged on the same
condition of wood, and a good infestation was secured. While the author
was absent for a month from the field station in the summer of 19 18
these larvae were all killed by a fungus. The sticks were on the ground
and so were caught in a period of rainy weather and were water soaked.

Experiment IV, IV ^. — In 1920 the pine and spruce strains were
continued in the same wood.

CONCI^USIONS

The pine form shows a decided preference for that host.

It can live in spruce and then shows a decided preference for that
host.

It will not live in juniper or maple.

In producing a new host strain a high mortality occurs in the young
larvse.

2o6 Journal of Agricultural Research voi. xxn. No. 4

A colony can be produced in a host which is in an unfavorable condition,
but the resulting adults are below normal size. Owing to the failure to
continue the colony it can not be stated whether or not such a strain
would show preference for the new condition of the host by selecting it
voluntarily.

CALLIDIUM JANTHINUM. EXPERIMENT V

Under Callidium antennatum reference was made to C. janthinum
Lee. It is distinguishable from the former by its smaller size, shining
surface, and bluish green color of the adult, by the fact that the larva
feeds only in juniper, and that the adults emerge about four weeks later
in the spring. It requires wood which was cut during the late fall and
which has not seasoned in contact with the ground. It will oviposit in
greener wood than C. antennatum although the inner bark should not
be sappy.

The first flight occurs during the first to third week in May and con-
tinues about two weeks. One year is required to complete the develop-
ment. The larvae feed beneath the bark until half grown, then excavate
long pupal chambers, the ends of which are plugged off for the trans-
formation cell.

These experiments were started with a lot of infested juniper branches
from Hummelstown, Pa., collected by J. N. Knull in April, 1916. May 2,
six adults emerged and were caged on juniper cut in April and rapidly
dried in the house. Since then the colony has been continued each
year in September and November cuts of juniper, which are preferred.

PiNUS. Experiment V^ — May, 191 7, two pairs were isolated on pine
cut in November, 19 16. A few eggs were laid from which larvse hatched
and entered the bark. By July 10 all had died. The same test was
repeated in 19 18 with similar results.

CONCLUSIONS

This species shows a decided preference for jumper and will not develop
in pine from early stages. Larval transfers to pine with nearly matured
larvae were not made.

CAIvWDIUM ANTENNATUM AND C. JANTHINUM. VXIV; IV XV

Because of the taxonomic confusion between these species (cited pre-
viously) and with the idea that a crossing of these two forms might possi-
bly influence the selection of a host, attempts were made to cross the
species.

VxIV. — In May, 19 17, four females of the juniper form were crossed
with two males of the pine form and were caged on juniper. Both
species had been previously isolated from the pupal cells to avoid all
possibility of mating. These insects immediately mated, and the eggs
were laid on the juniper from which a good infestation was secured.

Oct 23, 1931 Hopkins Host-Selection Principle 207

April 17, 19 1 8, the first adult emerged from this cross. The second
adult emerged April 25. May 3 and 4 eight adults emerged. All the
19 1 8 adults resembled the juniper form in color. These adults were all
isolated in a cage containing juniper and pine cut in November, 19 17.
An examination in July showed only the juniper to be infested, but by a
very light brood. During the remainder of the summer all died except
three larvae which constructed pupal cells. May 5, 19 19, two males and
one female emerged and one pair was caged on juniper cut in November.
A light infestation occurred. May 12, 1920, five adults emerged and
were recaged on juniper. The selection of pine and juniper was not
again tested. t;"- ,.,) hf>q<f:?"

IV X V. — ^These same species were mated in 19 17 by making the recip-
rocal cross (males from juniper and females from pine) and caged on
pine. The sexes did not mate readily, not noticing one another for some
hours after being caged together. However, several matings finally took
place and eggs were laid on the pine. Young larvae developed but all died
later. The same experiment was repeated in 19 18 with the same results.
Conditions were similar to those in the previous experiment.

CONCLUSIONS

The crossing did not influence the selection of a host in the first genera-
tion of resulting adults.

These two forms, even though they may be crossed successfully, should
be regarded as distinct species based on adult characters and biological
differences.

The successful cross-mating produced progeny in the first generation
that emerged over the period of emergence of both parents — a few early
when the pine form emerges, the remainder some two weeks later when
the juniper form appears. In later years they emerge as the juniper
form.

The juniper color pattern of the adults is dominant.

HYLOTRUPES UGNEJUS, JUNIPEIR FORM. EXPERIMENT VIII

The adult forms generally included under Hylotrupes ligneus Fab. show
a great variation of color patterns. Many of these varieties have been
described as distinct species by Col. T. L. Casey. In the experiments
conducted all color varieties, however, have been kept distinct only
by the host in which they were found in nature and not by the color
variations. The experiments were primarily conducted to test these
variations in color patterns, but certain results bearing on the host-
selection principle were obtained and are here described.

Hylotrupes ligneus, juniper form, has a wide selection of hosts. Speci-
mens in the Forest Insect Collection of the Bureau of Entomology have
been recorded from all genera of coniferous trees indigenous to North
America. It uniformly prefers wood that has not seasoned a great deal.

2o8 Journal of Agricultural Research voi. xxn, no. 4

Later winter or fall cuts in which the inner bark has remained sappy are
most suitable. Species of wood which season more slowly, due to thick
bark, must be cut earlier.

The time of first emergence varies greatly with locality, but the species
is everywhere one of the first cerambycid beetles to fly in the spring.
The larvse in all cases feed immediately beneath the bark, only entering
the sapwood in late summer to make a shallow pupal cell. Pupation
and transformation to the adult usually take place in the fall.

The present experiment was started at Kanawha Station, W. Va, Dr.
Hopkins felled a juniper (Juniperus) in October, 1914. This tree was
infested the following spring and shipped to East Falls Church, Va.
The colony has since been continued in juniper and one host strain has
been produced in Douglas fir (Pseudotsuga) . This particular color
variety had never been recorded from Douglas fir.

Pseudotsuga. Experiment VIII^— April 11, 191 7, three females and
two males were isolated on a piece of Douglas fir cut April i . Eggs were
laid and young larvse entered the bark, but many died during the summer
and only two constructed pupal cells. One adult was secured next
spring. The fact that this wood was too green and that it seasons very
slowly may have caused a higher mortality than would otherwise have
occurred.

May 29, 191 7, twelve larvae and June 15, nine larvse were transferred
to this host, the wood then being better seasoned. March i, 1918, five
adults — four females and one male — were removed from pupal cells.
The remainder of the larvae had died. Two females and one male were
used to continue the colony by caging on Douglas fir, cut in October,
19 1 7. A good infestation was secured. A piece of juniper cut during
January, 1918, had also been placed in tliis cage but was not infested.
In January, 1919, four adults were removed from the logs — three males
and one female. The remainder had all died and these were very weak
and below normal size. One pair was recaged on juniper cut in January
and Pseudotsuga cut in November. July 30, 19 19, the sticks were
examined, but no infestation was found in either wood.

CONCLUSIONS

This juniper form of Hylotrupes ligneus, after feeding part of a year in
a new host, showed a preference for the new host.

A high percentage of mortality occurred in producing the new host
strain, which finally died out.

HYEOTRUPES EIGNEUS, PSEUDOTSUGA FORM. EXPERIMENT XXXV

This form of Hylotrupes ligneus is much darker and more hairy than the
preceding. It has been recorded only from Douglas fir. Its biology is
essentially similar to that of the juniper form except that the adults
emerge somewhat later. It is known from the Rocky Mountain region.

Oct. 22. I92I Hopkins Host-Selection Principle 209

The colony was started from a small tree collected at Colorado Springs,
Colo., and shipped to Falls Chm'ch. This tree had been killed by Scolytus
in the fall of 19 16 and infested by Hylotrupes in the spring of 191 7.

In 19 1 8 adults did not emerge until May and were caged on Douglas
fir to continue the colony. They were recaged on Douglas fir in 1919,
but all the larvae died from a fungus attacking the bark.

HYI,OTRUP:eS WGNBUS, PSEUDOTSUGA STRAIN VIII ^ AND PSEUDOTSUGA

STRAIN XXXV

Two females from VIII ^ (the juniper form in Douglas fir) were held
in a cool cellar until adults of this XXXV variety emerged. May 29,
1918, they were caged on Pseudotsuga with two males from the true
Douglas fir form (XXXV) . The two sexes absolutely avoided each other
and were never observed to mate. The females died without laying
eggs. Many attempts were also made to mate the original juniper form
with the Douglas fir form but without success.

HYLOTRUPES UGNEUS, SEQUOIA FORM. EXPERIMENT XL

The form of Hylotrupes ligneus occurring in sequoia is slightly larger
but otherwise resembles that in juniper very closely, although the speci-
mens reared in the experiments show a much greater variety of color
pattern than do those from juniper.

April 2, 1 91 8, a large series of these adults were removed from their
pupal cells in Sequoia sempervirens (Lamb.) Endl. and isolated in small
vials by F. B. Herbert at Laurel, Calif. April 13, 191 8, they were re-
ceived at Falls Church, Va.

JUNIPERUS. Experiment XL ^ — Three prominent color forms were
paired and each was caged on a piece of juniper cut in January, 191 8,
since no sequoia was on hand. They all oviposited, but about half of
the larvae died by July. The remainder made pupal cells and emerged.
The strain has since been continued in juniper.

Pseudotsuga. Experiment XL^. — April 20 one pair was caged on
a piece of Douglas fir cut in October, 191 7. Eggs were laid and a better
infestation secured than with the juniper form (VIII). All larvae died
and no adults were secured in the spring of 191 9. A fungus growth
under the bark was responsible in a large measure.

hylotrupes ligneus, JUNIPERUS STRAIN VIII X SEQUOIA STRAIN XL

April 13 several males from redwood (XL) were separately caged
with females from juniper (VIII) held over in a cool cellar since they
were isolated from the cells. One of these males mated with two females
(first and third tried) immediately on being isolated with them. This
same male would not mate with the second female tried, nor would any
males of XL mate with females of VIII. Many juniper (VIII) males

2IO Journal of Agricultural Research Voi. xxn. no. 4

were isolated with redwood (XL) females, but in no case did copulation
take place.

The females of the j'uniper form mated with males of the sequoia form
were caged on juniper cut in January, 19 18, and good infestations were
secured.

CONCLUSIONS ON THE ENTIRE HYLOTRUPES EIGNEUS GROUP

The experiments on the Hylotrupes ligneus group, as mentioned above,
were conducted primarily for the study of its color variation, and not a
great deal of attention was devoted to the host-selection principle. The
experiments cited show that among all the color varieties of this group
there are probably two good species, the darker and more hairy Douglas
fir form representing one species and all the other forms another. These
two species absolutely refused to mate, but the varieties from sequoia
and juniper were successfully crossed.

NEOCLYTUS CAPRAEA. EXPERIMENT VI

Neoclytus capraea is known to inhabit the eastern and central western
United States, extending its range south and west into Arizona. It has
been recorded from only two hosts, ash (Fraxinus) and white oak
(Quercus alba of the Rocky Mountains). In the eastern United States
it has never been found in oak. The condition of the wood necessary
for oviposition by these beetles must be exactly right. It must have
been freshly cut and the inner bark must be still moist and sappy. Should
this inner bark be slightly dried the females will not oviposit on it unless
forced to do so. Logs cut about two months before the flight period
are preferred to older cuts or those cut during flight. Trees cut as
early as November 15 are sometimes infested, but not commonly.

The adults fly very early in the spring in this locality (Falls Church,
Va.), about the last week in March and the first two weeks of April. The
larvse feed chiefly in the wood proper. Mining beneath the bark for a
short time, they then enter the sapwood and later the outer heartwood,
extensively honeycombing it. Pupation and transformation to the adult
take place in the early fall.

VI.— March 26, 1915, twelve adults were taken as they emerged from
an ash log and were caged on freshl}' cut wood. A good infestation was
secured, and the colony has since been continued in ash.

In the spring of 19 19 no adults emerged. All the larvae remained
over as larvae in their pupal cells until the fall of 19 19, when they trans-
formed to adults and emerged in 1920. No explanation for this can be
offered unless the logs were too moist in the early part of the summer
so that the larvae did not develop properly. Excess humidity or exces-
sive desiccation have both been found to produce retardation in devel-
opment of larvae in small isolated cages. This insect is one of the most
regular of those reared, in the time of emergence and development of

Oct. 22, I92I Hopkins Host-Selection Principle 211

the broods. Attempts have been made to start colonies in hickory and
white oak.

HicORiA. Experiment VI ^— March 31, 19 17, seven adults (four
females and three males) from ash were isolated on hickory cut Feb-
ruary I, 191 7. No infestation occurred. May 31 of the same year
fifteen larvae, 2 to 4 mm. long, were transferred to hickory. Again
on June 15, seven larvae, 4 mm. long, were transferred to the same
piece of wood. July 11 one larva was living and five more, over half
grown, were transferred.

April 6, 191 8, three adults (two males and one female) emerged from
the hickory. They were caged on hickory and ash cut January, 19 18.
These adults were very weak and inactive, not at all characteristic of
normal adults.

An examination in July showed neither wood to be infested.

July 24, 19 1 8, twenty larvae, one-half to three-fourths grown, were
transferred from ash to hickory cut April 15, 19 18.

April II, 19 19, one female emerged, one adult had died in its pupal
cell, and the remainder of the larvae had died before pupating. This
female was mated with a male from ash and caged on hickory and ash
of optimum cuts. An examination in July showed no infestation in the
hickory, but the ash contained a few larvae. These died later in the
summer.

QuERCUs AiyBA. EXPERIMENT VI ^. — ^April I, 1917, four pairs of
adults from ash were isolated on white oak cut in March, 191 7. Eggs
were laid on the wood, and the small larvae bored through the bark, but
all died before May 31. On this date fifteen larvae, 2 to 4 mm. long,
were transferred to white oak. July 1 1 one larva was living. Septem-
ber 17 all were dead.

In April, 19 18, three pairs were caged on wood cut in January, 19 18.
July 18 many larvae were still alive but under size. Several lived to
pupate, but all died before the following spring.

Seasoning. Experiment VI. ^ — ^April 4, 19 17, four pairs of adults
were isolated on ash cut September i, 19 16, and white oak cut in March.
The females laid eggs on the white oak, but the larvae did not live. On
Ma7 3 1 neither wood contained larvae.

CONCLUSIONS

The foregoing experiments show that this species feeding in ash
(Eraxinus) has become decidedly accustomed to that host. Several
attempts, both by oviposition and larval transfers, to produce strains in
Quercus alba Linn, and Hicoria have resulted in failure. In Hicoria
the few adults secured were incapable of continuing the colony, and in
both woods a high or total larval mortality occurred.

Even with this decided preference for a host, the adults laid eggs on
a new host rather than on an unfavorable cut of the normal host.
65583°— 21 3

212 Journal of Agricultural Research voi. xxn, no. 4

MOLORCHUS BIMACUI,ATUS. EXPERIMENTS IX, X, AND XXXVI

Two forms included under Molorchtis bimaculahis Say have been
caged in these experiments, a large form from hackberry {Celtis occi-
dentalis Linn.), and a smaller form from dogwood (Cornus florida Linn.)
and maple (Acer) . They both are found throughout the eastern half of
the United States. From the observations on the biology of these two
forms they are regarded by the writer as distinct species. Both forms
prefer early fall cuts of wood, but the Celtis form requires much drier
seasoned material.

MOLORCHUS BIMACULATUS, CORNUS FORM. EXPERIMENT IX

The Comus form feeds in a great variety of eastern hardwoods. It
has been reared from Hicoria, Acer, Juglans, Ouercus, Liriodendron,
Comus, Cercis, and Castanea. The larva feed beneath the bark, making
a long, curved pupal cell in the wood. By September they have trans-
formed to adults, which emerge early in May at Falls Church, Va. The
flight is very regular, nearly all emerging at the same time. The adults
are much smaller than those of the hackberry form.

In May, 19 16, adults were reared from dogwood collected at Falls
Church, Va. They were recaged on dogwood cut in April, but a poor
infestation was secured from which only five adults emerged in 191 7.
These five adults were caged on September and November cuts of dog-
wood and redbud (Cercis canadensis Linn.) . A good infestation occurred
in the dogwood, but no larvae were found in redbud.

May I, 191 7, five adults were isolated in a cage containing November
cuts of dogwood and maple. The maple was not infested, but many
larvae were found in the dogwood.

In April, 1918, 1919, and 1920, the colony was continued only in dog-
wood. No selection tests were made.

MOLORCHUS BIMACULATUS, ACER FORM. EXPERIMENT XXXVI

This form in all respects is similar to the dogwood variety IX.

Infested limbs collected at Falls Church, Va., were caged in the sum-
mer of 1916.

May I, 191 7, five adults were caged on branches of maple and dog-
wood cut in September and November. The maple was infested but no
larvae entered the dogwood.

In 19 1 8 many adults emerged from the maple and were recaged on
October cuts of maple and dogwood. Eggs were laid on the maple, but
the cage unfortunately was overlooked and became so dry that none of
the eggs hatched.

MOLORCHUS BIMACULATUS, CELTiS FORM. EXPERIMENT X

The form in hackberry, in which the adults are much larger, has been
reared only from this host. The larvae feed as in the dogwood or maple

oct.22, I92I Hopkins Host-Selection Principle 213

forms, but only about half the brood emerges at the end of the first year,
the remainder going over in the larval stage to the following season. The
adults emerge about a month earlier, April i to 10.

Infested hackberry branches from Hummelstown, Pa., were collected
and sent to Falls Church, Va., in December, 1915, by J. N. KnuU.

In April, 19 16, 20 adults were caged on January cuts of hackberry,
dogwood, and redbud, but only the hackberry was attacked.

In April, 191 7, eight adults were isolated on sticks of redbud, dogwood,
and maple, all cut in September and November. No eggs were laid in
any of these woods. The form has since been continued in hackberry.

CONCLUSIONS

A very decided predilection for the original host is exhibited by the
host strains of this species. It is not surprising in the case of the hack-
berry form, as this is the only host from which it has been found. How-
ever, this form would not even lay eggs on any hosts other than the
original. In the dogwood strain adults were not isolated on maple alone,
nor were adults of the maple strain isolated on dogwood alone. If this
had been done, it is veiy likely that infestations would have resulted.

NEOCIvYTUS ERYTHROCEPHAI^US. EXPERIMENTS XI, XII, AND XIII

The adult and larva of Neodytus erythrocephalus Fab. are quite different
from those of Neodytus capraea, but the range and habits are much the
same. The species attacks wood in a greater variety of conditions, but
the most favorable condition is an early spring cut. It has been collected
in almost all eastern hardwoods.

The first flight occurs at Falls Church, Va., in late May or early June;
consequently, that the wood may be sappy for infestation it must be
cut during April. The species overwinters in the larva stage, pupation
not taking place until early April. Farther south two or more genera-
tions occur each season.

Three host strains were collected in nature and experimented with.

NEOCLYTUS ERYTmiOCEPHALUS, HICORIA FORM. EXPERIMENT XI

June 9, 19 16, adults emerging from hickory at Falls Church, Va., were
recaged on wood cut in late March. A good infestation was secured.

June 8, 191 7, the colony was continued in April cuts of hickory. Two
pairs were isolated in a cage containing hickory and redbud cut in April
and dogwood and tulip {Liriodendron tulipifera Linn.) cut in May. In
July an examination showed hickory to be the only wood infested.

May 23, 19 18, two pairs were isolated on hickory, dogwood, and redbud
cut on April 15. When examined on July 18 hickory was found to be
lightly infested, dogwood heavily, and the redbud contained no larvae.

May 24, 19 18, six pairs of adults were caged on two pieces of hickory
and one of dogwood, cut April 15, of the same size as those of the

214 Journal of Agricultural Research voi. xxn.No. 4

experiment of May 23, 19 18. When examined on July 18, both woods
were heavily infested. Redbud was unintentionally omitted.

This experiment was repeated in 19 19, two pieces of hickory, one of
dogwood, and one of redbud being used. Two cages were prepared; in
one, a single pair was isolated, the resulting infestation being, hickory
heavily infested, dogwood and redbud uninf ested ; in the other cage three
pairs were isolated, the resulting infestation being, hickory and dogwood
both heavily infested, redbud uninf ested.

NEOCLYTUS ERYTHROCEPHALUS, CORNUS FORM. EXPERIMENT XII

June 13 to 15, 1916, adults emerging from dogwood at Falls Church*
Va., were recaged on this wood cut in April, 1916. A good infestation
was secured. June, 1917, the colony was continued in dogwood and two
pairs of adults were isolated in a cage containing dogwood and tulip cut
May 30 and hickory and redbud cut April 18.

In July it was found that both redbud and dogwood contained few
larvae while hickory and tulip contained none.

For some unknown reason the larvae continued in dogwood did not
develop very well, and in 1918 only one female emerged. May 25, 1918,
this female was mated with a male from hickory and isolated in a cage
containing dogwood, hickory, and redbud cut April 15.

July 18, 1 91 8, the dogwood was heavily infested, the redbud lightly,
and the hickory contained one larva.

In June, 1919, one pair was caged on pieces of dogwood, redbud, and
hickory. An examination in July showed dogwood to be very heavily
infested, the redbud and hickory containing seven and six larvae, respect-
ively.

NEOCLYTUS ERYTHROCEPHALUS, CERCIS FORM. EXPERIMENT XIII

Redbud infested with this species was collected at Hummelstown,
Pa., by J. N. Knull and sent to Falls Church, Va., in April, 1916. Adults
emerged in June and the colony was continued in redbud. June, 1917,
the colony was again continued in redbud, and two pairs of adults were
isolated in redbud and hickory cut in April and tulip and dogwood cut
in May.

An examination in July showed the redbud to be heavily infested;
the dogwood and hickory contained several larv^ae, and the tulip none.

In May, 191 8, two pairs were again caged on redbud, dogwood, and
hickory, all cut April 15. In July it was found that the redbud and the
dogwood were heavily infested while the hickory contained but three
larvae.

The same experiment was repeated in 1919, and the results showed the
redbud to contain eight larvae, the dogwood five, and the hickory two.

The selection tests of 191 7 were all carried out with the same quantity
of wood ; in each case the pieces were i }{ inches in diameter and i foot
long. Bach cage contained two pieces of the wood from which the

Oct. 22. I92I Hopkins Host-Selection Principle 215

adults emerged and only one each of the others. This amount of the
original host for two females was considered sufficient for oviposition
without bringing in the quantity factor.

These adults are extremely active and run rapidly over logs when
ovipositing in nature. They have very long hind legs. It was noticed
that in the glass cylinder used for cages in 19 18 these long legs were a
disadvantage. The adults could not get a foothold on the glass and
had difficulty in climbing up on the wood from the glass surface. They
crawled awkwardly about and when coming in contact with any stick
maneuvered until they managed to get on it. Such conditions may
have influenced the wood selected, as the adults could only with difficulty
go from one stick to another. In 19 19 wire boxes were used, the wood
lying flat on the bottom. In 1920 only ash and dogwood strains were

continued.

CONC1.US10NS

These experiments up to 19 19 did not seem to show results in any defi-
nite direction. Selections of the various host strains occasionally gave
results in conformity with those generally obtained, while again just
opposite results were recorded.

The experiments of 19 19 showed results in closer conformity to those
of other species. This may have been due to the different method of
caging, which gave the adults more opportunity to move about and select
the host.

UOPUS AIvPHA. EXPKRIMBNTS XXV AND XXX

Two color forms of Liopus alpha have been experimented with, a brown
form from sumac (Rhus) and a gray form from hickory (Hicoria) . These
color forms are very distinct and easy to recognize as adults. They are
not known from any other hosts. The sumac form has been collected
throughout the eastern United States and as far west as the Rocky Moun-
tains. The hickory form follows the range of the hickory trees.

The adults fly in late May and continue flying through June at Falls
Chiu-ch, Va. One year is required to complete the life cycle. The larvae
feed beneath the bark and pupate in the wood. They are found only in
small branches.

LIOPUS ALPHA, RHUS FORM. EXPERIMENT XXV

The sumac form prefers branches cut in the early fall and dried standing
in the air, although it will attack later cuts, provided they have dried
considerably.

April 26, 1916, Mr. Champlain sent from Long Island, N. Y., a lot of
infested sumac twigs which were caged at Falls Church, Va. In June the
first adults emerged, and 20 were caged on sumac cut in November, 1915.
Into the same cage were placed chestnut, hickory, and wild cherry twigs
cut during the winter, but none of these latter woods were infested. Since
then it has been continued in sumac.

2i6 Journal of Agricultural Research voi. xxn.No4

CastanEa. Experiment XXV^ — ^September 20, 1916, fifteen larvae,
one-half to nearly full grown, were transferred from sumac to chestnut
cut durmg March. July 10, 191 7, one adult emerged, the only one
from these transfers.

HicoRiA. Experiment XX V^ — July 25, 19 16, eleven larvae about
half grown were transferred from sumac to hickory. August 9, eleven
more were transferred. The larvae seemed to do quite well and by winter
many had made pupal cells.

During June, 191 7, twelve adults emerged and were caged on pieces
of hickory cut the preceding June, August, April, and February. No
infestation occurred in any of the wood. The cage accidentally dried for
a two- week period while the adults were ovipositing and this may account
for the failure of infestation, as they require considerable moisture.

In June, 191 7, adults from sumac were isolated in various cuts of
hickory but no infestation occurred.

July 23, 191 7, twenty-seven larvae were transferred from sumac to
hickory cut in September, 19 16, and March, 191 7. The larvae did well
and the following May and June 10 adults were reared and caged on
hickory sticks cut in September, 19 17. On several of these sticks bands
of thin outer bark of sumac were tied.

The adults oviposited only on those sticks and at those places where
the sumac bark was tied. July 30 they had not yet bored beneath the
hickory bark proper, but by fall nearly all had entered the bark. Only
one larva transformed to an adult in the summer of 19 19. One adult
emerged in 1920. Several larvae did not transform but continued feeding
beneath the bark during the summer of 19 19.

LIOPUS ALPH.\, HICORIA FORM. EXPERIMENT XXX

The Hicoria form was not successfully continued in confinement until
the summer of 191 7. It requires wood cut in August, dried in the air for
a month or so, and then placed on damp earth over winter. In addition
the adults must be well fed on fungus spores {Endothea parasitica was
used) before they will oviposit.

It was again continued in hickory in 1918, 191 9, and 1920. During
June, 19 19, many adults were caged on sumac branches and eggs were
deposited. Three larvae lived to construct mines under the bark, but
these died before November.

CONCLUSIONS

From the foregoing experiments and the fact that each of these two
color forms has been taken only in the host given, it is evident that each
has become restricted to that host and shows a strong predilection for
it. Even after having fed for one year in a new host (Hicoria) adults
developing from them showed a preference, in their oviposition, for that
part of the hickory twig surrounded by Rhus bark. A fairly high mortal-
ity of larvae occurred after transfer to the new host.

Oct. 22, iQji Hopkins Host-Selection Principle 217

HYPERPI/ATYS MACUIvATUS. EXPERIMENTS XXVI, XXVIII, AND XXIX

Hyperplatys maculatus Hald. occurs throughout the eastern United
States and west through the Rocky Mountain region. Two very similar
species have been described, H. maculatus Hald. and H. aspersus Say,
but the distinction is not drawn here, as each has many variations in
color and maculation. It feeds on a great variety of hardwood deciduous
trees. Probably any wood is attacked, provided it is in the proper
condition for infestation. Smaller twigs and branches are usually pre-
ferred. Those that have died during the fall and lain on the ground so
that a certain amount of fermentation has taken place in the bark give
the optimum condition.

The larvae feed entirely beneath or in the bark, only entering the sap-
wood to make a very shallow pupal cell. Adults fly in the early summer,
late May, and early June. Two distinct variations occur in the length of
the seasonal history. One form takes an entire year to complete the
development, only one generation appearing each year. Another ma-
tures from one-half to three-fourths of the brood in August and Sep-
tember, the adults emerging and infesting new wood. This may be a
basis on which to separate the two confused species. Four host strains
have been experimented with, chestnut (Castanea dentata), gooseberry
(Ribes), dogwood (Cornus florida Linn.), and yellow poplar (Liriodendron
tulipifera lyinn.).

HYPERPLATYS MACULATUS, LIRIODENDRON HOST STRAIN. EXPERIMENT XXVIII

The colony was started by collecting infested tulip branches in No-
vember, 1916, at Falls Church, Va. The following June adults emerged
and were isolated in a cage containing yellow poplar, maple, dogwood,
chestnut, and gooseberry cut in the fall of 1916. The original host,
yellow poplar, was well infested, and a few larvae were found in goose-
berry, but no other woods were attacked. In 1918, 1919, and 1920 the
colony was continued in yellow poplar; the selection was not repeated.

Only one generation of this form occurs each year.

Castanea. Experiment XXVIII ^ — ^June 4, 1917, ten adults from
yellow poplar were caged on chestnut cut in November, 1916. A very
good infestation was secured, forty-five adults emerging in 1918. Eight
of these adults were isolated in a cage containing yellow poplar and
chestnut cut in November, 191 7.

Examination in August showed the yellow poplar to be heavily
infested, while no larvae were present in the chestnut.

June I, 1 919, two pairs from yellow poplar were isolated on chestnut,
and in 1920 thirty-six adults emerged. Ten were caged on optimum
cuts of chestnut, and the yellow poplar was heavily infested.

HYPERPLATYS MACULATUS, RIBES HOST STRAIN. EXPERIMENT XXVI

This colony was started in December, 191 5, with infested gooseberry
stems sent to Falls Church, Va., from Colorado Springs, Colo., by G.

2i8 Journal of Agricultural Research voi. xxn,Na4

Hofer. It has since been continued in gooseberry cut at Colorado
Springs in the fall and shipped to Falls Church; in addition, several
other host strains were produced. Only one generation of adults occurs
each year.

May 20 to June 10, 1916, adults emerged and 47 were isolated in a
cage containing gooseberry, chestnut, and wild cherry, all cut in the
preceding fall. The gooseberry stems were heavily infested, a few larvae
were present in the wild cherry, but none were found in the chestnut.
The colony has since been continued in gooseberry.

Prunus. Experiment XXVI ^ — The infested wild cheiry twigs
(described above) were caged separately, and in June, 191 7, four adults
emerged. These were caged again on a fall cut of wild cherry. The
infestation was not very good, and only six adults were secured in 19 18;
these were recaged on the same wood, but no infestation occurred.

Castanea. Experiment XXVI^ — As previously stated, the chestnut
sticks were not infested in 1916 when caged with gooseberry. In June,
191 7, nine adults were isolated on chestnut cut in November, 19 16, and a
good infestation was secured. June, 1918, nine adults emerged and were
isolated in a cage containing chestnut and gooseberrry cut in November,
1917. Later examination showed only the gooseberry to be infested.

LiRiODENDRON. EXPERIMENT XXVIl — ^June 5, 191 7, eight adults
from gooseberry were isolated on tulip cut in November, 19 16. Five
adults emerged from these sticks in 19 18 and were isolated in a cage
containing tulip and gooseberry cut in November, 191 7. Neither wood
was infested.

HYPERPLATYS MACULATUS, CASTANEA HOST STRAIN. EXPERIMENT XXIX

In April, 19 16, at Falls Church, Va., branches of chestnut (Castanea)
containing larvse in the pupal cells were collected and caged. Some of
the adults emerging in June were isolated with chestnut cut in March,
1916, and the others isolated in a cage containing chestnut and dogwood
(Comus) branches cut in March, 19 16. Those isolated on chestnut
alone attacked this wood although it was a late cut. Those isolated on
the two woods infested both, but the dogwood more heavily. Nothing
more was done with the chestnut form. Many adults emerged that fall.

CoRNUS. Experiment XXIX ^ — ^The dogwood sticks were then caged
separately and adults secured in September, 1916, and more of them in
June, 191 7. Those emerging during the latter period were recaged on
August and November cuts of dogwood, but no infestation occurred.

CONCLUSIONS

In Hyperplatys maculatus host selection occurs to a certain degree ;
but this beetle behaves differently from most of the other species
tested. Thus the tulip form (experiment XXVIII) in 1 9 1 7 chiefly selected
the same host, but it also oviposited on gooseberry. This gooseberry
colony, however, was weak, and a high mortality in larvae occurred.

Oct. 22. X92t Hopkins Host-Selection Principle 219

Furthermore, although not selecting chestnut when the other host was

present, they produced a good colony when isolated on it; but in 19 18

these adults again selected tulip in preference to chestnut. The same was

true with the original gooseberry form which was transferred to chestnut

(experiment XXVI ^), for in 19 18 it returned to gooseberry in preference

to chestnut.

SUMMARY OF RESULTS

(i) In practically all the species experimented with the adults show a
marked predilection for the host in which they have fed as larvae,
provided they are not deterred by other factors, such as the unfavor-
able condition or the small quantity of the host.

(2) There is considerable variation in the degree of preference for the
original host, as between different species. Thus —

(a) Certain species are capable of living in only one genus or species of
plant, which consequently they select.

(6) Certain species, chiefly those living in nature in several hosts, can
be forced to adopt a new host.

(c) Certain species, chiefly those feeding in nature in a great variety
of plants, show little discrimination in the selection of hosts.

(d) Certain species feeding in nature in a great variety of hosts often
show a preference for a few of these.

(3) In forced transference of individual adults of a species to a new
host, a high mortality of the broods usually occurs, especially in the case
of eggs laid by beetles emerging from the original host, in which case
the mortality is often total. One-half to full-grown larvae, however,
usually can be successfully transferred to a new host and live and trans-
form to adults.

(4) With some species that can be reared in a secondary (new) host,
by the larvae feeding one or part of one year, preference for that host is
shown by the resulting adults.

(5) In general, the fewer the hosts in nature, the more marked the
predilection for a particular host, and vice versa.

(6) Continued breeding in a given host intensifies the preference for
that host.

(7) The condition of the host has a great influence on host selection, in
that every species prefers an optimum condition of the host which it
selects and will choose a new host in the optimum condition in preference
to an old host in which the conditions are unfavorable.

(8) The quantity of wood at the disposal of the ovipositing adults
may influence the insects in their choice between different kinds of host
wood, in that, if there are many adults to a limited amount of the primary
host, some species will select a secondary host if such is available. If
this is done, however, the resulting brood is weakened.

It is altogether possible that these experiments may indicate the origin
of certain closely related species or varieties of insects. For instance, a
species restricted to a very few plants may accidentally be forced to

220 Journal of Agricultural Research voi. xxn. no. 4

take a new host (as actually happened in the experiments with Cyllene
ia oak). A few individuals may survive and continue the strain so
that it becomes, after a time, at least physiologically different and may
also develop correlated differences of color or structure. It can hardly
be said that such forms are much less distinct than in the case of the two
species Callidium antennatum in pine and C. janthinum in juniper; for
even though these have a slight color distinction and each is absolutely
restricted to its own host, they interbreed. On the other hand, in the
different forms of Hylotrupes ligneus, of which the eastern form in juniper
is constant in marking, the western form in redwood is quite variable,
as is also the Rocky Mountain form in Douglas fir. The juniper and
redwood forms interbreed, but all attempts to mate either of these with
the Douglas fir form have failed. All these forms can be furnished with
substitute hosts, but in the experiments in which this has been done the
original color pattern has resulted thus far.

The grape and hickory strains of Cyllene pictus, although showing no
color differences, do not readily mate. Two species of Cyllene, C. pictiis
and C. rohiniae, are separable only as adults, by a slight difference in the
color pattern, yet in seasonal and biological habits they are strikingly dif-
ferent. It is conceivable that one of the two species originated through
the adoption of a new plant and continuous breeding in that plant.

It may be asked. If one or two years' feeding in a new host results in
individuals which prefer that host, thus giving rise at least to new physio-
logical varieties, why does not this occur more frequently in nature?
That it does occur must be granted, as we have species living in many
host plants as well as those restricted to a species or genus, but that it
is not of more common occun-ence is believed to be due to the high mor-
tality in first-stage larvae in a new host rather than to absence of oviposi-
tion in the new host. Although the adults show a decided predilection
for a favored host in ovipositing and even, in certain species, a preference
for the plants in which the larvae have fed for one or two generations,
the instinct to oviposit seems to overbalance that of host selection, con-
sequently new hosts are frequently selected — possibly more frequently
in nature than is generally realized. As an example of this, take
Cyllene pichis requiring hickory cut during the winter. This con-
dition would be fully met in tops left during logging operations. When
the timber cutting ceased, a concentration of adults would be left with
none of the favored host plant available in the right condition. The
grape, osage orange, and hackberry strains collected at Hummelstown,
Pa., were in reality taken in a woods v/hich had been logged for hickory
and in which operations had ceased three years prior to the finding of
these strains. At Falls Church, Va., in June, 1920, adults of Neoclytus
erythrocephalus were observed ovipositing on pine logs. Much infested
ash, from the previous year, was lying about from which they had
emerged in great numbers.

NOTES ON THE ORGANIC ACIDS OF PYRUS CORONARIA,
RHUS GLABRA, AND ACER SACCHARUM

By Charles K. Sando, Junior Chemist, Office of Plant Physiological and Fermentation
Investigations, Bureau of Plant Industry, United States Departjnent of Agriculture,
and H. H. BarTLETT, Collaborator, Office of Plant Physiological and Fermentation
Investigations, Bureau of Plant Industry, United States Department of Agriculture,
and Professor of Botany, University of Michigan

During the study of other compounds found in the plants in question,
we have incidentally isolated and identified the organic acids of the
fruits of the wild American crab apple {Pyrus coronaria L.) and the
smooth sumac {Rhus glabra L-.). We have also made an examination of
the product known as " maple sand " (found to be impure calcium malate)
which is formed as a granular deposit in the pans during the process of
boiling down sap of the sugar maple {Acer saccharum Marsh.) to make
maple sirup. Every precise record of the distribution of plant products
is distinctly worth while, and rather than hold our data on the acids of
these three plants for incidental mention in papers dealing with other
matters, we have thrown them together in the following notes.

OCCURRENCE OF MALIC ACID IN PYRUS CORONARIA, AND ITS
TRANSFORMATION INTO SUCCINIC ACID

As might have been predicted from the botanical relationship of Pyrus
coroiiaria to the common apple, the very sour fruit of the American crab
apple was found to contain malic acid. It was also found that in water
extracts of this fruit, made without heat, there is a transformation of
malic into succinic acid, apparently through the action of enzyms of
the fruit itself. This discovery will be of no little interest if further in-
vestigations substantiate our belief that microorganisms were not con-
cerned in the process.

Cold water extractions of crab apples collected near Ann Arbor, Mich.,
were made in the presence of both chloroform and toluol, with the
expectation of obtaining solutions of the fruit acids free from pectin
and other colloidal substances. The extractions were made in large
stone jars, tightly packed with sliced fruits and filled to the top with
water saturated with chloroform and toluol. At the bottom there was
an excess of chloroform and at the top an excess of toluol. The solution
quickly became intensely sour. The extraction was allowed to take
place for several weeks, at the end of which time the apple tissues were
as green and hard as when collected, showing no change whatever in
appearance. The infusion was brown at the surface layer, but clear and
of a pale straw color below. Only the non-oxidized lower part of the

Journal of Agricultural Research, Vol. XXJI, No. 4

Washington, D. C. Oct. 22, 1921

aad Key No. G-249

(221)

222 Journal of Agricultural Research voi. xxn,No.4

solution was drawn ofif through a tubulature at the bottom of the jar.
It was not until succinic acid instead of one of the usual fruit acids was
isolated from this solution that suspicion arose as to the possibility of
any fermentation, other than an autolytic one, having occurred, and it
was then too late to examine the solutions for microorganisms. It can
only be stated that there was no evidence that such were present. The
high acidity would have prevented bacterial action, and the perfectly
clear solution, saturated with chloroform and toluol, showed no evidence
of the presence of yeasts.

The infusion was neutralized by stirring with calcium carbonate.
During this process rapid darkening took place, with the formation,
presumably, of the same brown oxidation product that forms when a
cut surface of apple is exposed to the air. A dark precipitate separated,
which was not a salt of one of the fruit acids. It was filtered off. From
the filtrate it was possible to get relatively pure succinic acid by acidi-
fying with hydrochloric acid and shaking with ether, but the more econom-
ical and easy procedure, by which a larger yield was obtained, is
described below.

The neutralized filtrate was evaporated to a small volume and pre-
cipitated with several volumes of ethyl alcohol. An impure calcium
salt separated as a sticky, molasses-like mass. It was dissolved in dilute
hydrochloric acid, and the solution was concentrated until the succinic
acid crystallized out. It was purified by crystallization from lo per
cent nitric acid, and finally by repeated recrystallization from water.

The pure acid thus obtained agreed in all properties with succinic acid.
It melted at 184° to 185° C. (Rosenthaler (13) ^ gives 185° C). The
reaction mixture obtained by heating with concentrated sulphuric acid,
when diluted, boiled, and neutralized with ammonia, gave a red solution
with a strong green fluorescence, a characteristic reaction of succinic acid.

Combustions of the pure material dried at 110° C, resulted as follows:

(I) Weight of sample, 0.2378 gm. ; H.^O, 0.1047 g"!-! CO2, 0.3556 gm.

(II) Weight of sample, 0.3063 gm. ; HjO, 0.1356 gm. ; CO2, 0.4530 gm.
Calculated for C4H6O4; C, 40.66 per cent; H, 5.12 per cent.

Found: (I) C, 40.78 per cent; H, 4.94 per cent. (II) C, 40.33 per cent; H,
4.96 per cent.

Titration with sodium hydroxid (NaOH) gave the following results:

(I) 0.1776 gm. acid required 30.037 cc. NJio NaOH.

(II) 0.1453 g"^- ^cid required 24.424 cc. N/io NaOH.
Calculated for C4Hg04; replaceable H, 1.707 per cent.
Found: (I) 1.703 per cent; (II) 1.692 per cent.

A silver salt was prepared and analyzed, giving the following data:

(I) 0.4809 gm. salt gave 0.3119 gm. Ag.

(II) 0.4818 gm. salt gave 0.3126 gm. Ag.
Calculated for C4H404Ag2; Ag 65.02 per cent.
Found: (I) 64.86 per cent; (II) 64.88 per cent.

' Reference is made by number (italic) to "Literature cited," p. 22S.

Oct. 22, 1921 Acids of Pyrus coronaria, Rhus glabra, Etc. 223

It will be observed that if malic acid had been present in the cold water
extract, it would have been discarded with the mother liquor from the
first crop of succinic acid crystals, since malic acid is not only very deli-
quescent but likewise difficult to crystallize from solutions containing
sugars and other impurities. Some malic acid was doubtless lost at
this point, but the large yield of succinic acid indicated that it was the
chief acid derived from the fruits which had undergone autolysis.

It was of course a matter of interest to find out whether or not succinic
acid was present also in the living fruit. A new supply of crab apples was
therefore heated with water in an autoclave at 20 pounds pressure, and
the juice, after filtration through cloth, was evaporated to a small volume
and treated with several volumes of alcohol, to throw out pectin and
other colloids insoluble in alcohol. The alcohol was distilled from the
filtrate, which was concentrated, in vacuo, to a sirup. From this sirup it
was impossible to obtain even a trace of succinic acid, by either of the
methods which had been successfully used with the cold water extracts.
It contained, on the other hand, a large quantity of malic acid, identified
by the preparation and analysis of its silver salt. The concentrated sirup
mentioned above was diluted with water, which brought about a sepa-
ration of a small precipitate of red pigment, which was filtered off. When
lead acetate was added to the filtrate, the acidity of the solution was so
great that the first increment caused no precipitation of lead malate but
did throw out a small amount of dark precipitate, which was of course
removed. Further addition of lead acetate gave a voluminous precipi-
tate of lead malate (A), which was filtered off and washed. A second
yield of lead malate (B) was obtained from the solution by the addition
of alcohol. The two precipitates were separately decomposed with
hydrogen sulphid, neutralized with sodium hydroxid, and silver nitrate
solution was cautiously added. The first few drops of the silver nitrate
produced a dark precipitate which was removed by filtration. Further
addition of silver nitrate caused white silver malate to separate. The
precipitates were dried at 105 C. and analyzed as follows (two samples
each from A and B) :

(I) 0-5317 gin- salt from A gave 0.3315 gm. Ag.

(II) 0.7249 gm. salt from A gave 0.4518 gm. Ag.

(III) 0.3067 gm. salt from B gave 0.1882 gm. Ag.

(IV) 0.5374 gm. salt from B gave 0.3303 gm. Ag.

Foimd: (I) 62.34 per cent; (II) 62.32 per cent; (III) 61.36 per cent; (IV) 61.46
per cent.

Pure silver malate would have given 62.00 per cent silver. In view of
the fact that the acid itself was not purified before the silver salt was
formed, the analytical results are sufficiently close. Doubtless other
acids than malic are present in very small quantity in the crab apple.
The significant fact is that the fresh fruit contains malic acid as the pre-
dominant acid, and not enough succinic acid so that we were able to
isolate it.

224 Journal of Agricultural Research voi. xxn. no. 4

Although the old observations and experiments of Dessaignes (7) and
of Liebig ( //) showed that succinic acid was formed from malic acid when
calcium malate was present in mixtures being fermented by yeast, never-
theless succinic acid is generally found in plants in such small quantities
and always so intimately associated with asparagin that it is now cus-
tomary to look upon it as a degradation product of protein rather than
as directly related in metabolism to the other plant acids. The possi-
bility of amino compounds giving rise by enzym action in the plant to
succinic acid is sufl&ciently indicated by such discoveries as that of
Ehrlich (8), who has traced the production of succinic acid by yeast to
the fermentation of glutamic acid, and of Harden (jo), who has shown
that putrefactive bacteria {Bacillus coli communis), in the presence of
glucose, will transform aspartic acid almost quantitatively into succinic
acid. However, it must be stated that the whole subject of the place of
succinic acid in metabolism is much in need of investigation. It would
be a decided step forward to show that it is possible for malic acid to be
transformed directly into succinic acid by enzym action, as appears to
have taken place by autolysis in the crab apple.

We wish to indicate the possibility that such a transformation takes
place and to point out that green fruits containing malic acid afford ideal
material for a study of the problem. We do not wish, however, to give
the impression that the possibility of fermentation by microorganisms
was absolutely excluded in our work.

To anyone who may be inclined to take up the problem of acid trans-
formations in green fruits, a word of caution may not be amiss with regard
to the statements that have crept into general reference books such as
those of Czapek {6, p. 434) and Wehmer (14) with regard to the distri-
bution of succinic acid in plants. It is recorded from a number of unripe
fruits but has actually been isolated or satisfactorily identified in very
few cases. In 1876 Brunner and Brandenburg (2) isolated it from the
juice of uiu-ipe grapes (Viiis vinifera L.) . The source of most subsequent
reports is a paper published in 1886 by Brunner and Chuard (5). These
authors called attention to the earlier observation of Buignet (4) that
the juice of green fruits is capable of absorbing a large amount of iodin,
which enters into chemical combination with some constituent of the juice.
At the same time, a precipitate is formed, which Buignet erroneously
supposed to be the iodin compound. Brunner and Chuard, taking up
the problem at this point, showed that the iodin compound remained in
solution; and they obtained evidence which satisfied them that it was a
glucosid of monoiodosuccinic acid, derived from a naturally occurring
glucosid of succinic acid. Their investigation covered a considerable
number of green fruits and plant juices; and they actually isolated suc-
cinic acid, as such, from unripe gooseberries and from the petioles of
rhubarb. In the other instances it was merely inferred from analogy
that the supposed succinic acid glucosid was present. The procedure

Oct 23, 1921 Acids of Pyrus coronaria, Rhus glabra, Etc. 225

was to treat the juice with lead acetate, which supposedly threw out all
iodin-absorbing compounds except the succinic acid glucosid. Then the
presence of the latter, which was never isolated at all, was inferred from
two circumstances: (i) that the purified juice absorbed iodin, and (2)
that, after the absorption of iodin, a precipitate could be obtained with
basic lead acetate, supposed to be lead monoiodosuccinate, which when
treated with a mineral acid to liberate the free monoiodosuccinic acid,
and then with finely divided metallic silver, gave malic acid. The pro-
duction, under these circumstances, of malic instead of tartaric acid was
thought to indicate that iodosuccinic acid had been present rather than an
iodin derivative of the widely distributed malic acid.

The weakness of the whole argument is sufficiently obvious without
going into detail, since neither the putative glucosid of succinic acid nor
the iodosuccinic acid was isolated; and it was not shown that the basic
lead acetate precipitate was free from lead malate, which one would
naturally expect to be found there. To the physiologist who is interested
in the ripening of fruits it will be clear that the whole problem of the dis-
tribution and significance in metabolism of succinic acid is much in need
of more study. Especially, there can be no doubt that Buignet's iodin-
absorbing compound (4), whatever it may be, should be taken account
of in studies of fruit ripening. It exists in large amount in the unripe
fruit and disappears as ripening proceeds. As far as we are aware, it is
not even referred to in the recent literature of the subject.

ACIDS OF RHUS GLABRA

The acid of the sour, red pericarp of the sumacs (several species related
to Rhus glabra) has been variously reported by different investigators as
citric, malic, and tartaric. Gallic acid has likewise been reported.
The closely related species of true sumacs are doubtless alike as to acid
content. Our work, confined to R. glabra, has verified the findings of
Rogers {12) nearly a century ago, and Frankforter and Martin (9) that
the fruit acid is malic, nearly all in the form of the acid calcium salt.
We were also able to isolate free gallic acid, which seems not to have
been reported from this particular species. There are statements in the
older literature that free gallic acid occurs in the leaves of the European
sumac, R. coriaria L.

The berries of Rhus glabra were boiled with successive quantities of
distilled water. The water solutions were clarified and largely freed
from tannin by boiling with hide powder and o^gg albumen, and were then
shaken with ether. The combined ether extracts were evaporated to a
sirupy consistence and deposited gallic acid as a yellow powder. The
latter was filtered off on a Buchner funnel and crystallized repeatedly
from water. It was obtained in pure and almost colorless condition by
precipitation from solution in absolute alcohol by chloroform, or by
recrystallization from glacial acetic acid. As obtained by crystallization

226

Journal of Agricultural Research voi. xxn.No.4

from water it formed brown aggregates of large crystals containing one
molecule of water. (Calculated for CyHgOs. HjO, HjO, 9.57 per cent;
found, 9.39 per cent.) It was identified by the usual tests. Mr. N. A.
Lange made combustions of some of the purified acid and of its triacetyl
derivative, the results of which he kindly permits us to publish as follows :

I. The acid gave C, 50.19 per cent; H, 3.92 per cent. Calculated for gallic

acid: C, 49.40 per cent, H, 3.56 per cent.

II. The acetyl derivative gave C, 53.91 per cent; H, 4.13 per cent. Calculated

for triacetyl gallic acid, C, 54.39 per cent; H, 4.06 per cent.

The melting point of the triacetyl gallic acid, stated variously in the
literature from 151° to 165° and 166° C, was 162° to 163° C.

After the removal of tannin and gallic acid the aqueous extract from
the berries was largely neutralized with calcium carbonate and filtered
hot, after considerable concentration. Alcohol threw out a voluminous
precipitate, the first fractions taffy-like, later ones solid. These fractions
were treated with enough hydrochloric acid to form the acid calcium
salt, and were repeatedly treated with animal charcoal and recrystallized
from hot water.

The pure crystals were dissolved in water, exactly neutralized with
standard alkali; and normal silver malate was precipitated by the addi-
tion of silver nitrate. The four successive fractions of the crude calcium
salt were designated A, B, C, and D, and each was purified and converted
into the silver salt. In addition, a portion of fraction A was purified
by further recrystallization and was obtained in two portions called Aa
and Ab, from which silver salts were also prepared. The duplicate
analytical figures for all of the silver precipitates are given in Table I.

Table I. — Duplicate analyses of silver salts prepared from a series of precipitates obtained
by fractional separation with alcohol from an aqueous solution of calcium salts of the
organic acid of the sumac fruit

Fraction.

A-I...

A-II.

Aa-I.

Aa-II

Ab-I.

Ab-II

B-I...

B-II.

C-I...

C-II. .

C-III .

D-I. .

D-II.

Weight of
silver salt.

3.2895
•2133
•4385

^•1358
.6137
.9674
■4541
•6359
.4242

•5015
.3921

•4195
•5263

Weight of
silver.

0.1794
.1318
.2721
.7048

•3794
.5962
.2806

•3932
.2624

•3099
.2420
.2596
•3259

Percentage
of silver.

61.96
61.79
62.05
62.05
61.82
61.63
61.79
61.83
61.85
61.79
61.71
61.88
61.92

Oct 22. 1921 Acids of Pyrus coronaria, Rhus glabra, Etc. 227

The figures from all the fractions are in excellent agreement with each
other and agree fairly well with malic acid. The results prove beyond
much doubt that only one acid is present in any quantity. Rogers {12),
the first to show the presence of calcium malate in berries of Rhus glabra,
did not attempt to prove that malic acid was the only one present, and
subsequent work was less careful than his. Although convinced by the
identity of the silver salts that nothing but malic acid was present in our
material, we felt that the determinations should be closer to the calcu-
lated value. The average of 13 determinations makes the percentage of
silver in the silver salt 61.85, whereas the theoretical value is 62.00 for
pure malic acid. We, therefore, prepared silver malate, using a Kahl-
baum preparation of the acid, and made four silver determinations in the
same manner in which our other determinations were made. The four
determinations gave us 61.94 P^^ cent, 61.92 per cent, 61.81 per cent,
and 61.91 per cent, averaging 61.89 per cent silver in pure silver malate
by our method of preparation and analysis. There can, therefore,
remain no doubt that the acid of sumac berries is all malic.

MALIC ACID IN SUGAR-MAPLE SAP

It is no new observation that mafic acid is present in the sap of the
sugar maple. Cowles (5), for example, has published methods for the
estimation of malic acid in maple products. Although it might have
been anticipated that the granular precipitate known as "maple sand"
which is deposited in the pans during the concentration of the sap would
prove to be calcium malate, no one, as far as we know, has previously
reported an analysis. Bloor (j) used "sugar sand" as a source of acid
in his work on the transformation of malic acid into sugar by the tissue
of the maple ^ but gave no data to bear out the natural and perhaps
quite justifiable inference that the acid was actually malic. Our sam-
ple was kindly obtained for us from Ohio, by Dr. Clinton A. Ludwig,
now of Clemson College, S. C. It was only necessary to add to the
"maple sand " sufficient hydrochloric acid to transform the crude calcium
malate into the acid calcium salt. The latter was obtained pure by
repeated boiling with animal charcoal and recrystallization from hot
water. It was neutralized with alkali, and silver nitrate was added to
precipitate the insoluble silver malate. Three separate analyses for
silver gave the following results :

(i) 0.4129 gm. silver salt gave 0.2560 gm. Ag.

(11) 0.1922 gm. silver salt gave 0.1190 gm. Ag.

(ill) 0.2892 gm. silver salt gave 0.1796 gm. Ag.

Calculated for C4H405Ag2; Ag, 62.00 per cent.

Found: (i) 62.00 per cent; (11) 61.91 per cent; (iii) 62.10 per cent.

1 It may be noted that Bloor used tissues of "Acer saccarinum " for his work. Since he gives no authority
for the name, one is left in doubt as to whether he means the silver maple (A. saccharinum L.) or the
sugar maple (,A. saccharum Marsh.; A. saccharinum Wang., not I,.).

65583°— 21-^ — 4

2 28 Journal of Agricidtural Research voi. xxn, no. 4

SUMMARY

(i) The acid of the sour fruit of the wild American crab apple, Pyrus
coronaria, is malic acid. When the fruit undergoes autolysis under
anaerobic conditions, in the presence of chloroform and toluol, this acid
appears to be transformed largely into succinic acid. Further experi-
ments, however, will have to be made in order to repeat the observations
and to determine the exact process involved.

(2) The acid of the outer part of the red fruit of the smooth sumac,
Rhus glabra, is malic acid, occurring in the form of the acid calcium
salt. With it is associated a considerable quantity of free gallic acid.

(3) Malic acid is present in the form of calcium salts (both acid and
normal) in maple sap. The product known as "maple sand" obtained
from the evaporating pans is crude calcium malate.

LITERATURE CITED
(i) Bloor, W. R.

1912. STUDIES ON MALIC ACID. I. THE TRANSFORMATION OP MALIC ACID TO
SUGAR BY THE TISSUES OP THE MAPLE (aCER SACCHARINUm). In JoUt.

Amer. Chetn. Soc, v. 34, no. 4, p. 534-539-
(2) Brunner, Heinrich, and Brandenburg, Rudolph.

1876. UEBER DAS VORKOMMEN DER BERNSTEINSAURE IN UNREIFEN TRAUBEN.

In Ber. Deut. Chetn. Gesell., Jahrg. 9, p. 982-984.

is) ^^d Chuard, Ernest.

1886. PHYTOCHEMiscHE STUDiEN. 7» Bef. Deut. Chem. Gesell., Jahrg. 19,
p. 595-622.

(4) BlHGNET, H.

1861. RECHERCHES SUR LA MATI^RE SUCR^E CONTENUE DANS LES FRUITS
ACIDES; SON ORIGINS, SA NATURE ET SES TRANSFORMATIONS. In Ann.

Chim. et Phys., ser. 3, t. 61, p. 233-308.

(5) CowLES, H. W., Jr.

1908. THE DETERMINATION OP MALIC ACID IN FOOD PRODUCTS. In Jour. Amef.

Chem. Soc, v. 30, no. 8, p. 1285-1288.

(6) CzAPEK, Friedrich.

1905. BIOCHEMIE DER PPLANZEN. 2 vol. Jena.

(7) DessaignEs, v.

1849. NOTE SUR LA CONVERSION DU MALATE DE CHAUX EN ACIDE SUCaNIQUE.

In Ann. Chim. et Phys., s6r. 3, t. 25, p. 253-255.

(8) Erhlich, Felix.

1909. UBER DIE ENTSTEHUNG DER BERNSTEINSAURE BEI DER ALKOHOLISCHEN

garung. In Biochem. Ztschr., Bd. 18, Heft 3/5, p. 391-423.

(9) Frankporter, G. B., and Martin, A. W.

1904. A CHEMICAL STUDY OP THE SEED OP RHUS GLABRA. In Amer. JoUT.

Pharm., v. 76, no. 4, p. 151-158.

(10) Harden, Arthur.

19OI. THE CHEMICAL ACTION OF BACILLUS COLI COMMUNIS AND SIMILAR ORGAN-
ISMS ON CARBOHYDRATES AND ALLIED COMPOUNDS. In JoUt. Chem.

Soc. [London] Trans., v. 79, pt. i, p. 610-628.

(11) LiEBiG, Justus.

1849. UEBER DIE DARSTELLUNG DER BERNSTEINSAI7RE AUS APPELSAUREM
KALK. In Ann. Chem. u. Pharm., Bd. 70, Heft i, p. 104-107.

Oct. 2J.I92I Acids of Pyrus coronarta, Rhus glabra, Etc. 229

(12) Rogers, William B.

1835. ON THE EXISTENCE OP THE BI-MALATE OP LIME IN THE BERRIES OP THE
sumach; and the mode op procuring it prom them in the CRYSTAL-
LINE PORM. In Amer. Jour. Sci., v. 27, no. 2, p. 294-299.

(13) ROSENTHALER, L.

1914. DER NACHWEIS ORGANISCHER VERBINDUNGEN . . . I070 p., 3 fig., I pi.

Stuttgart. (Margosches, B. M. Die chemische Analyse, Bd. 19/20.)

(14) Wehmer, Carl.

191 1. DIE PFLANZENSTOFFE . . . xvi, 937 p. Jena. Literatur, p. x-xiv.

FERTILITY IN SHROPSHIRE SHEEP '

By Elmer Roberts
Illinois Agricultural Experiment Station

Heape (4) ^mentions that in some breeds young ewes bear fewer twins than
older ewes. Carlyle and McConnell {2) reported some observations which
they had made on the efifect of age on fertihty in sheep from which they
concluded that ewes from 3 to 6 years old averaged a larger percentage of
lambs than younger or older ewes, and also that i -year-old rams were not
so prolific as those 2 or 3 years old. The same conclusions were reached
by Humphrey and KJeinheinz (<5) from a study of later records of the
Wisconsin flock. Recently Jones and Rouse (7) showed that in sheep the
percentage of twins increased with age until 5 years, when there was a
decided drop.

The present paper gives the results of a study of the influence of age
and season upon fertility in American Shropshire sheep.

The source of data is the American Shropshire Sheep Record (j).
Individuals with registry numbers between 325502 and 344869 have
been used, date of birth noted, whether bom as single, twin, or triplet,
and age of dams and sires looked up.

AGE OF EWE AND FERTILITY

Table I shows the percentage of lambs born as singles, twins, and trip-
lets from dams of various ages. Ewes under i year and 6 months are
grouped in the i-year class, those i year and 7 months to 2 years and 6
months in the 2 -year class, and so on. The percentage in multiple births
increases to 4 years and remains fairly constant through 8 years. For
the older groups the numbers are too small to draw conclusions.

I Paper No. i6 from the Laboratory of Genetics, Agricultural Experiment Station, Urbana, III.
* Reference is made by number (italic) to "Literature cited," p. 234.

Journal of Agricultural Research, Vol. XXII, No. 4

Washington, D. C. Oct. 22, 192 1

aae Key No. Ill.-ii

(231)

232

Journal of Agricultural Research voi. xxn. N0.4

Table I. — Age of ewe and fertility

Age of dam in years.

Total
number of
offspring.

Percentage
of singles.

Percentage

of twins.

Percentage
of trip-
lets.

Percentage

in multiple

births.

379

2,299

2,025

1,762

1,256

942

506

405

157

23
3
4
I

4
5
I

77.0
66.4
63.6
57-6
58.0

53-7
56-3
54-8
62.4

38.5
60.9

100. 0
50.0

100. 0
75-0
20.0

100. 0

23.0
33-2
36.1
41.4
43 -o
46.0

43-1
44-5
37-0
61.5

39-1

23.0
33-6
364

42 .4

0.4

•3

I.O
I.O

•3
.6

•7
.6

44.0

46.3
43-7
45-2
37-6
61.5
391

I J

50.0

Z/i

25.0
80.0

20 *

9,868

60.8

38.7

39-2

" This may be a mistake in the record.

AGE OF RAM AND FERTILITY

Table II gives the percentages of lambs bom as singles, twins, and
triplets bom from sires of various ages. From these percentages one
can not ascribe to the ram any influence on fertility. Carlyle and
McConnell {2) thought that i -year-old rams were not so prolific as
older rams, but this is not borne out by the figures in Table II.

Table II. — Age of ram and fertility

Age of ram in years.

Total
number of
offspring.

Percentage
of singles.

Percentage
of twins.

Percentage
of triplets.

Percentage

in multiple

births.

1,101
3.265
2.552
1,460
650

434

244

118

58.7
60.6

59-1
65.8

55-5
66.1

70-5
74.6

63-4

68.1

100. 0

lOO.O

40.4
390
39-9
33-8
43-8
33-6
295
25-4
36.6

25-5

0.9

•5

I.O

.2
.6
.2

41-3
39-4
40.9
340
44-5
33-9
295
25.4
36.6

319

■I

4. ....

6^4'

9.947

61.2

38.2

38.8

TIME OF BIRTH AND TWINNING

Heape (5), who gathered information from flock masters, states that
55 per cent of them reported that twins were usually born early in the

Oct.

Fertility in Shropshire Sheep

233

lambing season. To test this point Table III was made, showing the
month of birth and the percentages of singles, twins, and triplets. It is
readily seen that a larger percentage of twins is bom early in the season
than is bom later. Of the 3,790 lambs bom in January, February, and
March 42.3 per cent are twins, while of the 4,617 born in April, May,
and June only 36.1 per cent are twins. If the triplets are added in with
the twins the percentages are 43.1 in multiple births for January,
February, and March, and 36.7 for April, May, and June. As Heape (5)
points out, this may be due to the ewes with the most vigorous and active
generative systems coming into heat earlier in the season. This may
be also affected by the fact that early in the breeding season more
green feed is available, a factor influencing the number of twins pro-
duced.

Table; lll.—Months of birth {Shropshires)

Month.

January. ..
February. .

March

April

May

June

August. .. .
September
December,

Total
number.

471

3,286

3.615
966

Percentage
of singles.

75-8
56.7
56.7
62.4
66.3

75-0
100. o
100. o

Percentage
of twins.

24.2

43-1
42.4

37-0
32.9
25.0

Percentage
of triplets.

In the hope that additional information might be obtained, a study
was made of the Dorset breed (j), which produces a large number of
young in the fall. Table IV gives the month of lambing and the percent-
ages of singles, twins, triplets, quadruplets, and of all multiple births.

Table lY.— Months of birth (Dorsets)

Month of birth.

January. .
February .

March

April

May

June

July

August. . . .
September
October. . .
November ,
December .

Total num-
ber.

Percentage
of singles.

1,818
2,386

3.919
2,366

857

296

102

925
1.546
1,088
1,418

16,634

61.5
54-3
52.7
51-7
';4.8

59-8
65.6
68.6

73-7
66.2
67.7
61.0

57-8

Percentage
of twins.

37-1
41.9

43-9

45-4
43-1
38.5
27.8

30-4
25-3
32.9
30-5
36.8

39-7

Percentage
of triplets.

1-3
3-8
3-2
2.6
2.1

1-7
6.6
i.o
•9
•9
1.8
2.2

2.4

Percentage
of quad-
ruplets.

Percentage

in multiple

births.

38.S
45-7
47-3
48.3
45-2
40.2

34-4
31-4
26.3
33-8
32.3
39-0

42.2

234 Journal of Agricultural Research voi.xxn.No.4

From Table IV it can be clearly seen that the percentage of multiple
births is greater in the spring. If the births occurring from February
to June, inclusive, are combined it is found that 48.2 per cent are in
multiple births, while for the other months the percentage is 34.9. In
Shropshires a larger percentage of twins or multiple births occurs in
January, February, and March than later. This condition does not seem
to hold for the Dorsets. Therefore, this condition in the Shropshires is
not likely due to more green feed early in the mating season. The
causes of these significant differences in multiple births at different sea-
sons among sheep are yet to be discovered.

SUMMARY

(i) Multiple births increase with age up to 4 years. From this point
they remain fairly constant until 8 years. Beyond this age the numbers
are too small to draw conclusions.

(2) The age of the ram has no influence on the percentage of multiple
births.

(3) Among Shropshire sheep more multiple births occur early in the
lambing season than later.

(4) Among Dorsets more multiple births occur in spring than in fall.

LITERATURE CITED
(i) American Shropshire Registry Association.

1912. AMERICAN SHROPSHIRE SHEEP RECORD. V. 25.

(2) CARtYLE, W. L., and McConnell, T. F.

1902. SOME OBSERVATIONS ON SHEEP BREEDING FROM THE EXPERIMENT STA-
TION FLOCK RECORDS. Wis. Agr. Exp. Sta. Bul. 95, 19 p.

(3) Continental Dorset Club.

1900-17. continental dorset club sheep record. v. i-16.

(4) Heape, Walter.

1899. abortion, barrenness, AND FERTILITY IN SHEEP. In JouT. Roy. Agr.
Soc. England, v. [60] (ser. 3, v. 10), no. 38, p. 217-248.

(5)

1900. note on the fertility of DIFFERENT BREEDS OF SHEEP, WITH RE-
MARKS ON THE PREVALENCE OF ABORTION AND BARRENNESS THEREIN,

In Proc. Roy. Soc. [London], v. 65, 1899, p. 99-111.

(6) Humphrey, George C, and Kleinheinz, Frank.

1907. OBSERVATIONS ON SHEEP BREEDING FROM RECORDS OF THE UNIVER-
SITY FLOCK. In Wis. Agr. Exp. Sta. 24tli Ann. Rpt. 1906-07, p. 25-40,

(7) Jones, Sarah V. H., and RousE, James E.

1920. THE RELATION OF AGE OF DAM TO OBSERVED FECUNDITY IN DOMESTI-
CATED ANIMALS. I. MULTIPLE BIRTHS IN CATTLE AND SHEEP. /«

Jour. Dairy Sci., v. 3, no. 4, p. 260-290. References, p. 288-290.

Vol. XXII OOXOBE)R 2^, 1921 No. 5

JOURNAL OF

AGRICUI/TURAlv

RESEARCH

CONTKNXS

Page

Relation of Soil Temperature and Other Factors to Onion
Smut Infection - ^ - - - - - - 235

J. C. WALKER and L. R. JONES

(Contribution from Bureau of Plant Industry and Wisconsin Agricultural Experiment Station)

A Physiological Study of Grapefruit Ripening and Storage 263

LON A. HAWKINS

(Contribution (torn Bureau of Plant Industry)

Absorption of Copper from the Soil by Potato Plants - 281

F. C. COOK

(Contribution from Bureau of Chemistry)

PUBUSHED BY AUTHOMTY OF THE SECRETARY OF AGRICULTURE,

WITH THE COOPERATION OF THE ASSOCIATION OF

LAND-GRANT COLLEGES

w:a.shingxon, d, c.

EDITORIAL COMMITTEE OF THE

UNITED STATES DEPARTMENT OF AGRICULTURE AND

THE ASSOCIATION OF LAND-GRANT COLLEGES

FOR THE DEPARTMENT

KARL F. KELLERMAN, Chairman

Physiologist and Associate Chief, Bureau
of Plant Industry

EDWIN W. ALLEN

Chief, Office of Experiment Stations

CHARLES L. MARLATT

Entomologist and Assistant Chief, Bureau
of Entomology

FOR THE ASSOCIATION

J. G. LIPMAN

Dean, State College of Agriculture', and
Director, New Jersey Agricultural Expert-
tnent Station, Rutgers College

W. A. RILEY

Entomologist and Chief, Division of Ento-
mology and Economic Zoology, Agricul-
tural Experiment Station of the University

of Minnesota

R. L. WATTS

Dean, School of Agriculture; and Dtrtctor;
Agricultural Experiment Station; The
Pennsylvania State College

All correspondence regarding articles from the Department of Agriculture should be
addressed to Karl F. Kellerman, Journal of Agricultural Research, Washington, D. C.

All correspondence regarding articles from State Experiment Stations should be
addressed to J. G. Lipman, New Jersey Agricultural Experiment Station, New
Brunswick, N. J.

JOIfflALOFAGltianmffiSEARCe

c — %..JS* "I at."— f"i<M~i- — ■ -

Vol. XXII Washington, D. C, October 29, i923e^ew YtpNo. 5

RELATION OF SOIL TEMPERATURE AND OTHER FAC-
TORS TO ONION SMUT INFECTION

By J. C. Walker, Pathologist, Office of Cotton, Truck, and Forage Crop Disease Inves-
tigations, Bureau of Plant Industry, United States Department of Agriculture, and
Assistant Professor of Plant Pathology, University of Wisconsin, and L. R. JONES,
Professor of Plant Pathology, University of Wisconsin

OCCURRENCE OF ONION SMUT IN RELATION TO CLIMATE AND
CULTURAL PRACTICES

The onion smut fungus, Urocystis cepulae Frost, was first reported by
Ware {11) ^ in the Connecticut River Valley in 1869. At this early date
it was causing some injury to the onion crop, and in 1888 it was reported
by Thaxter (jo) to be of much importance in the old onion soils of south-
em New England. During the years which have since elapsed it has
successively appeared and become an economic factor in nearly all the
more westerly regions of intensive onion culture of the northern States,
from New York to Oregon. It is possible that this fairly rapid distri-
bution of the parasite has been occasioned to some extent by smut spores
carried with the seed, as already noted by Chapman {2) and Munn (7,
p. 412). It has, however, more probably been brought about by the
increasingly widespread distribution of onion sets. Many of these sets
are grown in the northern States on smut-infested soils, and since they
are shipped in quantity to all parts of this country, and even exported,
their role in the wide dissemination of smut spores is obvious.

Chance introduction of the smut fungus in this way in the northern
commercial onion-growing sections seems almost certain to lead to its
permanent establishment. This evidently results from the fact that the
common intensive practice of continuous cropping with onions for an
indefinite term of years tends, when once the inoculum is introduced in
the soil, to favor its increase and wider distribution season by season until
it becomes a factor limiting further success with this crop. While this
holds true for the northern States, it does not seem to be so in the
southern sections. This is the more noteworthy since northern sets
grown on smutty soil are each year shipped into the southern onion
districts for propagation. This regional limitation of the smut fungus
was impressed upon one of the authors (Walker) in connection with a

1 Reference is made by number (italic) to *'I,iterature dted," p. 261,

Journal of Agricultural Research, Vol. XXII, No. s

Washington, D. C. , Oct. 29, 1921

aaf Key No. 0-250

236 Journal of Agricultural Research voi. xxii. no. s

survey which he made some two years ago of the chief onion-growing
centers of the entire country, as a representative of the Office of Cotton,
Truck, and Forage Crop Disease Investigations of the United States
Department of Agriculture. In connection with this, he personally
inspected the leading onion-growing sections of Texas and Louisiana
and conferred with the plant pathologists of these two States, Drs. C. W.
Edgerton and J. J. Taubenhaus. No evidence of the disease was found,
and it had not been reported to the Experiment Station of either State.^

In comparing the distribution and occurrence of onion smut in differ-
ent sections of the country, it is necessary to keep in mind that two
distinct types of onion culture are practiced in the United States. The
first is followed in practically all of the northern sections, the second is
the rule in the southern commercial growing regions, and in one or more
sections in the Pacific coast States. In the first, or northern, type the
seed is sown directly in the field as early in the spring as the soil can be
properly prepared — that is, in March, April, or May, according to local
climatic conditions. The bulk of the crop is harvested in these northern
districts in August and September. The Globe varieties predominate,
including the red, yellow, and some white. In the second, or southern,
type of culture the seed is sown in special beds in late summer. The
seedlings are tlien transplanted to the main field during the early winter
months and the crop is harvested during the period from April to July.
Here the Bermuda, Italian, and Spanish varieties predominate. The
survey previously referred to brought out the fact that onion smut has
become established in essentially all of the older onion-growing sections,
which practice the first type — with spring sowing of seed — while smut
is either entirely unknown or of no economic importance in those locali-
ties where the seed is sown in summer followed by transplantation.
Wherein lies the explanation? As already suggested, it can not be due
to the matter of chance introduction of the organism. This is certainly
being distributed frequently and widely throughout the South. It would
seem rather to be associated with some of the factors incident to the
southern type of culture. The conspicuously different factors as out-
lined above are three: (i) The varieties used, (2) transplantation vs.
direct seeding, (3) climatic differences associated with season of culture.

Greenhouse experiments, in which we have tested the different types,
have shown that the Bermuda and Spanish varieties which are used in
the South are as susceptible to smut infection as are the Globe varieties
of the northern sections. Hence varietal resistance does not furnish the
explanation. Turning to cultural methods, we find that in the South the
seed beds in which the onions are grown preliminary to transplantation
are usually of considerable size and are located as a rule in a portion of

1 The authors are indebted to Doctors Edgerton and Taubenhaus for continued cooperation in the search
for the smut in their respective States. They each reported again early in the current year that not a
single specimen had as yet been found.

Oct. 29, 1921 Relation of Soil Temperature to Onion Smut 237

the main field. Therefore, if the organism were present and environing
factors were favorable, it does not seem probable that this method of
culture would completely inhibit the disease. Indeed, judging from our
experience with cabbage transplantation in relation to clubroot and
other soil- or seed-borne diseases, this method, instead of reducing the
trouble, is likely to serve as a ready means of distributing the parasites
with diseased seedlings from localized centers to wider areas. We are
thus forced to turn for explanation of the absence of smut in the South to
the third suggestion, that relating to climatic differences, bearing in mind
the respective cultural seasons. The most evident environmental dif-
ferences associated with the two types of culture relate to soil tempera-
ture and moisture during the time of seed germination and early seedling
development, which constitute the smut infection period. In the north-
em type, the spring-sown seed develops in a soil which is comparatively
cool and which has in general a relatively high and constant surface
moisture content. In the southern type, the summer-sown seed must
germinate and pass the early developmental stages in a soil of relatively
high temperature and subject to superficial desiccation. Our problem
has, therefore, necessitated an attempt to analyze and evaluate the pos-
sible factors associated with variations in soil moisture and soil tempera-
ture during the seedling stage.

INFECTION PERIOD

It has been of obvious importance in this study to know quite defi-
nitely the period in the development of the host at which infection actu-
ally occurs. Thaxter {10) gave critical attention to the time and manner
of infection, concluding that the fungus always invaded the young seed-
lings below the surface of the soil and that, by subsequent growth of the
host, the infected cells were commonly carried above the ground before
visible signs of the disease appeared. He also noted that onion sets and
onion bulbs replanted for seed growing were not attacked and suggested
that the seedling was probably subject to attack in only the early stages
of its development. Sturgis (9) later found that seedlings half as thick
as a lead pencil and about 5 inches high, transplanted into smutty soil
did not contract the disease. Sirrine and Stewart {8) , in an experiment
started at Jamaica, N. Y., on May 2, sowed eight rows of onion seed,
each ID feet in length, in soil free from smut. Alternate rows were left
as controls. Soil from a smut-infected field was introduced in three
ways: (i) in the furrow with the seed in two rows; (2) on the surface of
the soil after the furrow was closed in one row; and (3) in a fourth row,
on the surface of the soil 1 1 days after planting, this being shortly after
the seedlings appeared above ground. The control rows remained
healthy. Heavy infection occurred with the first treatment, slight
infection with the second, no infection with the third. It seems possible

238

Journal of Agrictdtural Research voi. xxii, no.

that in the third treatment, where the inoculum was merely placed on
the surface of the soil when the seedlings were well started, the method
failed to insure a sufficiently intimate and immediate contact of germi-
nating spores with embryonic tissue to justify definite conclusions.
Reviewing the evidence as a whole, however, it is obvious that the
smut fungus is capable of invading the onion seedling for only a short
period after seed germination.

In order to define more exactly the limits of this period of smut infec-
tion, we carried through a series of greenhouse trials. In the first of
these 17 pots of sterilized greenhouse soil were planted with Red Globe
onion seed which had been treated with i to 25 formaldehyde solution
for 15 minutes. At two-day intervals beginning the eleventh day after
planting, two pots were inoculated by mixing smut-infested soil in the
upper layers of the pot, so that the inoculum was brought into close con-
tact with the embryonic region of the cotyledon. At the time of inocu-
lation, all retarded seedlings were removed, so that only plants of uniform
height were considered in each case. All plants were pulled and exam-
ined for signs of the disease three to four weeks after inoculation. The
results of this experiment, given in Table I, show that, under greenhouse
conditions at least, infection may occur until the cotyledon is about 2 }4
inches above ground, or for a period of two weeks or more after sowing.
Thus, the infection period appears to be slightly longer than that re-
ported by Sirrine and Stewart (8) .

Table I. — Relation of the stage of development of the onion seedling to infection by Uro-

cystis cepulae

Soil treatment.

Pot No.

Length of
period

between
sowing and
inoculation.

Height of

cotyledons

above

ground at
inoculation.

Number of
plants.

Percentage
infected.

I
2

Days.
II
II

Inches.

i-iK

28
18

89
67

iK-2

lK-2

Inoculated

5
6

lK-2

iK-2

17
16

7
8

17
17

2 -2>^
2 -2K

19
20

2K-3

f ^^

37
22
21
18

13
14
15
16

Uninoculated

25
45

i 17

Oct. 29, 1921 Relation of Soil Temperature to Onion Smut 239

The foregoing experiment was repeated in a somewhat cooler house,
in which the temperature remained close to 15° C. most of the time,
rising to about 20° during the middle of the day. Under these conditions,
the maximum length of the cotyledon was about 2)4 inches. The data
from this experiment are reported in Table II. The plants became im-
mune at approximately the same time as noted in the first experiment —
between the nineteenth and twenty-fourth days after sowing, when the
cotyledon had about attained its full growth and as the first leaf was
emerging. It will be recalled that the basal portion of the cotyledon, as
with each of the later leaves, forms a collar or sheath inclosing the lower
parts of the younger leaves. The question arose as to whether or not
immunity to smut infection is directly associated with maturity of the
tissues. If so, it would seem that the explanation of this later escape
of the onion plant from infection lies in the fact that the maturing basal
sheath forms a thin but normally complete barrier of resistant tissue
between the potentially infective soil and the deeper-lying embryonic
tissues of the younger developing leaves. The removal of this mechan-
ical barrier might, therefore, permit of infection at a later stage. In
order to determine whether this is the case, the following experiments were
undertaken. After the thirty-first day, when the onion seedlings had
passed the so-called susceptible period, the cotyledons were carefully
removed from the plants in one pot, and infected soil was placed around
the base of the exposed first leaf. Sixty per cent of the plants thus
treated became infected as shown in Table II, pot 9. This proves that
the first leaves are susceptible even after the cotyledon becomes immune.
On the fortieth day, a i-inch layer of infested soil was placed on top of
pots 10 and 1 1 , so as to surround the first leaves in proximity to the axils.
Pot 10 was left at the same temperature (15° to 20°) for 24 days and pot
II was removed to a temperature of 25° to 28° for the same period.
About 5 per cent of the plants in pot 10 showed infection of the first leaf
as compared with 28 per cent in pot 11. The reason for the increased
percentage of infection at the higher temperature has not been satisfac-
torily explained. It may simply have been consequent upon the stimu-
lated growth of the onion foliage. However this may be, it is evident
that the basal portion of the first leaf remains susceptible to infection
for a short time, at least, after it emerges from the cotyledon.

From a summary of these results it appears that our own experimental
data regarding the duration of tlie period of infection agree in the main
with those of previous investigators. The conclusion seems justified
that disease resistance is correlated with tissue maturity, and that the
onion cotyledon becomes immune to smut infection at about the stage
when growth ceases. The rate and nature of growth of the cotyledon
will naturally vary with environmental conditions; hence variation in
the actual length of the infection period is to be expected. The mature

240

Journal of Agricultural Research voi.xxii.no.s

basal sheath of the cotyledon thus protects the embryonic region of the
younger leaves from infection. That portion of the first leaf which
emerges from the cotyledon is susceptible to infection for some little
time after emergence, but since it ordinarily is not actually in contact
with infested soil, this fact is probably not of practical significance.

Table II.

-Relation of stage of development of the option seedling to infection by Uro-
cystis cepulae

Inter-

val be-

Pot

No.

Method of exposure to
inoculation.

val be-
tween
sowing
and in-
ocula-
tion.

Length of
cotyledon
at time of
inocula-
tion.

Condition of
first leaf at
time of inocu-
lation.

tween
inocu-
lation
and
final
exami-
nation.

Total
number

of
plants.

Per-
cent-
age
smut-
ted.

Days.

Inches.

Days.

1-3

Uninoculated

lOO-f
24

Infected soil around base

Not out

of cotyledon.

5
6

2 to 2X

2 to 2X
2 to 2%

24
24
24
24

34
41
19
36

35
8

24
31

... do

7
8

Just out. .. .
K to iK
inches

above axis.

Cotyledons removed and
infected soil placed
around base of first leaf.

2 to 2^

. ..do

One-inch layer of infected
soil placed on surface of
old soil so as to cover
lower inch of aerial por-
tions of plants.

2 to 2K

yi to 4 inch-
es above
axis.

Same as No. 10, except that
pot was transferred to a
temperature of 25° to
28° C.

2 to 2>2

...do

RELATION OF SOIL MOISTURE TO INFECTION

Since there are these well-defined limits to the time of smut infection,
the possibility becomes clearly evident that variable environmental
factors during this limited period may exercise a controlling influence on
the occurrence of the disease. As already stated, the problem seems to
resolve itself primarily into the question of the relations of soil tempera-
ture and soil moisture to infection. The results of several workers
upon the grain smuts, as summarized by Jones (5), have shown that soil
temperature may influence infection. Hungerford and Wade (4) have
published evidence that high soil moisture, also, may favor infection of
wheat by the smut fimgus Tilleiia tritici (Beij.) Wint. Variations in
the moisture content of the surface layers of soil are likely to be wide,
especially during the late summer planting season in the southern States
when high temperatures and low humidity may cause rapid desiccation.

Oct. 29, 1921 Relation of Soil Temperature to Onion Smut 241

An experiment was therefore carried out in which onion seedlings
were grown in smut-infested soil in pots which were held at different
degrees of soil moisture. Galvanized iron pots 5 inches in diameter and
4 inches deep were used for these trials. Greenhouse sandy loam soil
was used and its water-holding capacity was determined in advance by
the two standard methods recommended by soil physicists ^ — that is,
by means of the lo-inch cylinder and the i-cm. cup. The soils were
brought to the desired low and medium water contents before they were
placed in the culture pots ; and in those cases where the desired moisture
approached the water-retaining capacity, the water content was finally
adjusted after the soil was potted. Although these methods failed to
secure exact uniformity in the physical compactness of the soils in the
several series, they were considered satisfactory as to initial moisture
conditions. The pots were weighed daily during the progress of the
experiment, and water was added to replace the losses. Since it was
realized that the surface layer of soil would change in moisture content
through evaporation more rapidly than the lower layers, an effort was
made to reduce this surface evaporation so far as practicable. To this
end, tar paper covers were used until the seedlings came above ground,
when glass covers were substituted for a few days, and finally mineral
wool was packed on the surface about the seedlings to reduce evaporation.
Absolutely uniform moisture throughout the pot could not be maintained
even by this method, and the upper layers of soil unavoidably assumed
a somewhat lower water content than the average for the pot. There-
fore, at the end of the experiment, moisture determinations were made
of the upper inch of soil, since this was the important part from the
standpoint of smut infection.

The soil was inoculated at the outset by the introduction of spores
from diseased leaves and scales. The data from this experiment are
given in Table III. Good germination took place within the range of
10 to 15 per cent moisture content (45 to 70 per cent of the moisture-
holding capacity). A high percentage of infection also occurred within
this range. Above 15 per cent there was some decrease in germination
together with a gradual reduction in infection. At the extreme, how-
ever, where germination of seed was practically eliminated (pot i), one
of the two plants surviving became infected. It is evident from these
data that a good percentage of infection may be expected at a soil mois-
ture content up to the limit where good germination and growth of the
host plant occur. There was a gradual reduction of infection below 5
per cent (23 per cent of the moisture-holding capacity), but this was not
sufficient to insure good germination and support good growth. It
may be concluded, therefore, that soil moisture does not function as a
factor limiting infection with onion smut within the limits at either
extreme where good germination and growth, of the host occur.

1 We are especially indebted to Prof. E. Truog, of the Department of Soils of the University of Wiscon-
sin for advice in connection with this work.

242

journal of Agricultural Research voi. xxii, ^fo. s

Table III. — Relation of soil -moisture to infection by Urocystis cepulae

Pot No.

2.
3-
4-
S-
6.

7-
8.

9-
10
II
12

13
14

Original
moisture
content

(percentage
of dry

weight)."

Moisture
content of
surface lay-
er at end of
experiment.

19.6
17. 1
16.3
18.2
20.0
15.0
15.0
10. o
10. o
10.7

5-0
8. I

7-5
6.6

27. 1
24.7
23-7
23-4
14.0
12.4
II. 6

9-3
9.0
2.4

2-5

1.4
3-4
1-3

Number of

seeds

planted.

100
100
100
100
100
100
100
100
100
100
100
100
100
100

Total num-
ber of
plants.

52
48
50
60
66
66

71
66

32
66

Percentage
of plants
smutted.

71
90

85
94
59
50
19
35

« Water-holding capacity, as determined by lo-inch cylinders, was 22.3 per cent; as determined by the
i-cm. cup, it was 27.8 per cent.
The calculated wilting coefficient of the plants was 2.3 per cent.

TEMPERATURE RELATIONS

In calling attention to the importance of soil temperature as a factor
in the development of certain plant diseases, Jones (5) points out that
several investigators have stressed its bearing upon infection in the case
of stinking smut of wheat, Tilletia triiici (Beij.) Wint., and of the oat
smuts, Ustilago avenae (Pers.) Jens, and Ustilago Levis (K. and S.) Mag.
Heald and Woolman (j) showed that the amount of infection with the
stinking smut of wheat was reduced as the mean soil temperature rose
above 65° F. (18.3° C.) or fell below 40° F. (4.4° C). Humphrey (4)
states, for the same disease, that soil temperatues of 0° to 5° C. and
above 22° C. are decidedly unfavorable to infection.

In studying the relations of soil temperature to the developemnt of a
parasitic disease, consideration must be given to the possible influence
of such temperature upon the host and the parasite independently. This
may enable one to analyze with more confidence the effects when host
and parasite are subjected simultaneously to the experimental condition.
This has been done as far as practicable in connection with the present
work.

Unfortunately, germination of the fungus spores under artificial con-
ditions has been so scanty that the effect of temperature upon the fungus
has been necessarily limited to inoculation experiments with infested
soil. However, the disease is produced so readily and consistently by
the latter method that a very accurate index to the development of the
fungus can be secured by varying the condition of the infested soil dur-
ing the germination and early growth of the onion seedling.

The soil-temperature experiments were all carried out in the green-
house at Madison, Wis., during the winter months. The apparatus in use

Oct 29, 1921 Relation of Soil Temperature to Onion Smut 243

in the Department of Plant Pathology, University of Wisconsin, for the
control of soil temperatures has been described by Jones (5) . Briefly, it
consists of a series of water baths held at constant or nearly constant
temperatures in which the glass or galvanized-iron culture pots are
inserted.! s ^^-f?-' .'i.suuu-..

For these experiments galvanized-iron cylindrical pots 5 inches in di-
ameter and 8 inches in depth were used. In order to overcome the in-
fluence of the air temperature upon the upper layer of soil, the surface
of the latter was kept at >2 to i inch below the level of the water. Tar-
paper covers were placed over the pots until the seedlings came above
ground; these covers were then removed, and a layer of mineral wool
was placed on the surface of the soil. By this procedure the tempera-
ture of the upper inch of soil was kept reasonably close to that of the
deeper portions — that is, approximately that of the water in the tank.
In order to follow any minor variations, readings were taken three times
daily from thermometers inserted i inch below the surface of the soil.
At the beginning of the experiments the moisture content of the soil was
adjusted to two- thirds of the water-holding capacity. The pots were
thereafter weighed at intervals of one to three days, depending upon the
rate of water loss, and the moisture content was readjusted accordingly,
either by adding water directly to the surface or by introducing it at the
bottom of the pot through a glass tube. Obviously this method did not
secure uniform distribution of moisture throughout the pot, and una-
voidably the content of the upper layer of soil was somewhat lower than
the average for the whole pot. It is believed that this variation had little
if any influence, however, since other experiments, described earlier in
this paper, showed that infection is quite uniform over a much wider
range of soil moisture than here occurred. The seed was planted at a
depth of I inch. Since the chlorophyll in the tops in some cases ob-
scured the smut lesions, the plants were placed in alcohol acidified with
acetic acid until thoroughly bleached before final examination for the
disease was made.

EFFECT OF SOIIv TEMPERATURE UPON THE DEVEI.OPMENT OF THE HOST

Experimental studies to determine the relation of soil temperature to
the rate and character of seed germination and seedling development
were carried on in conjunction with those relating to infection, of which
the results will be presented in the next section. It will be simpler,
however, to discuss these two aspects of the problem separately, taking
up first the relations of temperature to host development.

Experiment I. — Seven pots of sterilized greenhouse loam soil were
uniformly planted with 50 Red Globe onion seeds in each pot. One pot
was then held at each of the following temperatures: 10° to 14°, 16.5°

1 since this description was published, numerous improvements have been made from time to time by
members of the Department. As now in use these are termed the "Wisconsin soil temperature tanks."

244

Journal of Agricultural Research

Vol. XXII, No. s

to 1 8°, 19° to 22°, 24° to 26°, 27° to 29°, 30° to 31°, 35° C. The moisture
content of the soil was held at two- thirds the water-holding capacity (22
per cent of dry weight). The air temperature of the greenhouse was
kept at about 15° with a rise to 20° during the middle of the day. These
conditions as to soil moisture and air temperature were such as had
proved favorable for both host and parasite development in the earlier
trials. The first seedlings to appear above ground were those at 27°
to 29°, those at 24° to 26° came up shortly afterward, then those at
19° to 22°. Good growth took place at these three temperatures, but
germination was very slow at lower temperatures. At the highest tem-
perature, 35°, a few seeds germinated, but growth was very slight. The
plants were all removed and the roots washed out on January 7,
1920, 29 days after the seed was sown. The data given in Table IV
summarize the condition of tlie plants at this date. It will be seen that
at this early stage in the development of the plants there was a tendency
for best root development at about 21° or below, while the best develop-
ment of tops took place at this point or above.

Table IV. — Development of onion seedlings in sterilized greenhotise soil held at 22 per
cent oj the dry weight or two-thirds the moisture-holding capacity, and at different soil
temperatures. Data on January 7, IQ20, 2Q days after sowing

Soil temperature.

"C.
10 to 14 . . .
16.5 to 18. .
19 to 22 . . .
24 to 26. . .
27 to 29 . . .
30 to 31. . .

Num-

Total

Per-

Per-
centage
with
first
leaf.

Average

ber of

num-

centage

dry weight

seeds

ber of

with 2

with 3

of tops per

of roots per

planted.

plants.

roots.

plant.

Gm.

0.00224

0 . 00046

.00293

. 00023

100

.00396

. 00043

•00307

.00028

0 14

.00285

.00014

.00212

Average

total dry

weight per

plan I. .

Gm.

0.0027
.0031
.0044
•0033
.0030
.002 1

» The reduced stand at 27° to 29° and 30° to 31° C. was due to damping-off fungi.

Experiment II. — ^The experiment was repeated, with some modifica-
tions, starting April lo, 1920. The Red Globe and Yellow Bermuda
varieties were used. Two pots of each variety were kept at each of the
following soil temperatures: 14°, 20°, 25°, 28°, 30° C. The air temper-
ature ran sHghtly higher (20° to 30°) during the middle of the day and
dropped to about 15° for the most of the night. Both the rate and the
percentage of germination were noted, and the data are recorded in
Table V. In both varieties the most rapid germination took place at 25°,
although the rate was only slightly less at 20°, 28°, and 30°. At 14° the
seedlings were distinctly slower in starting ofif. The plants from one pot
of each series were removed on the thirteenth day. The dry weights of
the tops and roots as given in Table V were so small at this age that com-
parison on this basis does not have any great value. The tendency for
rapid development of tops as compared with roots at 20° or above is,
however, shown very strikingly in Plate 25. The plants in the remaining

Oct. 29, 1921 Relation of Soil Temperature to Onion Smut

245

pots were removed on the thirtieth day. The relative dry weights of
roots and tops then secured are shown in Table V and those of the Red
Globe are graphed in figure i.

While the temperature relations of the two varieties were alike in their
main features, there was an interesting minor difference, possibly indica-
tive of the better adaptation of the Red Globe for northern culture and of
the Yellow Bermuda for southern. In both cases with these onions, as
indeed holds generally in our experiments with other plants, the best

.0020

.008

.00^4

\
I

\.ooz

.000

^a^zr

^r^o^^,^.

'^''■?'^rM>s\

.00/0 "1

.000s \

Z'^" 20'' 2S'=' 23*^

30''

.pooo

Fig. I. — Relative developments of dry weight in tops and in roots of Red Globe onion as shown at end of
30 days' growth in a series of culture pots kept at the several soil temperatures indicated, with all other
factors, including air temperature, alike for all. Note that the best root development occurs at the low
temperatures (12° to 15° C.) whereas the tops are forced more strongly at higher temperatures (20° to 25°).

root development occurs at relatively lower temperatures (12° to 20° C.)
while best top development occurs at higher temperatures (20° to 25°).
When the varieties are compared, it is seen that with the Globe both
roots and tops grew relatively better at somewhat lower temperatures
than did those of the Bermuda. Thus the data at 30 days show the
maximum root development of the Globe at 14° while that of the Ber-
muda was at 20° ; with tops the maximum was at 20° for the Globe and at
25° for the Bermuda.

246 Journal of Agricultural Research Voi.xxn. no. 5

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Oct 29, 1921 Relation of Soil Temperature to Onion Smut 247

EFFECT OF soil. TEMPERATURE UPON INFECTION

At present the chief interest in these data focuses upon the question of
any possible bearing of the rate of development of the host plant at
different temperattires upon predisposition to, or escape from, smut
infection, recalling that such infection is practically limited to the seed-
Hng stage before the maturity of the cotyledon. It has just been noted
that the promptest seed germination and most rapid growth of tops
during this early seedling stage occur at fairly high temperature, 20° to
25° C, with a rather pronounced drop in rate of aerial growth at tempera-
tures below 20°. It is to be expected, therefore, that in the northern
onion-growing sections where the seed is planted in early spring there
will be a rather tardy germination and slow early development of tops,
the growth energies during the seedling stage being directed under this
climatic environment to a relatively stronger development of the root
system. In the South where the seed is planted in the comparatively
warm period of early autumn, we should expect a more rapid top growth
at the outset, with correlated strength of root development coming later
in the autumn as the soil becomes gradually cooler.

The naturally infested soil was secured near Racine, Wis., from a badly
diseased field of sandy loam rich in organic matter. The soil which was
artifically inoculated consisted of a greenhouse mixture of loam and sand
to which were added fresh spores from smutty onion leaves. In order to
test the efficacy of this method of soil inoculation a preliminary planting
of onion seed was made in advance of the final experiments. This gave
a high percentage of smut infection, showing that the method of introduc-
ing the inoculum was satisfactory. Several early trials indicated that
below 25° C. soil temperature variations have little effect on the relative
amount of infection. The results of two such experiments, nearly cover-
ing the range of onion seed germination, are given in Table VI (experi-
ments I and 2). It is evident from these figures that abundant infection
occurred between 10° and 25°, both with naturally infested and with
artificially inoculated soil. The number of pustules per plant as shown
in Plate 25 proves that the fungus was very active even at low tempera-
tures. Above 25° infection is reduced very rapidly, as indicated by
both the percentage of infected plants and the number of pustules per

plant.

In order to determine more closely the point at which infection is
inhibited four more serial experiments were conducted (experiments 3
to 6) in which the temperature was kept as constant as possible at 2-
degree intervals between 25° and 31° C. The results given in Tables
VII and VIII, and illustrated in Plates 26 and 27, show that abundant
infection took place at 25° to 26°, while it was greatly reduced at 27° to
28° and completely inhibited at 29° or above. The infected seedlings
from experiment 5 show the great reduction in the amount of disease per

248

Journal of Agricultural Research vo1.xxii,no.s

plant at 27° to 28° as compared with 25° to 26°. It is interesting to note
that infection was reduced more abruptly at 27° to 28° in the artificially-
inoculated soil than in that naturally infested. This may be due to the
age of the inoculum, a smaller percentage of the spores being functional
in the former soil, or perhaps to the presence of a smaller amount of
inoculum.

Table VI. — Relation of soil temperature to infection of onion seedlings by Urocystis

cepu lae

Experiment i.

Experiment 2.

Naturally infested soil. Begim May 3, 1919; com-
pleted May 30, 1919. Records not kept as to soil
moisture nor as to air conditions.

Artificially inoculated soil. Begim Dec. 10, 1919;
completed Jan. 6, 1920. Soil moisture held at
22 per cent or two-thirds the water-liolding capac-
ity. Air temperature 13° to 23° C, relative hu-
midity 45 to 75 per cent.

Soil temperature.

Total ntmi-
ber of
plants.

Smutted
plants.

Soil temperature.

Total num-
ber of
plants.

Smutted
plants.

"C.
10 to 13

25
5"

47
25

Per cent.

100

15
0

10 to 14

64
49
63
56
52
36

Per cent.
98

18 to 20

16 5 to 18

22 to 24

25 to 30

2 2 to 26

28 to 34

27 to 29

93
8
0

20 to •?!

" stand reduced by damping-off fungi.

Table VII. — Relation of soil temperature to infection of onion seedlings by Urocysti^

cepulae

Experiment 3.

Naturally infested soil
pleted Jan. 10, 1920

Begun Dec. 20, 1919; com-
Soil moisture held at 25 per

cent or two-thirds the water-holding capacity.
Air temperature, 13° to 2?° C: relative humiditv.

Air temperature, 13
4S to 75 per cent

Soil temperature.

19 to 22

23 to 26

26 to 27

27 to 28

Total num-
ber of
plants.

100
86

Smutted
plants.

Per cent.

93
96

S7
12

Experiment 4.

Artificially inoculated soil. Begun Dec. 18, 1919;
completed Jan. 12, 1920. Soil moisture held at 13
per cent or two-thirds the water-holding capacity.
Air temperature, 13° to 25° C; relative humidity,
AS to 75 per cent.

Soil temperature.

23 to 26
26 to 28
29 to3i ,

Total num-
ber of
plants.

50
40

Smutted
plants.

Per cent.

12
O

Oct. 29, 1921 Relation of Soil Temperature to Onion Smut

249

Table VIII. — Relation of soil temperature to infection of onion seedlings by Urocystis

cepulae

Experiment 5.

Experiment 6.

Naturally infested soil. Begrun Jan. 16, 1920; com-
pleted Feb. 10, 1920. Soil moisture held at 25 per
cent or two-thirds the water-holding capacity. Air
temperature, 13° to 25° C; relative humidity, 40
to 80 per cent..

Artificially inoculated soil. Begun Jan. 16, 1920;
completed Feb. 10, 1920. Soil moisture held at
15 per cent or two-thirds the water-holding capac-
ity. Air temperature, ij° to 25° C; relative hu-
midity, 40 to 80 per cent.

Soil temperatiu-e.

Total num-
ber of
plants.

Smutted
plants.

Soil temperature.

Total num-
ber of
plants.

Smutted
plants.

T.
25 to 26

89
103

77
43

Per cent.
100

47
0
0

°C.
25 to 26

106
98
47
30

Per cent.

27 to 28

29to3o.s

2Q to ^0. <

30 to •?2

It may be concluded from the foregoing experiments that a high per-
centage of infection may be expected up to 25° C, above which there is
a rather abrupt reduction, leading to complete inhibition at 29°. There
appears to be no lower limit of temperature for infection within the range
where onion seeds will germinate and normal growth occur.

After it was clearly shown that no infection v/ould take place at 29° C,
the question arose as to how long seedlings must grow at this temperature
to become entirely immune. It has been shown that at moderate tem-
peratures the plant becomes immune in about 20 days, or at about the
time when the cotyledon has reached its maximum growth. To deter-
mine whether or not this condition is altered when the plants are grown
at 29°., several pots each of the naturally and the artificially inoculated
soil were started off at this high soil temperature. Pots were then
transferred from time to time to a lower temperature favorable for
infection (15° to 20°) where they were held for about three weeks before
they were examined for signs of the disease. The results of these experi-
ments are summarized in Table IX.

It is quite evident that the amount of infection was markedly reduced
by an exposure of 15 to 18 days at 29° C. Complete inhibition of infection
by even more protracted exposure to this high temperature was not
attained. However, where infection did occur there was usually not
more than one lesion per plant, which in the majority of cases was so
situated that subsequent infection of newly forming leaves would be
impossible. It is thus quite certain that where seedlings develop at
about 29° for the first 20 days the amount of damage from smut will
be negligible, especially in an area where the amount of inoculum is
slight.

250

Journal of Agricultural Research voi.xxn.Ncs

Table IX. — Effect oj different soil temperatures upon onion smut infection. In all
cases except the fifth, tenth, and eleventh, the pots -were held for the stated period at 20°
C, where infection was inhibited, then transferred to 15° to 20°, a temperature favor-
able for infection. In the fifth and tenth, where the continuous temperature was high,
note that practically no smut developed; in the eleventh, where the soil temperature was
continuously low, note that practically all the plants were smutted; in the remaining
series, where the plants were transferred from the higher temperature (29°) to the lower
(75° to 20°) after varying periods, note that long exposures at the higher temperature
tended to reduce the amount of infection.

Extent of infection at end of

experunent.

Type of soil inocu-
lation.

Length
of ex-
posure

t0 29''C

Size of plants at time
of removal to low
temperature.

Pot

No.

Total

number

Per-
centage
dis-

Extent of infection.

plants.

eased.

Days.

Cotyledons i inch
long.

63 per cent dis-
eased first leaf.

Cotyledons 2 to
2>^ inches long.

12 per cent dis-
eased first leaf.

) ^T3

25 per cent dis-
eased first leaf.

.2^

t-fl "^

First leaves i-f
inches high.

II per cent dis-
eased first leaf.

, 35

Continuous expo-
sure at 29°.

Cotyledons iK to
2 inches long.

14 per cent dis-
eased first leaf.

1 6 Coty le dons 2 ^to

3 per cent dis-

3K inches long.

eased first leaf.

First leaves out
in about one-

8 per cent dis-
eased first leaf.

half plants.

First leaves out
in most plants.

6 per cent dis-
eased first leaf.

Continuous at

0 per cent dis-

29°.

eased first leaf.

Continuous at

Most of these

15° to 20°.

plants died in
cotyledon stage.

It is interesting to note also in this connection that continued expo-
sure of onion roots to a temperature of 29° C. led to the gradual slowing
up of groM^th. With the transfer of the pots to the lower temperatui-e
(15° to 20°) both root and top development were greatly stimulated.
In attempting to correlate these results one must keep in mind the
fact that in nattire the temperature conditions under which the onions
develop are much different from those in the experimental pots.
Whereas in the pots the soil temperature is uniform throughout their
depth, in the natural soil there is a gradual decrease in temperature
at progressively greater depths. The temperature of the upper inch runs
extremely high during the summer months because of direct exposure
to the sun's rays, and this is the area critical for infection by onion smut.
The young roots, on the other hand, as they develop progressively
reach strata of lower temperature, which are more favorable for their
growth.

Oct. 29, 1921 Relation of Soil Temperature to Onion Smut 251

EFFECT OF HIGH AIR TEMPERATURE UPON THE DEVEI^OPMENT OF SMUT

The experiments reported above in which the soil temperature was
varied were carried on at an air temperature of 15° to 20° C. The latter
is considerably lower than the air temperature which prevails in southern
onion sections at the time when young seedlings are starting off. This
is shown for one section (Laredo, Tex.) in Table XIII, where the mean
air temperature is about 30° during most of the onion-planting time.
The question arose as to what efifect these high air temperatures might
have upon the development of smut in the aerial portions of the plant.
Five clay pots of onions in naturally infested soil were started off in a
greenhouse running at about 25°. Previous observations had shown
that the pustules become evident in the cotyledons on about the twefth
day under these conditions. Accordingly the plants were allowed to
grow at this temperature for nine days, at which time a few lesions were
barely visible. In order to prove that good infection had aheady taken
place, 10 plants were removed from infested soil and washed thoroughly
in running water to remove any external inoculum, after which they were
transplanted to clean soil. Within two days lesions were distinctly
visible in these plants, and smut developed in 8 out of the 10.

As a control on this method of removing the external inoculum 11
plants grown in clean soil were moistened and covered thoroughly with
infested soil. They were then washed in running water and trans-
planted to clean soil. No smut developed. On the ninth day after sow-
ing, 4 of the 5 pots were removed to a greenhouse running at 30° to 33° C,
one being allowed to remain at 25°. One pot was then transferred from
the higher temperature back to 25° at the end of each the second, fourth,
ninth, and fourteenth days. This exposure to the higher temperature
resulted in a stimulation of host plant growth for a few days. When
the plants were allowed to remain at this high temperature, however,
for three weeks distinct stunting became evident, while more prolonged
exposure resulted in death. The plants so transferred were allowed to
continue growth at 25° for three weeks or more, when they were examined
for the presence of smut. The final results are given in Table X. It
was evident that the gradual elimination of smut which took place was
proportional to the length of exposure to the higher temperature (29°).
After 14 days of exposure only small lesions developed on 16 per cent
of the onion plants, although presumably 80 per cent or more of these
plants were originally infected while they were growing at the lower
temperature. This experiment was repeated several times with prac-
tically the same results, namely, that exposure of plants bearing in-
cipient infections to a temperature of 30° to 33° for 12 to 15 days almost
entirely checked further development of the parasite.
65768°— 21 2

252

Journal of Agricultural Research voi. xxii. No. 5

Table X. — Effect of high temperature, following infection, in inhibiting the further
development of smut. The plants were from a series of pot cultures started at 25° C.
and held there until incipient infection had occurred, then transferred for the period
indicated to a high temperature, J0° to 33°, and finally brought back to the original 25°.

Length of exposure to temperature of 30° to 33°.

None (25° throughout)

2 days

4 days

9 days

14 days

Percentage
smutted.

94
45
37
33
17

It is to be noted that in the experiments just reviewed potted plants
were used. The entire pot, thus including roots and tops of the experi-
mental plants, was exposed to the stated temperature condition.

The question then arose whether the results secured were due entirely
to the effect of high air temperature upon the fungus or to an indirect
effect of the changed conditions upon the metabolism of the host. In
order to throw some light upon this point two experiments were con-
ducted in which seedlings were grown in infected soil at three constant
soil temperatures and each of two air temperatures, 25° and 30° to 33° C.
The results (Table XI) at the lower air temperature (25°) coincided
closely with those previously secured at air temperatures of 13° to 25°,
inasmuch as abundant infection occmred when the soil temperature was
25°, while complete inhibition was attained at 30°. It is, however,
significant to note, in comparison with the results in Table X, that, with
the soil temperature held at 20° or 24°, the 30° to 33° air temperatm-e
did not greatly check the development of the disease. It appears, then,
that roots as well as tops must be exposed to the inhibitive higher tem-
perature, 30° to 33°, in order fully to check the parasite after incipient
infection has taken place. This suggests that the inhibitory effect may
be due in part at least to the influence of the environmental conditions
upon the metabolism of the host and not entirely to a direct effect upon
the fungus itself.

Table XI. — Effect of different combinations of soil and air temperature upon onion smut

infection

Air temperature.

30 to 33 .

Soil
tempera-
ture.

20
25
30
20
24
30

Experiment i.

Number
of plants.

23
68

73
24

Percentage
smutted.

100
97

86
o

Experiment 2.

Number
of plants.

8S
49
22

41
.■50
30

Percentage
smutted.

95
92

o
46
60

1 stand reduced by damping-o£f fimgi.

Oct. 29, 1931 Relation of Soil Temperature to Onion Smut 253

^FFBCT OF MODERATEI/Y HIGH TEMPERATURES UPON SYSTEMIC INVASION

OF THE PI^ANT

Thaxter (jo, p. 134) observed that in some instances the smut fungus
may infect and develop in the cotyledon without invading the first leaf,
with the result that the plant eventually outgrows the disease. Obser-
vations lead us to believe that this may vary with different tempera-
tures. It has been pointed out that at a temperature of about 25° C.
the most rapid top growth of the onion seedling occurs, while at tem-
peratures below 20° the top growth is much retarded. Two pots of
infested soil were sown with onion seed and placed in greenhouses,
one at 24° to 28°, with a maximum of about 36°, for one or two hours
on sunny days, the other at 15° to 20°. A high percentage of cotyledon
infection occurred in both pots. After 31 days 24 out of 29 plants at
the high temperature were infected, but the pustules were all confined
to the cotyledons and no infection of first leaves had developed, although
the plants were now in the second leaf stage. At the low temperature,
on the other hand, of approximately the same number of plants, only
9 had survived on the thirty-seventh day, and 8 of these showed infec-
tion in the second leaves. It appears, then, that rapid growth of tops
at about 25° may result in a large percentage of plants outgrowing the
disease after the cotyledons become infected. The results of successive
field plantings, discussed in the next paragraph, seem to confirm this
judgment. The importance of the practical bearings of this matter are
such as to justify further critical attention.

EFFECT OF SUCCESSIVE PLANTINGS THROUGHOUT THE GROWING SEASON

UPON INFECTION

The laboratory experiments described early in this paper have shown
that onion smut infection is greatly reduced where a constant soil tem-
perature of 27.5° C. is maintained during the susceptible period of the
plant's growth, while a temperature of 29° thus applied completely
inhibits infection. Moreover, as explained in the last paragraph, when
plants are growing in infested soil with temperature of air and soil
held at about 25°, although a high percentage of cotyledon infection
may occur, there is a greater tendency than at lower temperatures for
the plants to outgrow the disease, owing to the rapid growth of tops.
These results combined to justify the expectation that successive field
plantings of onion seed throughout the growing season might show
considerable variations in the percentage of smut infection. In the
onion field the soil temperature usually varies widely during 24 hours,
often reaching a maximum considerably above 29° during the day and
descending to a minimum much below this at night. Under Wisconsin
conditions the daily mean temperature gradually rises during the spring
and early summer months and falls during the latter part of the growing

254

Journal of Agricultural Research

Vol. XXII, No. s

season. It seemed possible, therefore, that by making successive plant-
ings a period might be found for this latitude when the mean soil tem-
perature is sufficiently high to materially check or completely inhibit
onion smut infection.

In order to test this out, a series of plantings at intervals of from 8 to
14 days was begun on June 18, 1920, at Madison, Wis. Onion seed
was sown in smut-free soil in an open trench about i inch deep and was
then covered with naturally infested soil similar to that used in certain
of the laboratory experiments. Two varieties. Red Globe and Yellow
Bermuda, were used, one lo-foot row of each variety being put in at

S/ 5

Fig. 2. — Graph showing the daily mean soil temperature at a depth of i to 2 indies as it occurred in the
"successive planting" plots. Since the weather continued rather cool, one bed was covered with glass
to insure a higher temperature. The temperature of the uncovered bed is shown by the solid line,
the temperature of the glass-covered bed by the broken line. For further details see Table XII and
and accompanying text.

each planting. Temperatures of the soil at a depth of i to 2 inches were
obtained by means of a self-recording thermograph. The hourly mean
temperature for each day was then secured by adding temperatures as
recorded for each hour and dividing the sum by 24. These computa-
tions are represented graphically in figure 2. Since the weather in
July was unusually cool, a higher mean soil temperature was secured for
some of the plots by covering them with an ordinary glass cold frame.
Inasmuch as the dry weather and high temperature would cause a rapid
desiccation of the surface layer of soil, the plots were watered thoroughly
on alternate days or oftener during the early growth of the plants. The

Oct. 29. 1921 Relation of Soil Temperature to Onion Smut

255

data collected from this field plot are tabulated in Table XII. Obser-
vations were made by pulling plants at several points in each plot, and
examining for smut lesions after the chlorophyll had been removed by
means of alcohol and acetic acid. The first observation was made on
the twenty-first to the twenty-third day after planting. Subsequent
observations were made as indicated in the table.

TabIvE XII. — Development of onion smut in successive plantings in the field at Madison,

Wis., ig20

Date of

Variety.

Treatment.

First observa-
tion, 21 to 23
days after
planting.

Second observa-
tion, 29 to 31
days after
planting.

Third observation, 44
to 65 days after planting.

planting,
1920.

Ntim-
ber of
plants
exam-
ined.

Per-
cent-
age
smut-
ted.

Num-
ber of
plants
exam-
ined.

Per-
cent-
age
smut-
ted.

Time

after
plant-
ing
(days).

Num-
ber of
plants
exam-
ined.

Per-
cent-
age
smut-
ted.ff

June 18

Red Globe...
[Red Globe. . .

Uncovered
. ..do

108
62
45

41
20

46
26

S3
73
49

10
10

0
13
14

0
0

& SO

47
28

2>7>

C40
«86

/;3

52
44

31
SI

3
39

June 26

Yellow Ber-
muda.
[Red Globe . . .

...do

. . .do

65
65

65
53
53

53
53

61
24

135

July 10

Yellow Ber-
muda.

Red Globe . . .

Red Globe...

Yellow Ber-
muda.

Red Globe...

Yellow Ber-
muda.

...do

Covered . .
Uncovered
...do

Covered « .
Covered a .

0
II

July 19

" Covered for 15 days only.
•6 Observation 39 days after planting.

«^ Extent of infection: Systemic, 26 per cent; confined to dead cotyledon, 14 per cent.
<* Extent of infection: Systemic, 10 per cent; confined to dead cotyledon, 66 per cent.
« Extent of infection: Systemic, 5 per cent; confined to dead cotyledon, 81 per cent.
/ Extent of infection: All cotyledon infections.
0 Extent of infection: All systemic infections.

An analysis of the data secured can be made by referring to Table XII
and figure 2. It will be seen that the soil temperature mean gradually
rose until July 23 to 29, after which there was a gradual drop. At no
time did the mean in the uncov red plot reach the inhibiting temperature
(29° C), but it closely approached this point during the warmest portion
of the season. In the covered plot, however, the mean remained above
29° continuously until the cover was removed on August 3. The two
important points to be considered in the respective plantings were (i)
the amount of original infection which was determined three to four
weeks after planting (see first and second observations in Table XII)
and (2) the extent to which the disease either became systemic or was
entirely outgrown by the plants during the following four or five weeks
(see third observation in Table XII).

256 Journal of Agricultural Research voi. xxii. no. s

Considering first the amount of original infection, it will be seen that a
high percentage of disease resulted in all the plantings of June 18 and
June 26. The somewhat lower infection in that of June 18 may be ex-
plained in part at least by the fact that a smaller quantity of inoculum
was used than in subsequent plantings. The next two plantings (July
10 and 19) were so made that the resulting seedlings were exposed during
early growth to the maximum soil temperature of the season. By refer-
ring to Table XII it will be seen that associated with this higher tem-
perature there was a decided reduction in the amount of infection in
even the uncovered plots, while in the covered plots, where the mean
temperature remained continuously above 29° C, no infection whatever
occurred.

Considering, secondly, the extent to which the disease became sys-
temic or was outgrown, it will be seen that in the planting of June 18 a
majority of the infected plants showed systemic invasion at the second
observation (thirty-ninth day). In the next planting (June 26), how-
ever, by the time of the second observation most of the external signs of
the disease were confined to the dead cotyledons. The amount of
systemic infection increased somewhat, however, at the third observation
(39 per cent).

In the third planting (July 10) it is interesting to note first that the
plants in the covered plot remained entirely free from infection. In the
uncovered plot, although some cotyledon infection was noted at the sec-
ond observation, no disease whatever was found at the third observa-
tion. This indicates that the time when the temperature was at its highest
point the infected plants succeeded best in outgrowing the disease.

The field data secured in the foregoing experiments at Madison are
thus in general accord with the experiments performed under controlled
conditions. In such controlled experiments the amount of smut infec-
tion falls as the soil temperature rises toward 29° C. and is totally inhib-
ited above this temperature. Likewise in the field trials with successive
plantings there was a gradual reduction in the amount of infection fol-
lowing the rise in the mean soil temperature, with omplete inhibition of
infection where the mean was kept above 29° for two or three weeks after
planting. Complete freedom from infection under these Wisconsin field
conditions was secured only by growing the plants under artificial condi-
tions in which by covering the plants with glass the temperature was
raised several degrees above the normal. It is, however, to be noted that
the summer of 1920, when the foregoing results were secured, was some-
what cooler than the average. The weather records of other years indi-
cate that in a hot summer complete inhibition of smut infection would
be secured by such summer plantings.

Oct. 29, 1921 Relation of Soil Temperature to Onion Smut

257

CORRELATION OF EXPERIMENTAL RESULTS WITH FIELD CONDI-
TIONS OF THE SOUTHERN STATES

These results obtained in both greenhouse and field experiments justify
the question as to the part played by soil temperature in determining
smut infection in the onion fields of the more southern States. As
noted at the beginning of this article, a recent survey of southern onion
sections indicates that smut is not prevalent in the southern fields — for
example, in Texas — as it is in the northern onion sections. As was
earlier explained, it is the practice in these southern fields to plant the
onion seed in late summer or early autumn. It is thus quite possible
that the mean temperature for the surface inch of soil in southern onion
sections is considerably above the maximum for onion smut infection
during and immediately following the sowing of seed. According to
Mally (6), onion seed is sown in the Laredo district of southern Texas
as early as August i , while most of the seed is planted about September
10 to 25. The mean air temperature as recorded at Laredo, Tex., by
the United States Weather Bureau for August, September, and October,
1917, is given in Table XIIL

Table XIII. — Mean air temperatures for August, September, and October, iQiy, at

Laredo, Tex.'''

Day of
month.

August.

September.

October.

Day of
month.

August.

September.

October-

"F.

" F.

°F.

"F.

°F.

89-5

85.5

71.0

90. 0

79.0

80.5

88.5

86.0

80.0

90-5

83.0

81. s

88. s

86.5

80.5

91. 0

7!?-5

76. 0

88.5

87.0

80.5

92. 0

79-5

67..-;

89.0

88.0

80. s

90. 0

79-5

62. 0

88. <;

87-5

78.0

90. 0

80.0

63-5

89. i

88.0

81.0

90. 0

79-5

68. s

88.5

82.5

91. 0

79.0

61. 0

89.0

89-.=;

64-5

89-5

80. ■;

66.5

90-5

88.0

64-5

87.0

81. 5

76.5

90- .■;

83-5

70-5

89-5

81.5

74-5

88.0

85.0

73-.';

86.5

71- S

78. s

89-5

86. =;

7S-0

89.5

71- 5

75- 0

88.5

77.0

84.5

71- S

SO-.";

I c

90- S

88.5

83-5
83-5

76. 0

80.0

86. 0

0
16

° Obtained by averaging the daily maximum and minimum temperatures.

Table XIII shows that the air temperature ranged very high during
August and September, the onion-planting period. In this connection
it is to be noted, moreover, that the records of Bouyoucos (i) in Michigan
indicate that surface soil temperatures may considerably exceed air
temperatures. Thus, his observations showed that the maximum tem-
perature for the upper quarter inch of all the soils he studied was about
16° C. higher during hot, clear days than that of the air at an elevation

258 Journal of Agricultural Research Voi. xxu. No. s

of 4 feet, while the minimum temperatm-e of all the soils used, except
peat, was 0.5° to 1.0° C. higher, as a monthly average, than that of the
air. Our own observations in Wisconsin are in general accord with
these Michigan records. Assuming that the temperature of the surface
layer of soil under Texas conditions likewise averages several degrees
higher than the air, it is evident that the mean never went below the
point where infection is entirely inhibited (29° C, or 84° F.) during
August and seldom below it during September. Continuing up to
October 8 there were only a few days when the air temperature fell
below 27.5° C. (81.5° F.), the point at which our experiments have shown
smut infection to be markedly reduced. It seems probable, therefore,
that even if onion smut were introduced into this Laredo soil, it would stand
small chance of infecting onion seedlings to the extent of establishing
the disease as a permanent factor. The data available are not sufficient
to justify the attempt at more detailed geographic correlation of onion
smut occurrence with the temperature factor. We believe, however,
that the conclusion is justified that soil temperature during the early
seedling stage must be considered as a limiting factor in determining the
occurrence of the disease in any locality. It must be left with local
observers to make use of this fact in interpreting conditions as they
occur in any particular region.

SUMMARY

Onion smut was first noted in the Connecticut River Valley in 1869.
Since then it has successively appeared and become an economic factor
in nearly all of the northern onion-growing sections from New York to
Oregon. This has probably resulted from chance introduction of the
organism with seed or bottom sets, followed by its accumulation in the
soil where continuous cropping with onions is practiced. The disease
has not appeared in the southern onion-growing sections of Texas
and Louisiana, although they are exposed to similar chance introduction
of the parasite and the continuous cropping method is common.

These facts have raised the question as to wherein lies the explanation
of the regional limitation of the disease. The southern method of cul-
ture, characterized by special seed bed and transplantation of seedlings,
does not offer sufficient explanation for the absence of smut. No differ-
ence in susceptibility between northern and southern varieties has been
found. Is regional limitation explained by differences in environ-
mental factors in the North and the South at the time when the seedling
is susceptible to infection, that is, during the first two or three weeks after
germination? An analysis of certain of these factors in relation to
infection has been the object of the present investigation.

The cotyledon of the onion is susceptible to attack by the smut organ-
ism up to the time it attains full growth, a period of about three weeks.

Oct. 29, 1921 Relation of Soil Temperature to Onion Smut 259

varying somewhat with environment. Cotyledons remaining free from
infection during this period become resistant and serve as a barrier to
subsequent invasion of the embryonic region of the true leaves. Conse-
quently, if infection is prevented by environing conditions during this
period of susceptibility, the plant will remain free during the remainder
of its growth.

Experiments were conducted in which seedlings were grown on smut-
infested soil held at various soil moisture contents. A high percentage
of infected plants resulted over the entire range in which good germina-
tion and growth of the host occurred. At either extreme, very high or
very low moisture, there was some reduction in amount of infection, but
with it occurred a corresponding decrease in seed germination and rate
of growth of the plants. Soil moisture, therefore, does not appear as a
serious limiting factor in onion smut infection. .^rj v/ot;

The relation of soil temperature to the development of the host and
the parasite was studied by growing plants in pots held experimentally at
a series of constant soil temperatures in the special apparatus known as
the "Wisconsin soil temperature tank."

Seed germination and growth took place over a range of soil tempera-
ture from 10° to 31° C. Most rapid seed germination and development
of tops occurred at soil temperatures of 20° to 25°, while as a rule the
best development of roots occurred below 20°.

A high percentage of plants grown on smutted soil were infected at
soil temperatures ranging from 10° to 25° C. A decided reduction in
infection was noted at about 27°, and complete freedom from the disease
resulted at 29°. In these experiments all plants were under uniform
conditions of air temperature, which ranged from 15° to 20°.

The relation of variations in air temperature to the development of
the disease was then studied.

Exposure of plants bearing incipient infections of the fungus in the
aerial parts to an air and soil temperature of 30° to 33° C. so disturbed
the relations between parasite and host as to preclude any further develop-
ment of the disease. This was shown by growing plants at a tempera-
ture favorable for infection (15° to 20°). Then, just as the pustules of
the disease were beginning to appear (tenth to twelfth day), the plants
were removed to a room held at 30° to 33°. This stimulated top growth
for a few days, which was followed by a decided checking of the plants
and death after three or four weeks. However, if after 12 to 15 days
at the high temperature the plants were returned to the original tem-
perature (15° to 20°), they grew normally, but the fungus in nearly all
cases failed to produce spores, and the plants remained free from further
invasion.

Experiments were then performed in which seedlings were grown on
infested soil held at 20°, 25°, and 30° C. with a uniform air temperature
of 30° to 33°. A high percentage of infection resulted at soil temperatures

26o Journal of Agricultural Research voi. xxii, no. $

of 20° and 25°, but none at 30°, showing that high air temperature
alone is insufficient to check the development of the disease. It appears
probable that the failure of the fungus to complete its development in
the case described above (where the plants after infection were exposed
to an air and soil temperature of 30° to 33°) was brought about at least
in part by some marked disturbance of the metabolism of the host and
not simply by the direct effect of the high air temperature upon the
fungus in the aerial parts of the seedling.

Comparison between the development of the disease in plants grown
at 15° to 20° and at 24° to 28° C. (air and soil) was made. A high per-
centage of cotyledon infection occurred in both cases. At the lower
temperature the disease proceeded as usual to the infection of the true
leaves. At the higher temperature, however, the plants tended to out-
grow the disease, this being associated with a more rapid rate of top
development which apparently enabled the plants to slough off the
smutted cotyledons before infection of the first true leaf occurred.

The foregoing conclusions as to the dominant influence of soil tem-
perature upon onion smut infection, while primarily based on greenhouse
experiments, have been found to accord well with field developments.

Successive out-of-door plantings at Madison, Wis., made in inoculated
soil during the growing season, resulted in a gradual reduction of infection
as the season advanced and the soil temperature rose. Complete freedom
from smut was attained when the daily mean soil temperature at i to 2
inches depth remained at or slightly above 29° C. for two to three weeks.
There was also a tendency, as the temperature rose, for the seedlings to
outgrow the disease by the sloughing off of the diseased cotyledons before
infection of the first leaf occurred.

An examination of records from one of the southern onion sections
(Laredo, Tex.) shows that during a good share of the critical period for
onion smut infection (August and September) the mean air temperature
is above that at which complete inhibition of infection was attained in
our experiments (29° C. or about 84° F.). If we assume, as observed
in northern sections, that the mean temperature for the upper layer of
soil is several degrees higher than that of the air at this time of the year,
it is reasonable to conclude that even though the smut organism were
introduced into southern onion sections, its development would be pre-
vented or greatly minimized, first, by the prevention of infection due to
high temperatures, and, secondly, by the rapidly developing tops out-
growing the disease, should occasional infections occur.

In general we believe, therefore, that the regional distribution of onion
smut in the United States is conditioned upon the soil temperature
during the seedling stage of the plant's growth, the infection and develop-
ment of smut being favored by the relatively low temperatures and
inhibited by the high temperatures, with approximately 29° C. as the
critical point.

Oct. 29, 19" Relation of Soil Temperature to Onion Smut 261

It is hoped that the evidence here recorded may lead to the accumula-
tion of further field data bearing upon this particular problem by inves-
tigators in various places, especially in the southern States. It is also
believed that these results illustrate well the importance of more per-
sistent inquiry by the experimental method into the relation of environ-
mental factors to the occurrence of disease of plants in general.

(I
(2

(10

LITERATURE CITED
BouYOUCOS, George J.

1916. SOIL TEMPERATURES. Mich. Agr. Exp. Sta. Tech. Bui. 26, 133 p.
Chapman, George H.

I910. NOTES ON THE OCCURRENCE OF FUNGOUS SPORES ON ONION SEED. MaSS.

Agr. Exp. Sta. 22 Ann. Rpt., 1909, pt. i, p. 164-167.
Heald, Frederick D., and Woolman, H. M.
1915. BUNT OR STINKING SMUT OF WHEAT. Wash. Agr. Exp. Sta. Bui. 126,

24 p., 5 fig. (in text and on pi.).
HuNGERFORD, Charles W., and Wade, A. E.

1920. RELATION BETWEEN SOIL MOISTURE AND BUNT INFECTION IN WHEAT.

(Abstract.) In Phytopathology, v. 10, no. i, p. 53.
Jones, L. R.

1917. SOIL TEMPERATURES AS A FACTOR IN PHYTOPATHOLOGY. In Plant World,

V. 20, no. 8, p. 229-237, 2 fig. Literature cited, p. 236-237.
Mally, F. W.

1915. THE BERMUDA ONION. Tex. Dept. Agr. Bui. 46, 56 p.
MuNN, M. T.
1917. NECK-ROT DISEASE OF ONiONG. N. Y. State Agr. Exp. Sta. Bui. 437, p.
361-455, II pi. Bibliography, p. 4SO-455-
SiRRiNE, F. A., and Stewart, F. C.

1900. EXPERIMENTS ON THE SULPHUR-LIME TREATMENT FOR ONION SMUT. N. Y.

State Agr. Exp. Sta. Bui. 182, p. 145-172, i pi.
Sturgis, William C.

1896. TRANSPLANTING, AS A PREVENTIVE OF SMUT UPON ONIONS. In Conn.

Agr. Exp. Sta. 19th Ann. Rpt. 1895, p. 176-182, pi. i.
Thaxter, Roland.
1890. the "smut" OF ONIONS, (UROCYSTis cEPUOrAE FROST). In Conn. Agr.
Exp. Sta. Ann. Rpt. 1889, p. 129-154, pi. 1-2.
Ware, Benjamin P.
1870. EXPERIENCE OF A PRACTICAL FARMER. In 17th Ann. Rpt. Mass. Bd.
Agr. 1869, Appx., p. 1-16,

PLATE 25
Relation of soil temperature to the development of onion seedlings.

Upper row. — Seedlings of Red Globe variety 13 days old. Each cluster was the
entire crop from one experimental culture pot. All were grown in like virgin soil and
at the same air temperature (i5°to2o°C.) but with gradation in the soil temperature of
the respective pots as follows (left to right): 12° to 14°, 20°, 25°, 28°, 30°. Note that
there is a tendency for greater root development in relation to top growth at the lower
temperatures. This was especially marked at the lowest temperature, 12° to 14°.
For further details see Table V and the accompanying text.

Lower row. — Seedlings of Yellow Bermuda variety grown imder same conditions as
those in upper row.

(262)

Relation of Soil Temperature to Onion Smut

Plate 25

Journal of Agricultural Research

Vol. XXII, No. 5

Relation of Soil Temperature to Onion Smut

Plate 26

Journal of Agricultural Researcli

Vol. XXII, No. 5

PLATE 26

Relation of soil temperature to the infection of onion seedlings by the smut fungus,

Urocystis cepulae.

Representative seedlings taken from the experimental culture pots, showing the
influence of soil temperature upon the amount of smut. All the pots alike contained
smut-infested soil. The air temperature and other aerial factors were the same for all.
Soil temperature was the only factor varied experimentally, the temperature grada-
tions extending from about 10° to 30° C.

Note the abundance of smut at 10° to 22°, as shown in the upper row. A slight re-
duction occurred at 23° to 26°. At 27° to 29° the reduction is sharply marked. At
29° to 3 1 ° inhibition is complete. For the percentages of seedling infection and other
details see Table VI and the accompanying text.

PLATE 27
Relation of soil temperature to onion smut infection.

This shows the results from a series of experiments in which the methods described
for Plate 26 were repeated with the soil temperatures restricted to the critical limits
between 25° and 29° C. and controlled more exactly. Note the marked reduction in
infection at 27.5° and complete inhibition at 29°, thus establishing, but with more
exactness, the conclusions illustrated in Plate 26.

For the percentage of infected seedlings at these temperatures and other details, see
Table VIII and the accompanying text.

Relation of Soil Temperature to Onion Smut

Plate 27

\ V

( \

ZS^'C

27.5°C

Journal of AgriculturalResearch

Vul. XXII. No. 5

A PHYSIOLOGICAL STUDY OF GRAPEFRUIT RIPENING

AND STORAGE '

By IvON A. Hawkins

Plant Physiologist, Office of Horticultural and Pomological Investigations, Bureau of
Plant Industry, United States Department of Agriculture

In an earlier investigation (7) ^ the changes in Florida-grown grape-
fruit during storage were studied, particular attention being paid to the
sugar and acid content of the pulp or edible portion of the fruit as influ-
enced by some six different storage temperatures. It was found that the
acid content decreased in cold storage while the total sugar content re-
mained about the same. The percentage of cane sugar decreased and
the reducing sugar content increased. At the higher temperatures,
common storage (about 55° to 60°, 70°, and 86° F.) there was in some
cases apparently an increase in acidity and a reduction in the amount of
sugar, especially in fruit stored for long periods. The shrinkage, which
was very marked in the ventilated packages at these high temperatures,
made the obtaining of definite evidence on this point impossible.

The investigation described in the present paper is concerned with the
acid and sugar changes in the fruit on the tree as well as with the changes
which take place, both in warm storage and in cold storage, in fruit picked
at monthly intervals. The control of the pitting which occurs commonly
on grapefruit in cold storage is given some attention.

PLAN OF THE EXPERIMENTS

" Common Florida " ^ fruit from two trees was picked at monthly inter-
vals for four months, beginnmg July 27, making five different picks. At
the last three pickings fruit was also harvested from two additional trees
in the same grove. The fruit was expressed to Washington and sampled
on arrival. Part of the fruit was placed in warm storage at about
70° F. and part in cold storage at 32°. Analyses were made of the fruit
stored in the warm storage each month for two months and on fruit from
the last three pickings stored in cold storage, after it had been in storage
four months. By this plan it was possible to obtain data on the changes
in the fruit on the tree from a month or so before the fruit was in con-
dition to pick for market until the last of the season, and to compare the
changes which took place in cold storage in fruit picked from the same
trees at different times of the season.

1 This paper gives the results of a portion of the work carried on under the project " Factors affecting the
storage life of fruits."

2 Reference is made by number (italic) to " Literature cited," p. 278-279.

' The writer's thanks are due Mr. W. J. Krome for the picking and shipping of all the " Common Florida"
fruits used in these exijeriments.

Joimial of Agricultural Research, Vol. XXII, No. s

Washington, D. C. Oct. 29, 192 1

aag Key No. 0-251

(263)

264 Journal of Agricultural Research voi. xxii. no. s

METHODS OF ANALYSES

The fruit was prepared for sampling and sampled as in the previous
work. Analyses were made for acids, sugars, both reducing sugars and
total sugars, dry weight, shrinkage, thickness of peel, and percentage of
peel. In addition the acidity and specific gravity of the expressed juice
of the fruits were determined and the solids-acid ratio calculated after the
usual method. The acidity determinations were carried out as in the
previous work, as were practically all the other determinations with the
exception of the extraction of the sugar from the pulp. In the sugar
extraction a method was followed similar to that described in work on
potatoes (6). The weighed pulp was placed in a liter volumetric flask which
was then filled to volume with 85 per cent alcohol. It was allowed to
stand with frequent shakings for about three weeks, the losses from
evaporation, of course, being made up by adding alcohol. The alcoholic
solution of sugar was then separated from the residue by filtration, and
the sugars were determined in aliquots of the filtrate.

The first lot of grapefruit was of small size, green in color, with very
little juice in the pulp. No solids-acid determinations were made on
this lot. They were, however, maintained in warm storage for two
months. At the end of this period many of them had assumed the char-
acteristic yellow color of the ripe grapefruit.

The second pick, received August 29, was much further advanced,
being about 50 per cent colored and of good size. The third and fourth
picks, those of October 25 and November 28, respectively, were in fine
condition for shipping and are what would be considered midseason
fruit. The November 28 fruit was fair, possibly a little coarser than
the two picks immediately preceding. No sprouted seeds were found
in any of the fruits, however. The date of picking might be considered
as in the latter part of the grapefruit season for this locality and for this
variety.

The analyses of grapefruit picked from trees i and 2, from warm
storage at about 70° F. for one and two months, are shown in Tables I
and II. In the analytical work the analyses were usually made in
duplicate, and both analyses are given in the tables, as this furnishes
evidence on the experimental error in the method of sampling. The
tables are self-explanatory.

An inspection of Tables I and II shows that in the first four pickings
there is in all cases an increase in the acid content of the pulp, while in
the last picking from both trees there is no decided increase. A com-
parison of the acid content, as determined in the analyses of the
pulp and the acid content of juice, shows a similar behavior. The acid
content of the juice is, as a rule, markedly higher than that of the pulp,
due, of course, to the fact that in the last-mentioned case the weight of
fibrous material is taken into consideration in calculating the percentage

Oct. 29, 1921 Physiological Study of Grapefruit Ripening 265

of acid. In the fifth, pick from both trees there is no decided variation
in the acid content of the pulp during storage, and the percentage of
acid in the juice does not change as much as in fruit from any of the other
four picks. With the sugars, the percentage of reducing sugars and of
total sugars is always greater at the end of two months in warm storage,
except in the fifth pick. The reducing sugar increases most, due prob-
ably to the inversion of some of the cane sugar which is less in all cases
after two months in storage.

It was brought out in the earlier publication on grapefruit storage
that there was an indication that the acid content of the fruit was slightly
increased during a long period of warm storage. It was pointed out
also that definite evidence on this point was difficult to obtain because
the structure of the fruit prevented accurate calculation of the shrinkage
of the various portions. Further evidence, mostly of an indirect nature,
may be derived from the data on sugar and acid content of the fruit,
found in Tables I and II. As was mentioned above, there is in all cases
an apparent increase in the acid content and the total sugar content of
the pulp, due for the most part undoubtedly to loss of water during
storage. In the tables it is noticeable that the solids-acid ratio is usually
less after two months in storage. This indicates, of course, that the
increase in soluble solids is not proportional to the increase in acidity
and that some soluble substance or substances other than titratable
acids decreased in the storage period. This occurs in five cases out of
seven on which data were obtained. The other two cases, tree 2, third
pick and fifth pick, show slight increases, 0.07 and o. 1 1 , respectively.

These data are corroborated in the total sugars-acid ratios, which are
calculated by dividing the percentage of total sugar as dextrose by the
percentage of acid as citric. In the 10 cases the ratio of sugar to acid
is less in 7, practically the same in 2, and greater in i. Indications
are, then, that there is usually an increase in the ratio of sugar to acid
under the conditions of the experiment. This could be brought about
by either decreasing the sugar content of the fruit or by increasing the
acid content or by a combination of these two factors. It is notice-
able that in 6 cases out of 10 the acid-sugar ratio is greater after one
month in storage than it is after two months at the same temperature.
The acid and sugar in the fruit from warm storage will be considered
later in comparison with the changes taking place in cold storage.

There is in most cases not much variation in the percentage of dry
matter during storage, though there seems to be a tendency, more
marked in some cases than in others, toward an increase. This seems
probable, as the shrinkage where determined is from 14.4 to 23.3 per
cent for the full two months in storage. The percentage of peel always
decreases during storage at this temperature, due to the loss of water and
wilting. This is evident in the decrease in thickness of the peel, which is
very marked, especially in the earlier picks.
65768°— 21 3

266

Journal of Agricultural Research voi. xxii, no. s

CHANGES IN FRUIT ON TREES

The analyses of fruit from the various pickings at the time it was placed
in storage (Tables I and II) show marked differences in composition.

PfRl
con

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fts

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Fig. I. — Graphs showing changes in percentage of dry matter, acid, and sugars calculated as dextrose in
fruit on two Common Florida trees from August to December.

For convenience in comparison the data for sugars, acids, and dry matter
are shown graphically (fig. i). In these curves the percentage is plotted
on the ordinates and the time interval between pickings on the abscissae.

Physiological Study of Grapefruit Ripening

267

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Oct. 29, 1921 Physiological Study of Grapefruit Ripening 269

From an inspection of the curves it is evident that there is a decrease
in acidity as the season advances, the acid being highest in both cases at
the beginning of the season. The acid content is lowest at the fourth
pick and rises shghtly at the fifth pick. ColHnson ( j) shows a somewhat
similar decrease in acidity. This writer analyzed the fruit at more
frequent intervals but apparently did not begin sampling so early in
the season. According to his work there is a general tendency toward
lower acidity, though in a few of his series of analyses there is a higher
acidity toward the end of the season than in the midseason fruit.

There is a rise in the percentage of total sugars during the season, the
total sugar content of pulp of the fifth pick being about double that of
the first pick. Collinson shows an increase in the total sugar content,
but it is not so marked, due probably to the fact that his series begins
later in the season. As showm in the curves (fig. i), the rise in total sugar
content during the first month is very sharp. The increase in percentage
of reducing sugar during the season is much more gradual and regular
than that of the total sugars. The percentage of this sugar in tlie pulp
a little more than doubles in the four months of the experiment. Much
the same ratio of increase is found in the total sugars. The cane sugar
curves are not so regular. There is, however, a marked increase in the
percentage of cane sugar. The mean of the two sucrose curves is always
higher than that of the reducing sugars except at the last sampling.
Collinson records a series of analyses in which the reducing sugar is
markedly higher than the sucrose during the latter part of the season.
The irregularities in the total sugar curves in the present work are due
to the variation in sucrose content.

The percentage of dry matter, as determined in this work, is highest
at the first of the season between 11 and 11.5 per cent but drops in the
first month to between 9.5 and 10 per cent, the third and fourth analyses
giving about the same results. There seems to be, however, an increase
in the dry weights in the last month.

A comparison of the percentage of peel (Tables I and II) at the time
the five different lots of fruit were placed in storage shows there is a de-
crease from 2)Z-Z to 21.2 and 45.6 to 18.2 per cent of peel by weight for
trees No. i and 2, respectively. The percentage of peel decreases much
more rapidly in the first month than in the succeeding month. In fact,
in the case of tree i there is only a slight decrease in the proportion of
peel to pulp in the last three pickings. At the time these last three
pickings were made, the fruit was ready for market.

As might be expected, the decrease in thickness of the peel, as meas-
ured in these experiments, parallels the decrease in percentage of peel.
The peel was found to be 6.8 mm. and 8.1 mm. thick, respectively, for
trees i and 2 when the first pick was placed in storage, while it measured
4.5 mm. and 3.2 mm. at the first samphng of the fifth pick. This is a
reduction of 34 per cent and 60 per cent in the thickness of the skin fca:

270 Journal of Agricultural Research voi. xxii, no. s

the season. It is evident that the proportion of peel to pulp and thick-
ness of the peel decrease as the fruit matures.

A comparison of the acid and sugar changes in grapefruit in growth
and ripening with the acid and sugar changes of other fruits in the same
period of their life history brings out some interesting correlations and
differences. The total sugar content of deciduous fruits usually increases
during the growing and ripening period. This has been shown for
apples by Lindet, (8) Bigelow, Gore, and Howard (2), and others; for
pears by Ritter (12), Riviere and Bailhache (11), Magness (10), and by
Bigelow and Gore (i), for peaches. Numerous other investigations cor-
roborating this point might be mentioned. The literature pertinent to
this subject may be found in the works referred to here. With certain
vegetables a somewhat similar increase in total sugars is found. This
was brought out by Hasselbring (5), working with sweet potatoes, and
Sando (14) with tomatoes. This evidence would seem to indicate that
in fruits or vegetables where sugar is stored the percentage of sugar
calculated on a wet-weight basis increases during the growing season —
that is, there is not only an absolute increase but an increase in proportion
of sugar present as compared to the sum of the other constituents. This
increase in some cases is due to an increased content of reducing sugars, as
in the tomato, or may be due to an increase in both reducing sugars and
cane sugar, as in the apple, pear, and peach, or for the most part to an
increase in cane sugar, as in the sweet potato.

In regard to the acid content of fruits which contain both sugar and
acids in appreciable quantities, there is sometimes an increase and some-
times a decrease in acidity as the season advances. In pears there is
generally a decrease. Magness (10), however, found that pears from the
Yakima district, Washington, and Medford district, Oregon, showed an
increased acidity as the season advanced. Apples, according to the
analyses of Lindet (8), and Bigelow, Gore, and Howard (2), exhibit a
decrease in acidity as the growing season advances. Peaches, on the other
hand, increase in acid content as they approach maturity. The decrease
in acidity of grapefruit during the growing season is comparable to the
usual behavior of the acidity in pears and in apples.

COLD STORAGE EXPERIMENTS

As mentioned earlier in this article, experiments on the cold storing of
grapefruit were carried out during the 1920-21 season. Fruit from four
trees was used. These trees included the two from which fruit was
obtained for the warm storage work, the fruit being from the lots desig-
nated third, fourth, and fifth picks in the experiments aheady described.
Not sufficient fruit was available for this work from the fifth pick from
tree i , so only two experiments were possible with fruit from this tree.

Table III gives the results of analyses made at the time the fruit was
placed in storage and four months later. As was pointed out, because
of the structure of the fruit it is very difficult to obtain definite evidence
on the changes of the various constituents of the pulp. While the

Oct. 39, 1921

Physiological Study of Grapefruit Ripenivg

271

fruit for each experiment was carefully selected from a lot of fruit all
harvested at the same time from a single tree, the variation in composi-
tion of the fruit on this tree introduces a possible error which it is hardly
possible to calculate. It is only by obtaining a large amount of evidence
that a clear indication of the direction of the change can be obtained.
It was accordingly deemed advisable to give in this table all the data
obtained in the analyses in the 1 1 different storage experiments carried
out in this portion of the investigation. The table is self-explanatory.

Table III. — Percentage of acid, sugar, and dry matter in pulp, thickness of peel and per-
centage of peel, acid and soluble solids in juice, and solids-acid ratio of " Common Flor-
ida" grapefruit before and after storing four months at J2°F.

TREE I

First lot.

Second lot.

Third lot.

When
placed in
storage.

After 4

months in

storage.

When
placed in
storage.

After 4

months in

storage.

When
placed in
storage.

After 4

months in

storage.

f T rn

0.88
.90

2-55
2. 74

3- II
2. 72
=;. 66
5-46
9-43
9-37
26. 9

5-5
5

I. 02
8.87
8.66

I. 00

1. 02

2. 96
3- 04
3-13

3. 22
6. 09
6.26
9-45
9-54

21.8

0.88
.92
3-48
3-09
3- II
3- SI
6. ."59
6.6

9-74
9-74
21. 6

4-3
4.9

I. 01
II. 25
II. 16

citric 1 I. 08

Percentage of reducing f 2. 62

Percentage of cane sugar

1 2.61
I 2. 93

as dextrose \ 5. 51

Percentage of dry mat- 1 f 9. 49
ter il 9. 63

Thickness of peel (in

5-S

Percentage of shrinkage
Acidity of juice as per-
centage of citric

Soluble solids (Brix) . . .

1.27
9-45

7.38

I. 14

ID. II
8.81

TREE 2

Acid as percentage of
citric

Percentage of reducing
sugar as dextrose

Percentage of cane sugar
as dextrose

Percentage of total
sugar as dextrose

Percentage of dry mat-
ter

Percentage of peel

Thickness of peel (in
mm.)

Percentage of shrinkage

Acidity of juice as per-
centage of citric

Soluble solids (Brix) . .

Solids-acid ratio

1. 06
2.47

2.44

2. 74
2. 52

21
96

54
S

5-6

1.28
9.40
7-31

I 2.61

}2.6s

} 5-26

9.46

23-3

4.4
3

I. 19

10. 04

8.44

I. 06

3. 01
. 2.92

3-3°

3-38

6.31

. 6.30

' 9.66

. 9-43
21. 2

4. 2

I. 18

IO-33

8.73

I. 00
3-37
3-54
3-03
2-75
6. 40
6. 29
9.86
10. 16
21. 7

4-3
4

I. 09
10. 16
10. 29

I- 15
3-73
3.66

3-24
3-25
6.97
6. 91

10.30

18.2

I. 19

10.79

9- 03

I. 10

3-29

3-59

6.88

9-77
9.82
22. 4

4.8
3-9

I. OS

11-35
10. 84

272

Journal of Agricultural Research voi. xxii. no. s

Table III. —Percentage of acid, sugar, and dry matter in pulp, thickness of peel and per-
centage of peel, acid and soluble solids in juice, and solids-acid ratio of "Common Flor-
ida" grapefruit before and after storing four months at 32° F. — Continued

TREE 3

First lot.

When
placed in
storage.

After 4

months in

storage.

Second lot.

When
placed in
storage.

After 4

months in

storage.

Third lot.

When
placed in
storage.

percentage of

Acid as
citric .

Percentage of reducing
sugar as dextrose

Percentage of cane
sugar as dextrose

Percentage of total sugar
as dextrose

Percentage of dry mat-
ter

Percentage of peel

Thickness of peel (in
mm.)

Percentage of shrinkage .

Acidity of juice as per-
centage of citric

Soluble solids (Brix) . . .

Solids-acid ratio

|- 1.06

\ 2. 46
I 2.58
I 2.83
f 3.01
L 5-29
I .?• 59
L 8.77
[ 8.78
24

5-3

I. 26
8.6s
6. 92

2-55
2-35
2.58
2.77

S-I3
5.12
8. 10

8-53
24. 2

4-7
5

I. 17

II. 19

9- SI

I. 10

1. 09
2.77

2. 50
2.81
2.81
5.58
5-31
9-56
9. 16

4-9

03
07
22

50
16

75
38
25
19
67

21.8

I. 29
I. 18
3-36
3- 51
3.01

3-2?

6-37
6.76
9.82
9.68

22. 2
4-3

1.25
9.66
7.71

08
46

I. 29

10. 29

7-94

TREE 4

percentage of

Acid as
citric .

Percentage of reducing
sugar as dextrose

Percentage of cane
sugar as dextrose . . . .

Percentage of total sugar
as dextrose

Percentage of dry mat-
ter

Percentage of peel

Thickness of peel (in
mm.)

Percentage of shrinkage .

Acidity of juice as per-
centage of citric

Soluble solids (Brix) . . .

Solids-acid ratio

I I. 18
[ I. 10
f 2. 70
1 2.66
f 2. 62
^ 2.56
^ 5-32

I 5-22
9. 28

I 9-53
22

5-4

1.36

9-35
6.86

94
03
,18
80
31
69
49
49

I. 17
ro. 01
8.55

1.24

3-04
1.78
.^.82

58
57
3

4-S

I- 31
10.23

7-79

I. 04

4.9
2,- 2,

I. 04

10. 16

9-73

I. 21
I. 22
3-75
3-85
3. 10
3.02
6.85
6.87
9.87
9.90

I. 26

10.86

8.60

From Table III it is evident that in every case there is a lower acidity
in the fruit after it has been held in storage four months than in fruits
from the same tree and picking when placed in storage. This is in accord-
ance with the findings reported in the previous publication and would
seem to establish this point definitely. The fact that in the present
experiments fruit was picked at three dififerent times during the growing
and ripening season strengthens the evidence.

Oct. 29, 1921 Physiological Study of Grapefruit Ripening 273

The total sugar content is usually slightly higher at the end of the four
months' storage period, though there are several instances in which it is
lower. These cases are mostly in well-matured fruit of the last pick.
The increase in total sugars is due for the most part to an increase in
the reducing-sugar content, as there is usually a marked decrease in the
percentage of cane sugar during storage. There is never more than 5 per
cent shrinkage during these four months. This shrinkage is doubtless
partly from the peel and partly from the interior portion or pulp.

The fact that in most cases there is an apparent increase in total sugars
can be accounted for by the loss of water and consequent shrinkage. It
is very evident from these data that there is no appreciable diminution
in the amount of sugar in the grapefruits in four months at 32° F. On
the other hand, there is without doubt no considerable increase. It is,
of course, probable that some of the pectins and other hemicelluloses or
the glucosid in the fruit break down slowly, and it is possible that some
reducing substance is formed from these decomposition products.

A comparison of the behavior of the acids and sugars in grapefruits
stored in warm storage (Tables I and II) with the results obtained in the
cold storage experiments just considered brings out some rather striking
differences. In the data obtained from the warm storage experiments
there is evidence of an increase in acidity or a decrease in total sugars or
both — that is, in most cases the ratio of total sugar to acid decreases, while
in the cold storage the reverse is true. This is corroborated by the acidity
and soluble solids of the juice. In the warm storage experiments the
solids-acid ratio is in most cases less after two months in storage, while in
the cold storage there is always a decrease in acidity and an increase in
solids-acid ratio. It is evident that there is an increase, or at least not
a decrease, in acidity in warm storage and a decided decrease in cold stor-
age. It would, therefore, seem probable that some of the processes which
go on in the fruit stored in the warm are modified when the fruit is placed
in cold storage. It is possible, of course, that in respiration the acid is
used up in cold storage while the sugars are used in warm storage. There
is an indication that the sugar content may decrease slightly in the fruits
held in warm storage, while there is no evidence of change in the per-
centage of sugar in the cold-stored fruits. Magness (9) has shown that the
composition of the gases in the interior of apples and potatoes varies
with the temperature at which they are held. For example, he found
that the gas from the interior of Yellow Newtown apples stored at 2° C.
(about 35° F.) analyzed 14.2 per cent O2 and 6.7 per cent CO2, while at
30° C. (86° F.) the extracted gas was 3.2 per cent O2 and 21.4 per cent
COj. The air surrounding the fruits used in these experiments was practi-
cally the same in both cases. The oxygen content was low and the carbon-
dioxid content high in the fruit at high temperatures because the oxygen
was used up in respiration faster than it could diffuse in from the outside.
While no such determinations have been made on grapefruits, it seems

274 Journal of Agricultural Research voi. xxii. no. s

probable from the size of the fruit and the thickness and structure of the peel
that in fruit held at high temperatures for any considerable period there
would be a low oxygen pressure. This might result in some intermolecular
respiration and the formation of acid. At low temperatures the respira-
tion rate would be considerably decreased, while the rate of diffusion of
O2 through the tissues would not be so greatly reduced, and sufficient
oxygen might be present for the breaking down of the compounds used
in respiration of COj and HgO. A careful investigation of this point is
needed. The work of Gerber {4) is of interest in this connection.

The dry weights are about the same at the conclusion of the experi-
ments as at the beginning. There may be a slight diminution in the
percentage of dry matter, but this apparently lies within the experi-
mental error of the determinations. The variation in thickness of peel
and percentage of peel is so great that thera is frequently a higher per-
centage of peel after the fruit has been stored four months than when it
was placed in storage. This is undoubtedly due to the lack of uniformity
in the fruits and the low percentage of shrinkage.

The loss in weight during the four months' storage is from 3 to 4.9 per
cent, averaging around 4 per cent. The relative humidity of the storage
rooms was around 75 per cent. The fruit was not in the best condition
for merchandising at the end of this storage period, as it was in many
cases badly pitted. It is doubtful whether this method of storage would
be applicable to commercial conditions if the fruit were placed directly
in cold storage.

EXPERIMENTS IN THE CONTROL OF PITTING

As was mentioned in the earlier paper (7) , grapefruit tends to pit in
cold storage. This pitting begins as a small indentation of the skin in
practically any region of the surface. The sunken area gradually in-
creases in size, frequently becoming as much as i cm. in diameter. They
are usually, in the type of fruit used in these experiments, about i mm.
in depth. In time they may take on a brown color. This coloring occurs
more quickly if the fruit is removed to a warm room. These pits may be
very numerous on the surface of the fruit, in many cases coalescing in
irregular shaped patches.

Cross sections of these pits show that they are formed by a breaking
down of the layer of tissue containing the oil vesicles. There is appar-
ently no disintegration of the tissue. The cells and vesicles simply flatten
out as if subjected to local pressure, the layer of tissue becoming brown.
The injury apparently does not extend to any distance in the spongy
tissue ben-ath this oil-bearing layer, and it is only after a long period that
any evidence of the discoloration appears on the inside of the peel. The
pulp of the fruit is apparently uninjured. The affected fruit, however,
is very unsightly, and badly pitted fruit would hardly be salable in a
normal market. It was evident that unless some method of preventing

Oct. 29, 1921 Physiological Study of Grapefruit Ripening 275

this pitting was worked out the storing of grapefruit for any considerable
period would hardly be commercially practicable.

Experiments were, therefore, undertaken to see if it were possible to
treat or handle the fruit so that it could be cold-stored without this danger
of pitting. As was mentioned earlier, fruit stored in warm storage, 70°
to 86° F., or in common storage (about 55° to 60°) apparently does not
pit. It was considered possible that if fruit were cured for a time in warm
storage before being placed in cold storage the injury from this blotching
and breaking down of the surface of the peel might be obviated. Ac-
cordingly a lot of I dozen fruits from tree i , third pick, was maintained at
a temperature of 70° and a humidity of about 65 per cent for one month,
then removed to cold storage (32°) and xamined at intervals. At the
end of three months in cold storage none of these fruits were pitted, while
about 60 per cent of the fruit from the same lot olaced directly in cold
storage at 32° were badly pitted.

The experiment was repeated with grapefruits of the Duncan, Marsh
Seedless, and Silver Cluster varieties from Polk County, Fla., which were
placed in storage February 12, 1920. Part of the fruit of each lot was
placed directly in 32° F., and the rest of the three lots were placed in the
curing room and maintained at a temperature of about 70° with a relative
humidity around 60 per cent. Portions of the lots from the curing room
were removed to 32° cold storage at intervals. The entire storage period
was three months for all lots. The results of the experiment are shown
in Table IV, in which are given the length of time in curing, the time in
cold storage, and the percentage of pitting of the different lots. In
these experiments the pitting is given as slight and bad pitting. Bad
pitting is applied to pitting that would markedly injure the sale of the
fruit. Slight pitting refers to pitting that while noticeable does not par-
ticularly injure the fruit for sale. It is at most a few spots usually small.

It is noticeable in Table IV that most of the control fruit that was
placed directly in cold storage without curing is pitted and that there is a
high percentage of bad pitting. In the Duncan, 6 per cent was good,
while the poorest lot of cured fruit of this variety was about 90 per cent
good. There was more pitting in the cured Silver Cluster than in the
Duncan and somewhat more in the Marsh Seedless than in the Silver Clus-
ter. The data obtained in this one storage experiment are hardly suffi-
cient, however, to justify the conclusion that Duncan grapefruit store
better than Silver Cluster and Marsh Seedless. The experiments, how-
ever, seem to show that the pitting can be controlled by proper curing
before the fruit is placed at the low temperatures. The specific effect of
this curing, by exposure to warm temperatures from one to six weeks,
on the tissue of the peel so that the pitting is prevented has, of course,
received little attention. Pitting has all the external appearance of
injury considered to be due to Colletotrichum gloeosporioides (Penz.) by
Rolfs, Fawcett, and Floyd (zj) and figured by them. This fungus,

276

Journal of Agricultural Research voi. xxii, No.

however, has a high optimum temperature, and it seemed highly improba-
ble that its growth could be controlled by exposing to high temperatures
and that it caused most damage at temperatures around 32° to 40° F.
It was possible, however. This point was investigated by Dr. F. V.
Rand, of the Laboratory of Plant Pathology, Bureau of Plant Industry.
The results of this work are as yet unpublished. The following account,
however, is based on Dr. Rand's work.

Table IV. — Results of storage experiments with Duncan, Marsh Seedless, and Silver

Cluster grapefruit

Num-
ber of
fruits.

Num-
ber of
days in
curing
room.

Num-
ber of

days in
cold

storage.

Tem-
pera-
ture of

cold
storage.

Num-
ber of
good
fruits.

Per
cent-
age of
good

fruit.

Num-
ber
with
slight
pitting.

Per-
centage
with
slight
pitting.

Num-
ber
with
bad

pitting.

Per-
centage

with

bad
pitting.

5°
27

23
14
10

a 00

19
25

90
78
71
65
49

°F.
32

32
32
32
32

3
24

23
13
10

88.9
100

92.8
100

10
3

20
II. I

7-1

MARSH SEEDLESS

82
42
30
19

a 00
10
18
24
30

90
80
72
66
60

32
32
32
32
32

3
II
12
16
10

3-6
26.2
40
84.2
83-3

7-3

73
31
12

89
73-8
40

3
2

15.8

16.7

Sn,VER CLUSTER

46
16
17

a 00

90
81
72
65

32
32
32
32

23
14
15
17

87-5

88.2

5°

2
2
3

12.5
II. 8
15

" Controls placed directly in cold storage.

Cultures were made from the pits and from the tissue of the peel
between the pits. Cultures were also made from the peel of cured fruits
which had been in cold storage for three months after curing. The
results are shown in Table V.

It is evident from Table V that Colletotrichum was almost univer-
sally present in the peel of these Florida grapefruits and that while it
is usually to be found in the pit it is just as common in the normal peel
of the pitted fruit or the cured fruit. It is, of course, impossible to
assert from the evidence at hand that the fungus does not cause the
breaking down of the peel. The cold storage might so affect the physi-
ology of the peel as to make it susceptible to fungus attacks, while curing

Oct. 29. 192 1 Physiological Study of Grapefruit Ripening

277

and warm storage render it resistant. This, of course, is somewhat
doubtful. The case is somewhat analagous to that cited by Winston
(15) in regard to tear stain, which has up to now been considered to be
due to Colletotrichum gloeosporioides, mainly because this fungus was
usually found in cultures from the diseased areas. In the present work
it seems fair to conclude that whether or not the fungus causes the pit-
ting it is controlled at least to a large extent by curing before placing the
fruit in cold storage.

Table V.

-Results of cultural experiments with pitted grapefruit and with fruit from
same lot which was unpitted «

Date.

Source.

Num-
ber of
fruits.

Num-
ber of
pieces

of
tissue.

Col-
leto-

tri-
chum.

Clado-
spo-
rium.

Alter-
naria.

Pusa-
rium.

Peni-

cil-

lium.

Ster-
ile.

Mis-
cel-
lane-
ous
fungi.

Pits

Between
pits

Pits

Between
pits

Pits

JBetween
pits

Control . . .

[Pits

JBetween

1 pits

(Control . . .
[Pits

Between
1 pits

Control . . .

Pits

JB etween
1 pits

Control . . .

[Pits

JBetween
1 pits

Control . . .

I
2

4
5
3

2
I
3

3
3

2
2
5

2
4

7
3i

17
53

29-
70
32

27
14
38

17
23
38

24
20
93

38
84

I
17

10
30

15
42
21

12
14

I
16

3
3

3
4

22 i 4

1 TI

Feb. I, i92i(not sterilized)

Feb. 3. 1921 (sterilized 3

3
16
38

15
17
68

27
63

I
I
2

Feb. 3, 1921 (not sterilized)

4
10

Feb. 8, 1921

7
S

o Unless otherwise stated, all pieces of tissue were sterilized two minutes in i to 1,000 bichlorid solution
and were washed three times in sterile tap water before pouring plates. The control fruits were without
signs of the pitted spots under investigation. ' ' Between pits ' ' refers to sound tissue between the spots.

GENERAL DISCUSSION AND CONCLUSION

In the investigation of grapefruit storage described in the foregoing
pages it has been brought out that in warm storage the percentage of
acid calculated to the wet weight of the pulp increases markedly in two
months' storage. There is evidence that this increase is not due entirely
to loss of water from the pulp, but that there is an increase in the amount
of acid present. There is evidence indicating that there may be a slight
decrease in the sugar content in warm storage. In cold storage there is a
decrease in the acidity very marked after four months in storage, while
there is little change in the amount of total sugars present. A possible
explanation of this difference in the behavior of the sugars and acids in
warm and cold storage was pointed out. This phase of the problem
deserves further attention. The investigations on the changes in the

278 Journal of Agricultural Research voi. xxii, no. s

fruit during development on the tree showed that the total sugar con-
tent increased while the acidity decreased, the increase in sugar content
being very marked.

Fruit on the tree increases in palatability and food value. There is,
of course, always danger that the seeds will sprout in the varieties
containing seeds if the fruit remains on the tree too long. There is also
danger that the fruit will drop or be shaken from the tree by high winds.

It is of interest to note that the behavior of the acids and sugars
during growth and in cold storage is similar to the behavior of these
constituents of some of the deciduous fruits — that is, it is apparently
possible to remove the fruit from the tree after it is well along toward
maturity and to ripen it in storage. The result will be an apparently
sweeter fruit, due to loss of acidity and a reduced bitterness, the naringin
or bitter principle breaking down in storage. A period in cold storage,
then, renders the fruit more palatable. From the experiments detailed
above it seems probable that the pitting of grapefruit can be controlled
by curing at 70° F. before they are placed in cold storage. Investiga-
tions are in progress at the present time on this last-mentioned phase of

the Avork.

LITERATURE CITED

i) BiGELOW, W. D., and GorE, H. C.

1905. STUDIES ON PEACHES... U. S. Dept. Agr. Bur. Chem. Bui. 97, 32 p.
2) and Howard, B. J.

1905. STUDIES ON APPLES... U. S. Dept. Agr. Bur. Chem. Bui. 94, 100 p.,
30 fig., 5 pi.

3) COLLINSON, S. E.
I913. SUGAR AND ACID IN ORANGES AND GRAPEFRUIT. Fla. Agr. Exp. Sta.

Bul. 115, p. 1-23.

4) Gerber, Charles.

1896. RECHERCHES SUR LA MATURATION DES FRUITS CHARNUS. In Ann. Sci.

Nat. Bot., ser. 8, t. 4, no. 1/6, p. 1-280, pi. 1-2.

5) Hasselbring, Heinrich.
1918. BEHAVIOR OF SWEET POTATOES IN THE GROUND, /n Jour. Agr. Research,

V. 12, no. I, p. 9-17, I fig.

6) Hawkins, Lon A.

1916. EFFECT OF CERTAIN SPECIES OP FUSARIUM ON THE COMPOSITION OP THE

POTATO TUBER. In Jour. Agr. Research, v. 6, no. 5, p. 183-196.
Literature cited, p. 196.

7) and MagnESS, J. R.

1920. SOME CHANGES IN FLORIDA GRAPEFRUIT IN STORAGE. In JoUr. Agr.

Research, v. 20, no. 5, p. 357-373. Literature cited, p. 372-373.

8) LiNDET, L.
1894. RECHERCHES SUR LE D^VELOPPEMENT ET LA MATURATION DE LA POMME

A cidrE. In Ann. Agron., t. 20, p. 5-20.

9) Magness, J. R.

1920. COMPOSITION OF GASES IN INTERCELLULAR SPACES OP APPLES AND POTA-
TOES. In Bot. Gaz., v. 70, no. 4, p. 308-316, i fig. Literature cited,
p. 316.

Oct. 29, 1921 Physiological Study of Grapefruit Ripening 279

(10) Magness, J. R.

1920. INVESTIGATIONS IN THE RIPENING AND STORAGE OF BARTLETT PEARS.

In Jour. Agr. Research, v. 19, no. 10, p. 473-500, 8 fig. Literature
cited, p. 499-500.

(11) Rivi^rE, Gustave, and Bailhache, Gabriel.

1908. iSTUDE relative a la PROGRESSION ASCENDANTE DU SUCRE ET A LA
PROGRESSION DESCENDANTS DE l'ACIDITE;, DANS LES FRUITS DU
POIRIER, DEPUIS LEUR FORMATION JUSQU'A LEUR MATURITY. In

Jour. Soc. Nat. Hort. France, ser. 4, t. 9, p. 284-289.

(12) RiTTER, Georg.

1910. UEBER DEN CHEMISCHEN REIFUNGSPROZESS DER FRUCHTE, MIT BESON-

DERER BERUCKSiCHTiGUNG DES OBSTES. In Deut. Obstbauztg.,
Jahrg. 56, Heft 31, p. 429-435-

(13) Rolfs, P. H., Fawcett, H. S., and Floyd, B. F.

1911. diseases of CITRUS FRUITS. Fla. Agr. Exp. Sta. Bui. 108, p. 25-47,

fig. 10-23.

(14) Sando, Charles B-

1920. THE PROCESS OF RIPENING IN THE TOMATO, CONSIDERED ESPECIALLY

FROM THE COMMERCIAL STANDPOINT. U. S. Dept. Agr. Bul. 859, 38

p., 3 fig., 4 pi. (1-2 col.). I,iterature cited, p. 32-35.

(15) Winston, John R.

1921. TEAR-STAIN OF CITRUS FRUITS. U. S. Dept. Agr. Bul. 924, 12 p., 2 pi.

Literature cited, p. 12.

ABSORPTION OF COPPER FROM THE SOIE BY POTATO

PLANTS

By F. C. Cook

Physiological Chemist, Miscellaneous Division, Insecticide and Fungicide Laboratory,
Bureau of Chemistry, United States Departm.ent of Agriculture

RESULTS OF PREVIOUS INVESTIGATIONS

Some of the results obtained by a few investigators on the absorption
of copper by plants and cells may be summarized as follows :

Schander^ found that copper in a soluble form is a poison for plant
cells of both high and low order.

Tschirch - believes that living plants are able to absorb copper through
their roots and also through the epidermis of the leaves, the amount of
copper absorbed being very small, however.

Haselhoff ^ stated that soluble copper salts are injurious to plants at
a concentration of lo mgm. of cupric oxid per liter. When soluble cop-
per salts are added to the soil the plant materials, especially the potash
and the lime, are dissolved and washed away, as a consequence of which
the fertility of the soil is decreased. The action of copper sulphate is
more severe on some crops than on others. The presence of calcium car-
bonate in the soil prevents or decreases the toxicity of solutions of copper
sulphate.

True and Gies ^ have shown that when lime is used with copper sul-
phate solutions the toxicity of the copper is decreased. They state that
when there is lime in the soil four times the amount of copper that can be
allowed when no lime is found may be present in a soil without exerting
a toxic action.

Forbes ^ found that com grown in soil containing copper held most of
the copper in the roots rather than in the tops. He states also that the
toxicity of copper depends on the combination in which it exists in the
soil, the physical characteristics of the soil, and the chemical composition
of the soil, and on climatic and moisture conditions, as well as on the crop
grown.

iScHANDER, Richard, uber die PHYSIOWJCISCHE WIRKTTNG DER KT7PFERV1TR10UCAI.KBRUHE. In
Landw. Jakrb., Bd. 33, Heft 4/5, p. 517-584. 1904.

2 Tschirch, A. das kxtpfer vom standpunkte der gerichtuchen chemie, toxicologie und hy-
giene. 138 p., 2 fig. Stuttgart. 1893. Bibliographical footnotes.

'HaSELHOFF, Emil. UEBER DIB SCHADIGENDB WIREtTNG VON KUPFERSULFAT UND KUPFERNITRAT-

MALTlGEM WASSER AUF BODEN UND PFLANZEN. In Laudw. Jahrb., Bd. 21, p. 263-276, 2 pi. 1892.

* True, Rodney H., and GiEs, William J. on the physiologicai, action of some op the heavy
METALS IN mixed solutions. In Bvd. Torrey Bot. Club, v. 30, no. 7, p. 390-402. 1903.

^ FoBLBES, R. H. certain effects under irrigation of copper compounds upon crops. Ariz. Agr.
Exp. Sta. Bui. 80, p. 145-238, 16 fig., 4 pi. (i col.). Bibliography, p. 236-238.

Journal of Agricultural Research, Vol. XXII, No. s

Washington, D. C. Oct. 29, 1921

aah Key No. E-17

(281)
65768°— 21 4

282 Journal of Agricultural Research voi. xxii, No. s

OBJECT OF PRESENT EXPERIMENTS

The experiments discussed in this paper were undertaken to determine
what proportion of the copper present in standard Bordeaux spray, in
Pickering's limewater Bordeaux spray, and in a solution of copper sul-
phate, of equal copper content, is absorbed by potato plants when the
sprays or solution are applied directly to the soil in which the vines are
growing. The comparative distribution of the absorbed copper in dif-
ferent parts of the potato plants was also studied.

The copper in the Pickering spray was in an insoluble form, basic cop-
per sulphate, with no excess of lime present. The copper of the Bordeaux
spray was in an insoluble form, with a large excess of lime present. The
copper of the solution of copper sulphate v/as soluble. It was believed
that a comparative study of these three sprays, containing copper in equal
amounts, would show the extent to which the excess lime of Bordeaux
spray is instrumental in preventing the absorption of copper by the roots
of the potato plants, as well as the relation of the absorption of copper
from a soluble copper compound to that from an insoluble copper com-
pound when applied to the soil.

EXPERIMENTAL WORK

The tests were conducted on the Aroostook Farm of the Maine Agri-
cultural Experiment Sta.tion, at Presque Isle, Me., on Caribou type soil.
A single row, 8 feet long, of Norcross strain of the Green Mountain variety
of Irish potato plants was used for each of four plots which were treated
in the following manner : Plot i , sprayed with standard Bordeaux, 3-3-50
formula, containing 0.75 per cent of copper sulphate; plot 2, sprayed
with an "A ' formula Pickering limewater Bordeaux spray, containing
0.70 per cent of copper sulphate; plot 3, sprayed with a solution con-
taining 0.75 per cent of copper sulphate; and plot 4, a control plot, un-
sprayed.

The vines were 20- inches above ground when the first applications
were made. At each application i gallon of the spray or solution was
applied directly to the ground within 6 inches of the stems of eight potato
plants in each plot, each vine thus receiving i pint of the solution to each
treatment. An equal amount of water was applied to the roots of eight
control plants at the time the other applications were made. Applica-
tions were made on July 27, August 8, August 17, August 24, and August
30, 1917.

PREPARATION OF SAMPLES

Vines and tubers from each of the four plots were taken for analysis
at frequent intervals.

The vines from the various plots were dried in the air, then washed in
running water and held for 30 seconds in a 4 per cent solution of hydro-
chloric acid, after which they were immediately washed in water and

Oct. 29, 1921 A bsorption of Copper from the Soil by Potato Plants 283

finally in distilled water. The vines were next dried for 16 hours in an
oven at 110° C. Separate analyses of leaves, stems, roots, and tubers
were made.

Five or six tubers from each plot were thoroughly washed, rinsed in
distilled water, and dried with a towel. The tubers were pared, passed
through a grinder, well mixed, and transferred to a Mason jar with rubber
and top. Care is necessary in securing a uniform sample of the ground
tubers for analyses, as the water and solids separate very rapidly.

Samples of soil were taken 6 inches deep, near the roots of the treated
plants, from the various plots at the time the plants were sampled. The
soil samples were held in Mason jars with rubbers and tops until analyzed.
Before analysis the stones and other foreign matter were removed from
the samples.

DETERMINATION OF COPPER IN VINES AND TUBERS

From 5 to 10 gm, of the dried leaves and stems, and from i to 5 gm.
of the roots were taken for copper analyses. The samples were ashed
in 4-inch porcelain dishes, 30 cc. of 5 per cent nitri acid were added, and
the whole was allowed to remain overnight. The solutions were filtered
and washed, after which ammonia was added to faint alkalinity. They
were brought to a boil, cooled, and made to volume, usually 150 cc. The
precipitated iron and alumina were removed by filtration, and an aliquot
of the filtrate was taken for the determination of copper.

Table I. — Copper found in potato vines and tubers'^

Plants taken for analysis.

Aug. 8..
17-

24.

Sept. 3.
Average

Parts analyzed.

Leaves.
vStem.. .
Root...
Leaves .
Stem . .
Root...
Tubers.
Leaves.
Stem.. .
Root...
Tubers.
Leaves.
Stem . .
Root...
Leaves.
Stem . . .
Root.. .
Tubers.

Soil treated

with Pick-

with Bor-

with CuSOi

ering spray

deaux spray

solution

(0.7s per

(0.75 per

cent

CUSO4).

CuSOi).

CUSO4).

Per ceiit.

Per cent.

0. 004 s
. 001 15

0. 006=;

0. 0070

.0079

.0136

. 0052

. 0097

. 0100

. 0069

.0023

. 0042

.0047

.0036

.0030

. OIOI

. 0004
.0179

. 0001

.0258

. 0109

. 0029

. 0048

. 0067

.0128

. 0104

.0130

. 0001

.00-53

. 0069

.0225

. 0160

. 0069

. 0300

. 0107

.0179

. 0081

.0030

• OO.S7

. 0104

. 0081

. G085

. 0146

. C002

. 0001

Control
plot.

0017
o
0069
0027
0012
0002

<» Analyses made on dry basis.

284 Journal of Agricultural Research voi. xxu.no.s

As a rule, 25-cc. aliquots were evaporated to dryness in 50-cc. por-
celain dishes on the steam bath, and the residue was taken up in 5 cc.
of distilled water. Two drops of acetic acid and 3 drops of i per cent
solution of potassium ferrocyanid were added, and the color was im-
mediately compared with that of standard solutions of copper sulphate
which had been evaporated with ammonium nitrate and taken up in
5 cc. of distilled water.

Copper in the ground tubers was determined by the same procedure,
using 50 gm. of the moist sample. The analytical data are recorded in
Table I.

DieTERMINATlON OF COPPER IN SOEUS

One hundred gm. of the well-mixed soil samples were treated with a
mixture of 80 cc. of nitric acid and 20 cc. of sulphuric acid in large por-
celain casseroles. The mixtures were heated on the steam bath and
til en on the hot plate until the nitric acid fumes were removed. The
residues were extracted with 200 cc. of water, filtered, washed, and made
to 500 cc. volume. After evaporation to 200 cc, the iron was precipi-
tated with ammonia and the solutions were made to volume. They were
next filtered and aHquots were made acid with hydrochloric acid, through
which hydrogen sulphid was passed for 20 minutes, or until all the copper
was precipitated. The precipitated copper after settling was filtered
and dissolved in 10 cc. of nitric acid, the filter paper and precipitate being
transferred together. Ammonia was added to faint alkalinity, and the
solutions were evaporated to dryness in small porcelain dishes. The resi-
dues were taken up in 5 cc. of distilled water, two drops of acetic acid
and three drops of i per cent potassium ferrocyanid were added, and the
copper was estimated by colorimetric comparisons. In some cases after
evaporation to dryness it was necessary to take up in water, filter, wash,
and repeat the evaporation to remove precipitated material.

If present in large enough amounts copper may be determined electro-
lytically, by a method based on the procedure given by Forbes, Free, and
Ross.^

The results of the analyses of the first and last samples of soil taken
appear in Table II. This table gives also the results of a series of tests
on the soil around the roots of potato plants which had been commer-
cially sprayed with Bordeaux, with Pickering spray, and with a solution
of copper sulphate, to determine whether any appreciable amounts of the
copper occur in the soil beneath the sprayed vines.

> Forbes, R. H. certain effects under irrigation of copper compounds xtpon crops. Arlr. Agr.
Exp. Sta. Bui. 80, p. 145-238. 16 fig., 4 pi. (i col.). 1916. Bibliography, p. 236-238. Part 3, Appendix:
Methods of analysis, with the collaboration of E. E. Free and W. H. Ross, p. 229-233.

Oct. 29, 1921 Absorption of Copper from the Soil by Potato Plants 285

Tabl^ II. — Copper found in soil

SPRAYS APPLrED TO SOIL NEAR PLANT ROOTS

Sample
No.

Date of

sampling.

Description of samples.

Description of plots.

Total
copper

found
in soil.

July 26
Aug. 24
Sept. 3

Samples taken before any
copper was added to the
soils.

Samples taken just before
last application of copper
to soil.

Samples taken after the last
application of copper to
the soil.

Control

P. p.m.
2

3
4
5
6

7
8

9
10

Pickering "A" formula

Bordeaux ^-^-c;o

I
2

CUSO4 solution

Control

S
211
256
250

Pickering "A" formula

Bordeaux •?— •?— t;o

CUSO4 solution

Control

Pickering "A" formula

Bordeaux 3—3—50

225
243

449

CUSO4 solution

SPRAYS APPLIED TO VINES IN COMMERCIAL PRACTICE

July 16

Aug. 31

Samples taken before any
sprayings were made in
1917.

Samples taken after last
sprayings were made in
1917.

Control

Pickering "C" formula.

Bordeaux 5-5-50

Pickering "A" formula

Control

Pickering "C" formula

Bordeaux 5-5-50

Pickering "A" formula

DISCUSSION OF RESULTS

VINES AND TUBBRS

The leaves, stems, and roots of the plants from the soil receiving
the Pickering spray showed an increased copper content with each
successive analysis (Table I). The largest percentage of the copper was
held by the leaves. The roots held an appreciable part of the copper,
the amount increasing from o in the first sample to 0.0160 per cent in
the sample taken on September 3. The tubers contained only minute
amounts of copper.

The plants from the Bordeaux treated soil showed irregularities,
particularly with respect to the copper content of the roots and stems.
The leaves and stems contained more copper than those of the plants
from the Pickering treated soil, while the roots contained less copper
than the roots of the plants from the Pickering treated soil. The
amounts of copper found in the tubers were small.

The vines grown in the soil treated with a solution of copper sulphate
showed a marked progressive increase in copper content of the roots
with each succeeding analysis. The leaves contained somewhat larger

286 Journal of Agricultural Research voi. xxn.No. s

amounts of copper than the stems, but not as much as the roots. The
leaves contained less copper than the leaves of the plants grown on the
Bordeaux or Pickering treated soils. The tubers from the plot treated
with copper sulphate solution were as low in copper as those from the
other plots.

The analyses of the various portions of the conti'ol plants showed the
presence of copper, but in smaller amounts than in the plants grown on
soil treated with the copper sprays.

The results of the copper absorption experiments indicate that the
potato plants, with the exception of those grown in the soil receiving
the solution of copper sulphate where the roots were distinctly injured,
distributed the largest part of the absorbed copper to the leaves, while
the roots and stems contained appreciable amounts of copper. In all
normally sprayed potato plants the largest proportion of the copper is
said to be found in the leaves.

The plants grown on the soil treated with a solution of copper sulphate
were small and lacking in vigor. The roots had but few hairs, and
showed other signs of injury. The large percentage of copper found in
the roots, together with the small size of the roots, indicated some inter-
ference with the normal metabolism of the vines. The toxic effect of
the soluble copper salt was exerted primarily on the roots of the plants.
It was apparent that the soluble copper sulphate had injured the potato
plants, while the insoluble copper compounds had not.

The vines from the Bordeaux plot contained a little more copper than
the vines from the Pickering plot, indicating that the extra lime of the
Bordeaux spray did not aid in preventing the absorption of copper by

the plants.

soil,

The results of the analyses of the first and last samples of soil taken
show that no water-soluble copper was found in any of the samples
examined. The amount of copper in the first set of samples (Table II,
No. 1, 2, 3, and 4) which were taken before any copper had been added
to the soils, is practically the same in all cases. The sets of samples
taken before and after the final treatment of the plots show the pres-
ence of a large amount of copper in the samples receiving the copper
treatments. This means that copper in an insoluble form may be pres-
ent in the soil in marked amounts without exerting any apparent toxic
action on the growth of potato plants.

But little copper was found in the soil as a result of spraying with
copper sprays according to commercial practice.

On September 5, shortly after the last treatment of the soil, a few hills
of potatoes were dug. The weights and number of the tubers, the per-
centage of decayed tubers, as well as the notes taken on the size and
appearance of the vines are given in Table III.

Oct.29, I92I Absorption of Copper jrofyi the Soil hy Potato Plants 287

Table III. — Effect on potato tubers and vines of applications of sprays to soil

Tubers."

Spray used.

Weight.

stand and condition
vines.

Total.

Average.

Ounces.

Bordeaux (0.75 per

3 (i large and 2

Normal.

cent CUSO4).

small) in ihill.

Pickering (0.7 per

8 (5 large and 3

Do.

cent CuSOJ.

small) in 2 hills.

Copper-sulphate solu-

17 (all small) in 4

Small and stunted.

tion (0.75 per cent

hills.

CUSO4).

Unsprayed (control) .

8 (3 large, 3 medium,
and 2 rotten) in 2
hills.

Normal stand ;
blight.

a Rot found only on control tubers.

These data show tliat the solution of copper sulphate had a very dis-
astrous effect on the growth and yield of the tubers. The only decayed
tubers found were obtained from the unsprayed plot. These results are
so few that they can be considered only as suggestive.

vSUMMARY

Potato plants grown in soil treated with insoluble copper compounds
contained more copper in the leaves than in the stems, while but little
copper was found in the roots. The tubers showed only traces of copper.

When the soil was treated with the copper sulphate solution, the roots
were injured and the normal metabolism of the vines was disturbed.
The tubers from these vines were small and the vines stunted. The roots
of these plants held more copper than the leaves.

The soluble copper sulphate added directly to the soil caused injury
to the plants, while the insoluble copper compounds of the sprays did
not. The excess lime of the Bordeaux spray did not reduce the amount
of copper absorbed by the plants compared with the plants grown on
the Pickering plot.

Practically the same amounts of copper were found in all the soil
samples tested. Samples of soil from sprayed potato fields showed but
minute amounts of copper.

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Vol, XXII NOVKIvIBE^R 5, 1921 No. 6

JOURNAL OF

RE SEAJR.CH

CONXKNXS

Page

Pale Western Cutworm (Porosagrotis orthogonia Morr.) - 289

J. R. PARKER, A. L. STRAND, and H. L. SEAMANS

( Contribution from Montana Agricultural Experiment Station)

Biology of Embaphion muricatum - - - - - 323
J. S. WADE and ADAM H. BOVING

( Contribution from Bureau of Entomology )

PUBUSHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE,

WITH THE COOPERATION OF THE ASSOCIATION OF

LAND-GRANT COLLEGES

WASHINGTON, D. C.

GOVERNMENT PRINTING OFFICE

1921

EDITORIAL COMMITTEE OF THE

UNITED STATES DEPARTMENT OF AGRICULTURE AND

THE ASSOCIATION OF LAND-GRANT COLLEGES

FOR THE DEPARTMENT

KARL F. KEIyI<ERMAN, Chairman

Physiologist and Associate Chief, Bureau
of Plant Industry

EDWIN W. ALLEN

Chief, Office of Experiment Stations

CHARLES L. MARLATT

Entomologist and Assistant Chief, Bureau
of Entomology

FOR THE ASSOCIATION

J. G. LIPMAN

Dean, State College of Agriculture, and
Director, New Jersey Agricultural Experi-
ment Station, Rutgers College

W. A. RILEY

Entomologist and Chief, Division of EnU^
mology and Economic Zoology, Agrieui-
tural Experiment Station of the University
of Minnesota

R. L. WATTS

Dean, School of Agriculture, and Director,
Agricultural Exptrimenf Station; Tks
Pennsylvania State College

All correspondence regarding articles from the Department of Agriculture should be
addressed to Karl F. Kellerman, Journal of Agricultural Research, Washington, D. C.

All correspondence regarding articles from State Experiment Stations should be
addressed to J. G. Lipman, New Jersey Agricultural Experiment Station, Nev7
Brunswick, N. J.

JOURNAL OF AGMCETIMLEESEARI

Vol. XXII Washington, D. C, NovEm^'^*^?2i No. 6

PALE WESTERN CUTWORM (POROSAGROTIS
ORTHOGONIA MORR.)

By J. R. Parker, Assistant Entomologist, Montana Agricultural Experiment Station;
A. L. Strand, Assistant State Entomologist of Montana; and H. L. Seamans,
Special Assistant in Cutworm Investigations, Montana Agricultural Experiment
Station '

INTRODUCTION

Extensive injury to grain crops by cutworms was reported from north
central Montana during the period from 1915 to 1920. It was at first
supposed that well-known species were responsible for the damage and
the usual method of control, poisoned bran mash, was recommended.
The repeated failure of this method led to a study of the situation, and
from the results of numerous rearing records and the personal investiga-
tion of many infested fields it was found that the greater part of the losses
was caused by the pale western cutworm {Porosagrotis orthogonia Morr.),
a species previously not considered of economic importance in Montana.
The enormous damage which it has done during the last six years, the
rapidity with which it has extended its range, its unusually long period
of larval feeding, its comparative freedom from parasites, and the fact
that it works underground and can not be controlled by poisoned bran
mash, stamp P. orthogonia as the most dangerous of all our western
grain cutworms, not excepting even the army cutworm {Chorizagrotis
auxiliaris Grote).

HISTORY OF THE SPECIES

The species was given its specific name in 1876 by Morrison (15),^
who described it as A gratis orthogonia from specimens collected at
Glencoe, Nebr. In 1890 the species was placed under the genus Porosa-
grotis by Smith {16, p. 129), who also gave a description of the adult
and recorded its occurrence in the following new localities: Colorado,
New Mexico, Arizona, and Utah. Dyar {j, p. 139) lists the species and
gives its range as the Rocky Mountain region. In 1905 it was reported

1 The color plate and drawings for this article were done by Miss Helen Lund, with the remarkable
accuracy characteristic of her work. Mr. K. M. King, an undergraduate assistant in 1919, conducted the
rearing experiments during that year, and many of the observations recorded are based on his very complete
insectary notes.

^ Reference is made by nxunber (italic) to "Literature cited," p. 320-321.

Journal of Agricultural Research, Vol. XXII, No. 6

Washington, D. C. Nov. 5, 1921

aai Key No. Mont.-8

(289)

290 Journal of Agricultural Research voi. xxii, no. 6

(doubtfully) by Dod {2, v. 57, p. 53) from Calgary, and in 1908 by
Hampson (9, p. 102) from Prairie, Alberta.

The species was looked upon as a rare insect until 191 1, when Gibson
(5, 6) reported it under the name Porosagrotis delorata Smith as destroy-
ing large areas of wheat in southern Alberta, where one correspondent
claimed to have lost 320 acres before June 21. Hewitt {10, p. ijj) also
refers to this outbreak in his annual report for 19 12, and in his report
(jj, p. 506) for the following year the species is recorded along with
Euxoa ochragasier Gn. as having destroyed between 30,000 and 35,000
acres of crop in 19 12 in about the same territory where damage occurred
the previous year.

An account of the insect's first appearance, its depredations during
191 1 and 19 1 2, and a brief review of the control experiments carried on
the following year was given by Gibson (7) in 19 14. In this article he
states that Porosagrotis delorata Smith and P. orthogonia Morr. are the
same and records an adult of the species as having been taken at Regina,
Saskatchewan, on August 10, 1904. Hewitt {12, p. 861-862) states
that in 19 13 this cutworm caused much less damage to crops in southern
Alberta than in the preceding year.

The most complete published account of the species was written by
Gibson (<?, p. 30-31) in 19 15. A brief description of the larva and adult
is given, together with notes on its life history and habits. In this
article the common name "pale western cutworm" is used, apparently
for the first time.

During the season of 19 14 Porosagrotis orthogonia was again present
in Alberta, and experiments in its control were conducted by Strickland
(17), who found that surface applications of the bran mash were wasted
but that gratifying results were secured when a molasses-and-shorts
mixture was harrowed into the soil. The next account is by the same
author {18), who gives a brief statement of the life history and makes
recommendations for the control of the species by cultivation methods
and the modified use of a poisoned-shorts mixture.

According to Hewitt's 191 6 report {13), the pale western cutworm was
seldom seen in 19 15. Experiments, however, were conducted by Strick-
land which confirmed his earlier conclusions that shorts is preferable to
bran and that when the soil is moist harrowing in the poison is not so
advantageous as it is on dry soil.

In his 19 19 annual report (j, p. 8) Cooley points out the habits of the
species which make it such an important pest and places its control as
one of the most important entomological problems in Montana.

A review of the life history of the species, descriptions of the various
stages, and colored drawings of ^gg, larva, and adult, are given by Maxson
{14, p. 45-46) in his work on sugar-beet insects published in 1920.

Nov. 5>i92i Pale Western Cutworm 291

DISTRIBUTION

Published records of the occurrence of Porosagrotis orthogonia are as
follows: Glencoe, Nebr., by Morrison (15, p. 2jg) ; Colorado, New Mexico,
Arizona, and Utah by Smith {16, p. i2g) ; Rocky Mountains by Dyar
(j. P- 139) ; Calgary, Alberta, (doubtfully) by Dod {2, 37, p. 53) ; Prairie,
Alberta, by Hampson (9, p. 102) ; and southern Alberta by Hewitt
{10, p. 177), Gibson (7) and Strickland {17).

In Montana Porosagrotis orthogonia now occurs throughout the State
east of the continental divide. It has been most abundant in the tier
of counties which lies just east of the foothills of the main range of the
Rocky Mountains and which extends from the Canadian border to
within 100 miles of the southern border of the State.

Mr. E. H. Strickland has kindly given the following information on
the present distribution of Porosagrotis orthogonia in Canada :

Our records indicate that it is practically confined to southern Alberta, extending
as far north as latitude 51° and east to longitude 108°, although it has been recorded
as far as Regina, Saskatchewan. The maximum intensity, however, is confined to
an area that does not extend more than 100 miles east of the Rocky Mountains.

Dr. William Barnes, of Decatur, 111., who has an extensive collection
of western noctuids, has generously furnished the following records of
Porosagrotis orthogonia specimens in his collections: Denver, Oak Creek
Canyon, Lavetta, and Alamosa, Colo.; Deming and Fort Wingate, N.
Mex. ; Provo, Vineyard, and Eureka, Utah; Yellowstone National Park,
Wyo.; Reno, Nev. ; Redington, Ariz.; and Kern County, Calif.

Mr. George M. List states that Porosagrotis orthogonia is fairly com-
mon at Fort Collins, Colo., 430 moths having been taken at a trap dur-
ing the season of 1920. )<;■ ;0 I;

To Mr. C. N. Ainslie, of the Bureau of Entomology, United States
Department of Agriculture, we are indebted for a record of 63 Porosagrotis
orthogonia moths reared from a shipment of larvae from Dickinson,
N. Dak., on June 10, 1920.

From the foregoing records it is evident that Porosagrotis orthogonia
occiu-s at least in scattering numbers throughout the southwestern and
northwestern States with the possible exceptions of Oregon, Washing-
ton, and Idaho, where as yet it has not been collected. Correspondence
with entomologists throughout the territory where P. orthogonia has
been recorded indicates that it has never been of economic importance
outside of the heavily infested areas in Montana and Canada.

METHODS OF STUDYING
IN THE INSECTARY

Larvae were reared in individual tin boxes 1.5 inches in diameter.
The bottom of each box was covered with filter paper which was slightly
moistened at each feeding. This prolonged the freshness of the wheat

292 Journal of Agricultural Research voi. xxn.No. 6

and dandelion which were used as food, and the cans could be easily
cleaned by replacing the filter paper whenever it became soiled.

Pupae were placed in moist, well-pulverized soil in individual glass
vials I inch in diameter and 4 inches deep. Each vial was filled to a
depth of 2 inches with well-firmed soil in which a round hole i inch
deep was punched to receive the pupa, which was placed in it with the
anterior end uppermost. A small twig was placed in each vial so that
the moth upon emerging could hang from it and allow the wings to expand.
The vials were closed by cheesecloth held in place by rubber bands.
The best results were obtained by keeping the pupae in uniformly moist
and mellow dirt. Extreme dryness or excessive moisture often resulted
in the death of the pupse. Uniform moisture conditions were more
easily obtained by allowing water to run slowly down the side of the
tube instead of flooding it over the surface of the soil.

After the moths emerged they were placed in wire s,creen covered
tin cans 3.5 inches in diameter and 2.5 inches deep. An inch of moist
soil was kept in the bottom of the cans, and alfalfa or clover blossoms
were added each day for the moths to feed upon (PI. 30, A) and to
hide under. Cutworm moths of all kinds seem very contented in these
cans, and with Porosagrotis orthogonia no difficulty was encountered in
getting the females to mate and lay eggs.

Eggs were placed on filter paper in pint Mason jars with the caps
lightly screwed down. A few drops of water were placed on the filter
paper from time to time to provide the proper amount of humidity.

IN THE FIELD

A very good opportunity to watch the development of this insect
under natural conditions was afforded in a heavily infested field at Wil-
sall, Mont., in 19 19. This field was first examined on May i and was
visited several times a month all summer. Many fields in other parts of
the State were also visited, but that at Wilsall was the only one where
Porosagrotis orthogonia was followed through all stages of its develop-
ment. During the summer of 1920 a temporary field station was estab-
lished at Willow Creek, Mont., in a district where thousands of acres of
wheat had been destroyed during May and June. Moths appeared in
large numbers during August and September and were under observa-
tion at all hours, both day and night. Special attention was given to
the egg-laying habits, and for this purpose two observation cages were
set up. The cages were 2 -foot cubes with screen wire sides and solid
metal tops. They were placed over sunflower plants, and the ground
inside the cages was covered with soft dirt, stubble, clods, baked earth,
and green plants, thus offering the moths nearly all the natural condi-
tions of the neighborhood. Experiments with trap lights were also
carried on at the Willow Creek field station.

Nov. s. I92I Pale Western Cutworm 293

SEASONAL HISTORY AND HABITS
DURATION OF EGG-IvAYING PERIOD

During the season of 19 19 the eggs were found to be well developed
in the ovaries when the female moth first emerged, and egg laying began
and was completed within a short time under insectary conditions.
The first eggs were obtained from reared moths on August 17, the aver-
age period between emergence and the beginning of egg laying being
four days. Moths collected in the field on August 26, which appeared
to have just emerged, laid numerous eggs the following day and con-
tinued to lay until September 9.

In 1920 no records were available from reared moths, but a study of
moths in the field seemed to indicate that the eggs that season were
not fully developed when the moths first emerged. Thus out of 35
moths examined on August 24 only one had well-developed eggs in the
ovaries. On September i many moths were found with the ovaries
filled with well-developed eggs. These moths were mostly badly rubbed
specimens, indicating that they had probably emerged some little time
before.

Our field observations show that the height of the egg-laying period
is during the last week in August and the first week in September.
Eggs in smaller numbers may be laid during the first three weeks in
August and as late as October i .

WHERE EGGS ARE LAID

The first eggs obtained were from moths confined in tin rearing cans.
When the soil in the cans was dry and light most of the eggs were placed
from X iiich to i inch below the surface and could be found only by careful
searching. When the soil was hard and lumpy eggs were scattered
about on the surface and could be easily seen. In the rearing cans the
eggs were not always laid in the soil. Many of them were placed on
the stems, leaves, and flowers of alfalfa, and frequently scattering eggs
were found on the sides of the cans or on the screen covers. Thus out
of 243 eggs found in one can, 180 were found in the soil, 62 were found
on the stems, leaves, and flowers of alfalfa, and i was found on the
side of the can. Some were laid singly and others were in clumps of
2, 3, or 4, and sometimes as many as 40.

In outdoor cages, where a variety of soil conditions and various kinds
of vegetation were available, eggs were laid only in loose, dry dirt.

Under field conditions the eggs are very difficult to find, and the only
ones we have ever found were secured by carefully examining the soil
at the exact spots where females were seen in the act of egg laying.
Eggs are found most frequently in loose, mellow dirt from X to >^ inch
below the surface. This is an important point, and will be discussed
later in connection v^^ith the habits of the moths and from the standpoint
of control methods.

294

Journal of Agricultural Research voi. xxii. No. 6

NUMBER OF EGGS

The only females upon which we have complete recor ds are five moths
reared and mated in the insectary. They averaged 315 eggs, the lowest
number per moth being 248 and the highest 453. Ten moths brought
in from the field averaged 132 eggs, but these had probably laid numerous
eggs before they were caught. Under normal field conditions the aver-
age number per female is probably between 300 and 400. The records
of the individual moths are shown in Tables I and II.

Table I. — Time between emergence and egg laying, length of egg-laying period, and
number of eggs laid by reared specimens of Porosagrotis orthogonia

Moth
No.

August, 19 19.

Eggs

ovaries

at
death.

Total

28 29

ber
eggs.

E
E

243

D
E
E
E

0
249
0
0
3

248

258

96
66

30s

S4
2SI

2S
12

283

453

E= emerged. D=dead.

Average period between emergence and egg laying 4 days.

Average period of egg laying 3 to 4 days.

Average number of eggs i'^s-

Table II. — Period of egg laying and number of eggs laid by Porosagrotis orthogonia
moths brought in from the field and kept in rearing boxes

Moth
No.

August.

September.

Eggs

ovaries

at
death.

Total

ber
eggs.

Caught

63{
1

100
D
23

6
IS

6
16

]

91
0
88

103

I
0
0

152

167

116

74
5

43
3

1
D
fD

\ 3

\i4
/D
I 8

184

. .do

107

112

4
35
26

IS
20

25
30
32

12
22

134

127

8 ..

...do

(

116

. ..do

\26

106

/
D

D=dead.

Average period of egg laying 3 to 4 days.

Average number of eggs 132.

DURATION OF EGG STAGE

The length of the egg stage is exceedingly variable, depending largely
upon moisture conditions. It may be as short as 1 1 days or may extend
over several months. Eggs laid in the insectary August 19 hatched
August 3c. On September 30 several newly hatched larvae were found

Nov. s, 1921 Pale Western Cutworm 295

at Wilsall. Hundreds of eggs were laid in the breeding cans during the
last week in August and the first week in September, but with the ex-
ception of three larvae which hatched August 30 no eggs hatched unless
they were placed in a very humid atmosphere. The larvae mature
within the eggs in from 10 to 20 days and may remain for months in this
condition, waiting merely for proper moisture conditions to allow them
to break through the eggshell. Examples of this may be of interest.
Moth number 1427-G laid 160 eggs on August 24 and 25. These were
placed on a piece of filter paper in a Mason jar. They were allowed to
stand in the laboratory for 10 days and were then placed in an incubator
and held at day temperature of 80° F. and night temperature of 60°.
At the end of three weeks practically all the eggs had darkened, and the
black heads of the young cutworms could be seen through the eggshells,
but none had hatched. On October 4 a few drops of water were added
to the filter paper, and when the jar was opened 24 hours later 70 larvae
were found to have hatched. On October 1 1 water was again added to
this jar, and 40 more larvae hatched out.

On October 27 a few drops of water were added to another jar of 62
eggs which were laid August 29. In two hours 8 larvae had hatched,
but no more hatched during the next six hours. Twenty-four hours
later all of the eggs had hatched. On October 27 moisture was added to
eggs that had been kept in the greenhouse since August 30, and in two
hours many of them had hatched. On this same date one of these eggs
was placed on a block of plaster of Paris, and water was slowly dropped
upon it from a medicine dropper. At the second drop the larva began
to move within the egg. Soon it began to move its mandibles and after
several attempts the eggshell was punctured, and within 30 minutes
after the first drop of water was added the larva was free from the shell
and actively moving about. On November 20 eggs that were laid
August 30 and had been kept for a month in a small tin can on a shelf
directly over a radiator were examined, and living larvae were removed
from them by carefully breaking the eggshells with fine needles.

On November i twenty eggs which were laid on August 29 and had
been kept indoors were placed in two small wooden boxes and buried in
a pail of damp sand. The pail was set on the ground, outdoors, where
it was covered with snow practically all winter. The eggs were exam-
ined once a month, but none hatched until the second week in April
when all the eggs were found to have hatched and the larvae were alive
and vigorous.

From our studies of the egg it would appear that if there is sufficient
moisture and proper temperature condition the majority of the eggs
will hatch in the fall, while if it is unusually dry or cold weather starts
early the eggs will not hatch until the following spring. Strickland {18)
found eggs on frozen ground December 3, which would indicate that in
Canada some of the eggs at least do not hatch until spring.

296 Journal of Agricultural Research voi. xxii. no. 6

FIRST APPEARANCE OF I^ARV^E

Larvae may appear in the fall. This is proved by the fact that three
larvae were found at Wilsall on September 30 and that larvae hatched in
the rearing boxes during October and November whenever sufficient
moisture was added. Large numbers of larvae must have hatched at
Wilsall during the fall of 1918, for as soon as the snow left the ground
the following spring fourth- and fifth- instar larvse were found in large
numbers. We have no records of injury to wheat during the fall months,
but it seems quite probable that in years when there is considerable
moisture and mild weather during October and November great damage
may be done.

The larvae begin to feed shortly after the wheat begins to grow in the
spring. In 191 9 at Wilsall 80 acres of winter wheat were completely
destroyed by May i, which indicates that the worms must have been
active 10 days or 2 weeks previous to that date.

In 192 1 at Willow Creek first-instar larvae were found on March 3.
The weather had been warm for about a week, and winter wheat was
starting to grow again. No larger larvae could be found, and it seemed
as though the very small first-instar larvae must have just hatched.

PERIOD OF LARVAIv FEEDING

One of the reasons why Porosagrotis orthogonia is such a dangerous
insect is the unusually long period of heavy larval feeding which extends
until the middle of June or even to July 10 in the case of late-hatched
specimens. Judging from the reports of injury received, the larvae
attract most attention during the month of May and the first two weeks
in June, differing decidedly in this respect from Chorizagrotis aiixiliaris,
which generally has reached the height of its destructiveness by April
15 and has practically disappeared by May i.

The length of the larval stage as determined for 20 larvae, 5 from each
of 4 parent moths, under insectary conditions varied from 62 to 151
days and averaged 118 days, as shown in Table III.

All of the larvae were kept under very similar conditions, and no
reason has been found for the wide variation. They were always fed at
the same time and were kept on one tray in individual rearing boxes,
thus giving practically identical conditions of moisture, temperature, and
food. In spite of this similarity of conditions we find that larva 2ie
pupated 62 days after hatching from the egg, while larva 2 id from the
same parent and from the same egg cluster took 124 days, or just twice
as long, to reach the same stage of development. The number of instars
was also found to vary. Thus the number of larval instars for the five
larvae from each of the moths was as follows: Moth 21 — four had 7
instars and one had 8; moth D — all five passed through 8 instars; moth
42 — two had 7 instars and three had 8; moth 24 — one had 7 instars and

Nov. 5, 192 1

Pale Western Cutworm

297

four had 8. According to the rearing records of other larvae than those
shown in the table, several individuals passed through 9 larval instars
and one passed through 10. Eight instars, however, is the usual number
and the minimum is 7.

Table III. — Duration of larval instars of Porosagroiis orthogonia under insectary

conditions

Larva record No.

Number of days in each instar.

IV.

VII. VIII.

Num-
ber of
days in
larval
stage.

2ia

2lb

SIC

2 id

2ie

42a

42b

42c

42d

426

Ma.

Average

12
12

9
12

13
12
12

13
II
10

14
12

13
9

7
13
13

5
6
6
9
7
9
9
10

7
9
8

10
8
II
6
7
9
12

9
10
II

9
12
10

9
7
9
7
13
10

14
10
10

9
10
10
10

12
10
14
9
II
II
II

17
II

9
18
12
12

13
13
18

13
17
13
9
16
16
15
14
20

17
12

13
9
18
16
14
13

9.4

9.8

II- 3

14.4

44
29

32
16

14
14
14
21
16
19
25
41
34
14
20

18
18
26

37
48

45
51
32

2>Z
33
75
56
65
43

124

125
121

151
125
150
123
104
112
112
119
148

144
148

131
83

22. 6

29. 6

118

FEEDING HABITS OF LARVA

The larva differs from that of most cutworms in its feeding habit in
that it almost invariably attacks the plant below the surface of the
ground. The most frequent type of injury is the eating away of the
central stem and its surrounding sheaths from }i inch to i inch below the
surface of the ground. In many instances the stem is severed and the
lower part not eaten, and frequently it is only slightly chewed into before
the worm moves on to another plant. Even slight injury to the under-
ground portion of the central stem usually results in the death of the
plant. A very small portion of each plant is actually eaten by this
cutworm, and its capacity for destruction is thus greatly increased.
The first indication of injury is the presence of wilting or dried plants
which can be easily lifted out of the soil without bringing the roots.
The destruction of grain that is just pushing through the ground is par-
ticularly rapid, one worm being able to cut off plant after plant in quick
succession. The worm usually moves along the drill row, taking each
plant as it goes (PI. 30, D). Where one crop has been destroyed and

298 Journal of Agricultural Research voi. xxn.No. 6

the land has been reseeded the worms often attack sprouting grain and
sometimes even gnaw into the kernels before they germinate.

The larvae feed during both day and night. Freshly cut plants
have been found repeatedly during the day and cutworms have been
found with their heads inside the sheath of the plant in the act of feeding
on the central stem. In the rearing boxes no difference could be noticed
between the amount of day and of night feeding.

According to observations made by Strickland and reported by Gibson
(7) it is the habit of the larva —

to travel over the surface of the soil and when a suitable plant for attack is found
it immediately burrows and feeds just below the surface.

In our observations we have never vdtnessed this habit. Considerable
time has been spent during the late afternoon and evening in heavily
infested fields, and except in rare instances we have never seen Porosa-
grotis orthogonia larvse above ground. On the other hand, we have
closely examined hundreds of newly attacked plants where there was no
sign that the soil had been disturbed at the surface by the burrowing of
the larva. In order to find out whether the worms would work from
one plant to another without coming to the surface, wheat seedlings
were started 3 inches apart in a flat in the greenhouse, and when the
wheat was up about i inch 12 half-grown cutworms were placed in one
end of the flat. At the same time a line of plaster of Paris was placed
across the center of the flat so that any traveling of the worms over the
surface at the center would be indicated by lines through the plaster of
Paris. The seedlings at the end of the flat where the worms were intro-
duced were immediately attacked, and within a week all of the seedlings
in the flat had been cut off below the surface of the soil and without any
indication that any of the larvae had crossed the plaster of Paris line.

If it were the natural habit of Porosagrotis orthogonia to travel over
the surface of the ground in getting from plant to plant, it would come
in contact with poisoned bran mash scattered on the ground, and it
seems as if there would be no difficulty in controlling it by the ordinary
methods. The fact that poisoned bran mash is useless against Porosa-
grotis orthogonia, together with our field and insectary observations, leads
us to believe that it very rarely comes to the surface in getting from one
plant to another but instead moves underground, generally along the
drill row.

We have received occasional reports from farmers stating that pale
western cutworms had been seen feeding above ground during and im-
mediately following rains, but only one instance of this kind has come
under our own observation. This was at Willow Creek on June 27,
1920. A light thunder shower at 6.30 p. m. cooled the air and wet the
ground to the depth of i inch. As soon as the storm was over, numer-
ous cutworms were seen crawling over the surface in a wheat field that

Nov. 5. 1921 Pale Western Cutworm 299

was known to be badly infested with Porosagrotis orthogonia. It was
estimated that about 50 per cent of the total number of cutworms in
the soil were on the surface at any one time. They appeared in greatest
numbers in the spots where the grain had previously been cut off. At
first they merely wandered about over the surface, but later on, as it
became dark, they started feeding. Observations were continued until
II p. m., at which time the worms were still moving about and feeding
upon the leaves of wheat and grass. At 5 a. m. the next morning a
few worms still remained on the surface, but all disappeared as the
sun came up. The soil around stray wheat plants was noticeably
stirred up where the worms had come up and gone down. That this
habit of feeding above ground is not a common one is shown by the
fact that only in rare instances have we ever found any injury to that
part of the grain plant which is above the ground, and in such cases
there was always some doubt as to whether the injury might not have
been done by some other species.

LARV^ DO NOT LEAVE FlElvDS AFTER GRAIN IS DESTROYED

Another unusual habit of Porosagrotis orthogonia is that it seldom
migrates even though its food supply becomes exhausted. If, in fol-
lowing along the drill row, it fails to find a plant within a few feet, it
simply remains where it is, perhaps for several weeks, without feeding
or growing to any extent. In fields that are only partially infested the
injury shows up as scattered bare spots, and in such places the larvae
do concentrate along the edges of the standing grain, but we have never
known them to migrate more than a few rods. This habit of remain-
ing in the fields where grain has been destroyed has a very important
bearing on farm practice, as will be shown by the following example:
Eighty acres of winter wheat at Wilsall in 19 19 were completely de-
stroyed by May i, the ground being left entirely bare. The field was
reseeded to spring wheat the second week in May. On May 24 the
grain was just coming through the ground and was being rapidly cut
off by cutworms which had remained in the field since the winter wheat
had been destroyed, some three weeks before. The worms continued
to feed for several weeks and destroyed all the spring wheat.

The ability to go for a long time without feeding was well shown by
a half-grown larva which remained in a rearing can for 12 weeks with-
out food and was then fed and reared to maturity.

FOOD PLANTS

In Montana this cutworm has been most commonly found feeding
upon winter and spring wheat. Oats, barley, rye, flax, and alfalfa
have also been attacked. In the insectary, larvae have fed readily and
grown rapidly upon dandelion. In Canada, Gibson reports Porosagrotis

300 Journal of Agricultural Research voi. xxii no. e,

orthogonia larvae as feeding upon fall and spring wheat, oats, barley, flax,
beets, onions, cabbages and carrots.

PERIOD OF INACTIVITY BEFORE PUPATION

Although Porosagrotis orthogonia larvae are mature and have prac-
tically ceased feeding by the middle of June they do not pupate until
nearly a month later. During this period they occasionally feed
slightly, but for the most part they remain in a semidormant condition,
gradually turning whitish in color and shrinking in size just previous
to pupation. This was noticed both in the field and under insectary
conditions. Notes taken at Wilsall June 20, 1919, state that on that
date cutworms were decreasing in numbers and were nearly all full
grown. This field was visited again on July 4, when many whitish larvae
were found, some of which had formed earthen cells, but no pupae were
found in a two-hour search.

Records kept on 75 larvae in the insectary showed an average period
of 20 days of complete inactivity previous to pupation and a period
of 26 days in which only very slight feeding took place.

PUPAI^ PERIOD

Pupation generally takes place about the middle of July. Out of
80 specimens collected as larvae at Wilsall in May, 1919, and reared to
adults in the insectary, the average date of pupation was July 19, the
earliest date July 2 and the latest August 1 1 . This checked out almost
exactly with conditions in the field at Wilsall.

About a month is spent in the pupal stage. The average length of
pupal period of 80 specimens was 29^^ days, the shortest 2 1 days and
the longest 40 days.

The pupae are protected by a cemented earthen cell and are usually
found at a depth of 3 to 4 inches in the soil beneath the plants where
they last fed.

SEASONAL ABUNDANCE OF ADULTS

The earliest emergence of Porosagrotis orthogonia moths which we have
on record is July 31, although Gibson {8, p. 30-ji) reports the emergence
of a moth of this species on July 19, In general, the period of greatest
abundance is during the last two weeks in August and the first week in
September.

The field at Wilsall where Porosagrotis orthogonia larvae destroyed two
seedings of wheat during May and June, 19 19, was searched for moths
on August 7, but none could be found. A trap light was run until mid-
night on this date, and not a moth of this species was taken. On August
26 the field was again visited, and numerous moths were found during
the day, and at night they came to trap lights by the thousands. The
majority of the moths taken at this time were in prime condition and

Nov. s, 1921

Pale Western Cutworm

301

looked as though they had just emerged. One week later the number
of moths was greatly reduced ; the)'' were difficult to find during the day,
and very few came to lights at night. On September 30 an entire day
was spent in searching the same field, but not a moth could be found.
The ovvTuer of the field had been disking and drilling throughout the
month of September and during the first half of the month had frequently
seen moths fly up as the ground was disturbed but had seen none after
September 15.

Seventy-five larvae collected in this field May i and reared in the
insectary at Bozeman emerged as adults on the dates shown in Table IV.
In 1920 at Willow Creek the first moth was caught on August 9, the
heaviest flight was from August 19 to 24, and several moths were seen as
late as October 8.

Table IV. — Dates of emergence of Porosagrofis orthogonia moths in igiQ

July 31.
Aug. 4

5
6

7
8

10
12
13
14
IS
16

Number
of moths.

Aug. 17

18
19

23
24

25
26
27
28
31

Sept. I.

Number
of moths.

5
S
4
4
2
I
I
I
2

EGG-LAYING HABITS

Egg laying was first witnessed at Willow Creek in 1920. Several
gravid females were placed in outdoor observation cages in which a
variety of soil conditions and vegetation was offered and were closely
watched for several days. On the afternoon of September 4, at 4.45,
one of these females was seen laying eggs. She crawled over clods,
stubble, and plants, constantly feeling with the ovipositor the objects
which she walked upon. On reaching a patch of soft earth she stopped
and carefully worked the abdomen into the soil until the wings were flat
on the ground. After remaining quiet for a short time she moved and
repeated her actions in another spot. Three ovipositions were made in
15 minutes, and after each one the dirt was stirred as the abdomen was
withdra-'vn and the hole left covered with dirt. The dirt around these
holes was carefully removed with a teaspoon and eggs were found in
clusters of 3 or 4 about % inch below the surface of the ground. A total
of II eggs were recovered from the three ovipositions.

302 Journal of Agricultural Research voi. xxn, no. t

Porosagrotis orthogonia moths were seen laying eggs in the open at
Willow Creek on September 5, 1920. Just before dark moths were seen
flying over a freshly worked, summer-fallowed field, being most abun-
dant on the higher knolls and along the ridges where the soil was soft
and loose. One moth was followed for some distance. She would fly a
few feet, never getting over 10 inches above the ground, and would then
crawl a short distance, continuously feeling the surface with her ovi-
positor. On reaching soft dirt she stopped and laid eggs for six minutes,
going through the same actions as the moth observed in the cage. When
she left the ground she flew straight away for at least X ^^^ ^t a height
of 20 to 30 feet above the ground and was finally lost to view. Five eggs
were recovered from this oviposition. Other moths were seen flying to
the ridges and knolls, but it was too dark for further observations on this
date. A few days later another moth was observed laying eggs on a
knoll in the same field. One oviposition was made which lasted 23 min-
utes, during which time the moth was not in the least disturbed by any
movements of the observer. When the ovipositor was finally with-
drawn the moth swung around X iiich and started in again, this time
remaining quiet for 1 7 minutes, after which she crawled under a clod to
hide. This moth had oviposited for 40 minutes, and 12 eggs were re-
covered from the two holes. Moths were seen ovipositing along the
knolls and ridges in this field for several days.

Moths in egg-laying show their preference for spots in the field where
the soil is softest and also indicate a preference for freshly worked fields
over those which have become caked and hard. Across the road from
the freshly worked, summer-fallowed fields in which egg laying was
observed was another summer-fallowed field which was spotted with
Russian thistles and in which the soil was caked on the surface, due to a
rain some two weeks earlier. Moths were continually observed flying
into this field, but they usually flew on across it to the knolls in the
freshly worked field, even though it was ]4 mile farther. Very few
moths flew to similar knolls in the caked field, and those that alighted hid
under the thistles or clods of dirt and made no attempt to lay eggs.
Further evidence of this preference for mellow fields will be brought out
later in this paper.

ATTRACTION OF THE MOTHS TO LIGHTS

Our first experiments in attracting the moths to lights were conducted
at Wilsall on the evening of August 26, 19 19. A large Coleman gas lamp
was placed on the ground in the field where the grain had been destroyed
the previous spring. As soon as it grew dark Porosagrotis orthogonia
moths began to come to the light at the rate of one every two or three
minutes. The lamp was placed upon bare sandy soil and the ground was
well lighted for several feet on all sides. The moths usually struck the

Nov. 5, 1921 Pale Western Cutworm 303

ground from 2 to 15 feet from the light and then crawled toward it,
where they could be easily picked up. As soon as it became totally dark
the moths came to the light so rapidly that two men could not keep them
picked up, and from 9 o'clock until midnight 282 females and 164 males
of P. orthogonia were placed in rearing cans or killing bottles. This by
no means represented the total number that came to the light, for hun-
dreds escaped. Many different species of noctuids were attracted to the
light, but fully 95 per cent were P. orthogonia. Ten of the females thus
captured averaged 132 eggs. (Table II.)

In another part of the same field a Duro moth trap was run through-
out the nights of August 26 and 27, 19 19, and each morning the pan was
well filled with Porosagrotis orthogonia moths. During the two nights
4,900 moths were caught, of which 4,200 were males. It is difficult to
understand the preponderance of females caught at the larger light
between 9 and 12 p. m. and the very small percentage of females caught
at the smaller light during the entire night.

Experiments with trap lights were conducted on a somewhat larger
scale at Willow Creek. A trap was designed which was made up of
utensils commonly found on every farm and which would serve other
purposes when not in use as a trap light (PI. 30, B). It consists of a
No. 2 galvanized-iron washtub and a No. 2 barn lantern. In addition,
a galvanized-iron arch is made which fits across the tub and serves the
dual purpose of deflecting the moths and holding the lantern. When
the arch is wired firmly and the lantern swung in place the flame of
the lantern is just above the edge of the tub. "W^Tien set in place, the
tub is staked down to prevent its being blown over, and about 4 inches
of water are poured into it. About }i to }{ inch of kerosene is floated
on the water to kill the moths which fall into it.

Eleven such traps were put out at Willow Creek, and observations
were made during the flight period of Porosagrotis orthogonia. Two
traps for catching moths alive were also used, and when these showed
that P. orthogonia was beginning to fly the tub traps were put out on
fields that had been heavily infested with worms.

During the first few nights the catches were small and the moths
were counted. The numbers increased nightly until the height of
flight, which was from August 19 to 24, inclusive. The night flight
gradually decreased after the latter date. When the numbers became
too large to count they were estimated, and during the height of flight
they were measured in pints. As a pint measure holds from 962 to 1,000
moths, the measuring of moths gave a fairly accurate count. During
the heavy flight several of the traps ran as high as 4,000 moths in a
single night. The entire season 's catch of Porosagrotis orthogonia moths
in the 11 traps was 82,488. The catch on individual nights is shown in
Table V.

304

Journal of Agricultural Research

Vol. XXII, No. 6

Table V. — Number of Porosagrotis orthogonia moths caught at trap lights at Willow
Creek, Mont., during the season oj igzo

Aug.

Sept.

13
14
IS
16

17
18

23
24

27
28
29
30
31

Weather conditions and remarks.

Number
of moths
caught.

127

342

1,528

1,566

449

11,720

Rain in afternoon; night warm, cooler toward midnight

Early part of night warmer than usual; warm after midnight. . .

Warm all night

Night moderately warm

Cooler, especially so after 9.30 p. m. ; not as many moths flying .
High wind in afternoon blew over traps; cold and windy after

dark ; no moths out

Windy; cold soon after dark, almost frost; moths flew only a

few minutes

Warm at 8 p. m.; moths flying heavily; windy and cooler after

ir.30; fewer moths out

Warm west wind most of night; heavy flight of moths 13 , 990

Same as night before; height of flight 9.15 p. m 14.950

Warm, light west wind 13 , 650

Warm, west wind; wind strong and cold after 12.30 a. m 16,250

Cold and cloudy after 9 p. m. ; southwest wind ! 7 , 490

Windy and cold ; moonlight; no moths flying '

Windy until 8 p. m.; clear, cold; full moon; no moths flying.

Wind and heavy rain ; cold ; no moths

Rain all morning; cold, windy night; traps not lighted !

Cool; little wind; few moths flying early; too cold after 9 p.m. . . 68

Clear, cool; bright moon; no moths flying

Cloudy to 9.30; clear, cold; bright moon

Moths flying in daylight after noon; few flying after dark

No moths flying after dark; a few found feeding

Few moths out after dark ; not attracted to lights

Three Nociua c-nigrum caught; no others flying to light

No moths flying to traps; traps taken up

119

171

Total.

82,488

In all observations made at Willow Creek no moth was ever seen to
land on the ground on its way to a light trap as did the moths at Wilsall
the previous year. This may have been due to the fact that the tub
hid the light so it would not strike the ground, and in order to keep in
the path of light the moth had to fly straight to the trap. This was
usually the case, and for the most part moths flying to the traps came
on a straight line from 4 to 15 feet above the ground. They either struck
the arch or lantern or went straight on over the trap.

On a still, dark, fairly warm night the moths would come to the traps
in varying waves of abimdance for which there was no apparent reason.
There would be a cloud of moths for a few minutes and then they would
come in scattering two's or three's. If the wind was strong no moths
were caught in the traps and no moths could be found moving about on
the ground. No moths were caught during a rain or ever after a rain, as
long as the ground and vegetation remained wet. When the moon
was bright, moths were not caught nor were any seen flying, though they
would start the minute the moon went behind a cloud. Practically no
moths were caught after the temperature had dropped below 58° F.

Nov. 5, 1921 Pale Western Cutworm 305

MOTHS BOTH NOCTURNAL/ AND DIURNAL

During the last half of August when the nights were warm and night
flying was at its height the moths remained inactive during the day,
hiding under clods and weeds. As the nights grew colder, the moths
flew only an hour or two after dark, and on September i they were seen
flying during the day. On this date they began flying about 4.30 p. m.
and were seen in abundance feeding upon sunflowers, golden rod,
tumbling mustard, yellow greasewood and lamb's quarter. All but one
of the moths seen at flowers at this time were males, a search under
weeds and clods at the same time revealing only females. At 8 p. m.
when the flower patches were visited moths were still feeding in large
numbers, practically all of them being females. They paid no attention
to lights, and none were caught in a trap light set close by. As the night
grew colder all of the moths disappeared and could be found hiding under
clods or weeds. On the following day moths were found feeding at
flowers at i p. m., and at 3.30 p. m. a patch of yellow greasewood
{Chrysanthus jrigidus), which seemed to be the favorite flower, had
attracted dozens of Porosagrotis orthogonia, nearly all of which were
males. At 5.30 p. m. the patch was again visited, and it was found that
the males were then leaving and that females were flying to the flowers
from a nearby summer-fallowed field. On September 3 moths of various
species were found feeding during the morning, and at noon the flowers
of the yellow greasewood were covered with moths, none of which were
P. orthogonia. At 3 p. m. about 10 per cent of the moths present at
flowers were P. orthogonia. These gradually increased in numbers until
6.15 p. m., at which time practically all other species had disappeared.
After the moths had finished feeding they invariably flew toward the
higher ridges and knolls in neighboring cultivated fields. Many of the
moths in coming to the flowers were seen to fly from 200 to 500 yards
directly against a stiff breeze. Moths were seen flying to flowers in large
numbers until September 8, when a cold rain and wind occurred. The
males were always found feeding earlier in the day and the females later,
although neither was ever found before noon.

ECONOMIC IMPORTANCE

The record of this cutworm during the last 10 years has demonstrated
its capacity for doing enormous damage to grain crops. When in 191 1
Porosagrotis orthogonia, then an obscure insect, suddenly increased in
numbers and did considerable damage to grain in southern Alberta {10, p.
iji) little importance was attached to it. In the following year, however,
when 33 per cent of all the grain sown in the Lethbridge land district
was destroyed and an accurate estimate by the superintendent of the
experiment station at Lethbridge placed the actual loss from this insect
at from 30,000 to 35,000 acres (jj, p. 506) it was looked upon as a pest of

; 65769°— 21 2

3o6 Journal of Agricultural Research voi. xxn, no. 6

major importance. During the last two years P. orthogonia has been re-
sponsible for losses in central Canada amounting to several million dollars.

In Montana the pale western cutworm has been on the increase since
it was first noticed in 19 15 and is now the most destructive insect pest
with which the grain grower has to contend. In 19 15 at Conrad 80 acres
of wheat were destroyed and were reseeded to oats, which was also taken.
Flax was then seeded, but this also was so badly injured that the owner
plowed the field and summer fallowed it. This instance was typical of
scores of losses in the district now composed of the counties of Chouteau,
Teton, and Pondera.

During the next two years, especially in 19 17, great losses were sus-
tained throughout the north central portion of the State, due to cut-
worms which worked entirely beneath the surface of the soil and which
were doubtless no other than Porosagrotis orthogonia. In 1919 the pale
western cutworm appeared in destructive numbers farther south and
caused severe losses in Park and Jefferson Counties as well as in the
previously infested area. A conservative estimate of the losses for the
year based on the reports of county agents, hundreds of questionnaires
returned by farmers, and the personal investigation of many fields in
different parts of the State, is at least 200,000 acres. In 1920 the injury
in Jefferson and Park Counties was more widely extended, and there was
a decided increase in the damage done in many of the districts previously
infested. The loss over the entire State for the year is placed at 250,000
acres, valued at $3,000,000. In the Willow Creek district in Jefferson
County a careful survey conducted in 1920 showed that 29 per cent of
the total seeded area had been destroyed by this cutworm, and a similar
survey in several of the northern counties showed a loss of 35 per cent of
the grain crops planted.

To show perhaps a little more clearly what this cutworm has been
doing it may be stated that 100 fields personally inspected during the
summer showed a loss of 2,437 acres out of a total of 6,844 ^^ 1919, and
in 1920 a loss of 3,382 acres out of 6,844, or 35.7 per cent in 1919 and
49.4 per cent in 1920. Mr. George O. Sanford, manager of the Sun River
irrigation project, has stated to us that of the 15,300 acres seeded to crop
on the Greenfield Bench in 1920, 7,345 acres was a total loss and that
some damage was done to the remainder. Using the figures he has given
for the average yields on the undestroyed acreage — wheat 11.5 bushels,
oats 20.86 bushels, and flax 6.31 bushels — the average value of tlie prin-
cipal farm crops of that section was at least $15 per acre. Accordingly,
using that as a fair valuation per acre of the crops destroyed, the pale
western cutworm inflicted a loss of $110,175 in this one comparatively
small territory. Although these losses took place on irrigable land, no
water was available until after the first of June. Were it not that irri-
gation made it possible in some cases to reseed and grow a late crop on
part of the originally destroyed area, the loss would have been 55 per
cent instead of 48 per cent of the acreage in that district.

Nov. 5. 1921 Pale Western Cutworm 307

EXPERIMENTS IN CONTROL
ORDINARY CUTWORM CONTROI, METHODS NOT EFI^ECTIVE

Early in our study of Porosagrotis orthogonia it became apparent that
the ordinary method of scattering poisoned bran mash over an infested
field was not effective in controlling this species. On May 2, 19 19, pois-
oned bran mash was scattered over a heavily infested field in southern
Montana at the rate of 20 pounds to the acre and was followed by three
other applications on successive days. The field was exammed each
day by the owner, who reported that he could not find a single dead worm.
On May 7 the treated area was carefully examined by one of the writers
but no dead worms could be found, nor could any decrease in the number
of live worms be noted. On May 7, 19 19, poisoned bran mash was scat-
tered over 2 acres of heavily infested wheat in northern Montana. During
the next 10 days no results whatever were secured from this treatment.
County agents and numerous farmers have reported that attempts to
poison this species by the ordinary method of scattering poisoned bran
mash over the surface have always resulted in failure.

At Willow Creek in 1920 pale western cutworms were noticed crawling
over the surface of the groimd in the evening after a rain, and an attempt
was made to kill them by scattering poisoned bran mash during the
night. The bait was scattered soon after dark over an area which in-
cluded bare ground, scattering wheat, and a good stand of wheat, all
heavily infested. Observations were made during the night by the aid
of automobile headlights, and many of the worms were seen feeding upon
the bait. Two days later a search was made for dead cutworms. In
the area where there was no vegetation it was estimated that 60 per
cent of the worms were killed; where there was a scattering of wheat
the percentage of dead worms was 50; and where there was a good stand
of wheat 43 per cent were killed. It is possible that several night appli-
cations of poisoned bran mash during rainy weather might bring about a
satisfactory control, but as yet we have not had the opportunity to
try it.

POISONED BRAN MASH HARROWED INTO THE SOU.

Strickland reports {17) that poisoned bran mash harrowed into the
soil gave gratifying results. This method was tried out at Wilsall in
May, 19 19. Poisoned bran mash was scattered over 3^ acre of heavily
infested wheat at the rate of 25 pounds to the acre. On several square
rods where the worms were thickest the mash was worked well into the
soil with a hand rake, and the remainder of the treated area was thor-
oughly worked with a spike-toothed harrow. The plot was carefully
examined three days after the poisoned bran mash was applied, and it
was estimated that the treatment was not more than i per cent effective.
Very few dead cutworms could be found, and eventually all of the wheat
was destroyed.

3o8 Journal of Agricultural Research voi. xxn.No.6

POISONED BRAN MASH DRII,IvED INTO THE SOII#

Since Porosagrotis orthogonia very rarely comes to the surface to feed,
placing the poisoned bran mash beneath the soil was tried in the hope
that the cutworms would thus come in contact with it and feed upon it.
The most promising way of doing this seemed to be with a seed drill.
This method was tried out in northern Montana at Havre and in southern
Montana at Wilsall.

TESTS AT HAVRE

At Havre two formulae were used.

FORMULA NO. I

Shorts pounds . . 25

Paris green do ... . i

Oranges 4

Molasses quarts . . 2

Water gallon. . i

FORMULA NO. 3

Shorts pounds. . 25

Paris green do ... . i

Molasses gallon . . i

Water quarts . . 2

These mixtures after being prepared were spread out and allowed to
dry for 24 hours. When dry, No. 2 was distinctly stronger smelling,
although both had a good molasses odor. The reason for using the
large amount of molasses in these formulae w^as to secm-e a distinct odor
in the dried material which we hoped might attract cutworms in the
soil for some little distance.

The dried material was seeded into the ground with a Van Brunt
drill at the rate of 16 pounds to the acre and at a depth of about 1.5
inches. Six acres were ti"eated. The drill was run at right angles to the
rows of grain so that the worms in working from plant to plant would
only move a few inches before coming in contact with the bran. The
greatest difficulty encountered was in getting the bran to feed evenly
through the drill. When it was sufficiently dr}^ to be well divided it
was too light to force its way through and it was necessary to agitate
the mixture continuously in the seeder box to get any-where near an
even distribution.

The field was examined two days after the poisoned bran mash was
drilled in, and it was found that formula No. i had killed approximately
50 per cent of the worms while formula No. 2 gave slightly better results
with a kill of about 55 per cent, which was not enough to prevent the
destruction of the crop.

Nov. 5. 1921 Pale Western Cutworm 309

TESTS AT WILSALt

On June 8, 19 19, a similar test was conducted at Wilsall. The follow-
ing formula was used :

Shorts pounds. . 25

Paris green do .... ^

Salt :':v^...v;?..';:.:;v.r. : do %

Molasses quarts . . 2

Water. gallon . . i

After mixing, the mash was spread out to dry, which with a hot sun
and a fair breeze was accomplished in half a day. The mixture was
distributed over 25 acres of infested wheat at the rate of 12 pounds of
the dry mash to the acre. Sixteen acres were sown with all the spouts
of the drill working and 9 acres with every other one closed. The drill
was run across the old grain rows. The greatest difficulty encountered
was the same as in the test at Havre — the mixture was too light to feed
evenly through the drill. This was overcome by using two men on
the drill, one to drive and one to keep the bran shaken down where it
would come in contact with the disks of the drill. This was done by
frequently pounding the seeder box with a padded hammer and punch-
ing out packed masses with a small stick. Dead and dying worms were
found the second day after the poisoned bran was drilled in, and on the
third day a careful examination was made and it was estimated that
from 50 to 60 per cent of the worms had been killed. The field was
examined two weeks later, and there was a very noticeable difference in
the number of worms found in the treated and untreated areas, but this
did not prevent total destruction of the crop.

TEST AT WIL1.0W CREEK

During May, 1920, poisoned bran mash was distributed with a grain
drill over a very badly infested field at Willow Creek. Cutworms were
uniformly scattered over a 40-acre field of spring wheat, and at the time
the poisoned bran mash was applied had destroyed about half the plants.
Conditions were ideal for a good test of control methods. The following
mixtures were used :

FORMUI,A NO. I

Shorts pounds. . 25

Paris green do ... . i

Molasses quarts.. 2

Salt pound. . i

Water gallon . . i

After mixing, this was thoroughly dried out and was then seeded 2
inches deep through a Van Brunt drill at the rate of 20 pounds to the
acre. Two acres were sown. This mixture did not feed uniformly
through the drill unless constantly agitated.

310 journal of Agricultural Research voi. xxu. no.6

FORMULA NO. 2

Shorts pounds . . 25

White arsenic ■ do ... . i>^

Molasses quarts. . 2

Salt pound . . i

This was prepared and distributed in the same manner as formula
No. I. Two acres were sown.

FORMULA NO. 3

Shorts pounds . . 25

Paris green do .... i

Salt do .... I

This was mixed dry and seeded at the rate of 12^^ pounds to the
acre. It ran through the drill about the same as the mixtures which
were mixed wet and then dried. Two acres were sown.

FORMULA NO. 4

Shorts pounds . . 25

White arsenic ; .* ! .'.'.. 1 do ... . i}4

Salt do ... . I

This was prepared dry and then thoroughly mixed with an equal
bulk of wheat. This combination ran through the drill very evenly,
the wheat being heavy enough to carry the bran through the drill without
clogging. Two acres were seeded at the rate oi \2}i pounds of bran to
the acre. Three days after the poisoned bran was put out the field was
examined and it was estimated that about 10 per cent of the worms
in the treated areas had been killed. No difference could be seen in the
effectiveness of the various formulae, and numerous living cutworms
remained in all the plots. One week later the plots were again examined
and the number of dead cutworms had not materially increased. A
final examination of the field was made on June 14, three weeks after the
poisoned bran was put out. Cutworms were found in abundance on all
plots, and in plots 1,2, and 3 practically every spear of wheat had dis-
appeared. In plot 4, which was seeded with a mixture of wheat and
poisoned bran, the wheat was about 3 inches in height and was being
rapidly cut off, 50 per cent of the new stand being already destroyed.
From a practical standpoint the control on all plots was a complete
failure and an absolute waste of materials.

POISONED BAIT SPRAY FOR ADUI,TS

The presence of large numbers of Porosagrotis orthogonia moths at
flowers led us to try out the following poisoned bait spray:

Water gallon . . i

Molasses pint . . ^

White arsenic ounce . . }4

Amyl acetate do %

Nov. 5, 1921

Pale Western Cutworm

311

This was scattered in coarse droplets over flowers and vegetation
where moths were abundant. Many flies and bees were killed, but no
moths were observed feeding upon the bait, and dead moths were never
found in the vicinity of the sprayed vegetation.

CUI^TURAI, METHODS AS A MEANS OF CONTROI,

In our study of Porosagrotis orthogonia under field conditions we have
repeatedly noticed instances where crops in one field were completely
destroyed, while in an adjacent field the grain escaped unharmed. This
suggested that the manner in which the ground was worked before the
crop was put in might have been responsible for the great difference in
the amount of damage done in the two fields, and in 1920 a survey was
conducted with the object of determining the relation of cultural methods
to cutworm abundance. This survey was conducted in two ways : (1) By
an auto trip through the districts most heavily infested by means of which
hundreds of farmers were personally interviewed and the histories of
their fields obtained for the period 1919-20; (2) by questionnaires sent
to all farm bureau members in counties where Porosagrotis orthogonia was
known to be present.

The percentage of cutworm losses under various cultural methods as
shown by a study of fields, the owners of which were personally inter-
viewed, is shown in Table VI.

Table Vl. ^Percentage of Porosagrotis orthogonia injury in IQ20 under various methods
of cultivation in preparation for seeding

Cultivation between previous crop and 1920 crop.

Number of

Total

Acres

fields.

acres.

lost.

465

200

2,250

1,301

1.536

661

643

138

2,465

666

1.332

526

3. "4

267

Percentage
lost.

Fall double disked

Spring double disked

Spring single disked

Fall-plowed ; disked or harrowed be 'ore seed-
ing

Spring-plowed" disked or harrowed before
seeding

Spring-harrowed

Summer-fallowed

43- o
57- o
43- o

21. o

27. o
40. o

8.5

A study of the results shows a high percentage of cutworm injury in
all cases where the stubble was only disked or harrowed before seeding.
Fields which were plowed either in the fall or spring showed a somewhat
lower percentage, while summer-fallowed fields showed only the very
small loss of 8.5 per cent.

While the average cutworm loss in summer-fallowed fields was low,
yet several individual fields suffered severe losses. It was therefore
decided to make a study of the histories of summer-fallowed fields during
the two seasons of 19 19 and 1920. Since the moths were known to

312

Journal of Agricultural Research voi. xxii, no. 6

prefer loose mellow soil for egg-laying, it was thought that the condition
of the ground in summer-fallowed fields during the egg-laying period
might have considerable rafluence on the number of eggs deposited in
the field and on the percentage of loss the following spring. Since egg
laying does not begin until about August 15, fields which are not culti-
vated or disturbed in any way after July 1 5 become more or less crusted
and caked. Fields which are cultivated in any way during the last part
of July or during August, on the other hand, are very likely to be soft
and mellow during the egg-laying period, thus offering tlie very con-
ditions which the moths are seeking. Forty-eight fields, for which we
had data for both 19 19 and 1920, were therefore classified as crusted, if
they were worked only before July 15, or as mellow, if they had been
worked after that date. The percentage of loss for the variously worked
fields is shown in Table VII.

Table VII. — Percentage of Porosagrotis orthogonia injury during IQIQ and IQ20 in
"crusted" and "mellow" sumtTier-f allowed fields. Data secured by personal interview
with grower

Condition of field and time of cultivation.

Number of
fields.

Total
acres.

Number of

fields
infested.

Acres
lost.

Percentage
lost.

"Crusted" — worked only before
July 15

27
21

1,828
1,562

3
14

425

00. 7
27. 2

"Mellow" — worked after July 15. .

Farmers were asked in questionnaires sent to farm bureau members
in counties infested with Porosagrotis orthogonia whether they had
noticed any relation between the condition of the soil in summer-fallowed
fields during August and the amount of pale western cutworm injury
the following spring. Sixty-eight grain growers answered this question.
Fifty-three said that injury was most severe in fields where the surface
soil was well pulverized, or, as one farmer stated it, "The more mulch
the more worms." Seven reported that the greatest injury had oc-
curred in fields that had been crusted during August, and five stated
that they could see no relation between soil conditions and cutworm
injury.

The foregoing data, together with the fact that we have seen ovi-
positing females show a distinct preference for mellow fields, leads us to
the conclusion that the physical condition of the soil during the egg-
laying period has a very important bearing upon the amount of Poro-
sagrotis orthogonia injury that may occur the following spring. Accord-
ing to the data at hand greatest injury may be expected in fields in
which the surface soil is loose and well pulverized during the egg-laying
period. This loose, mellow condition may have been brought about in
summer-fallowed fields by tillage during late July and August or it may

Nov. 5. I92I Pale Western Cutworm 313

be a natural condition such as is found on knolls and ridges where the
soil is generally light and easily drifted. In fields where a crop is re-
moved during July or August the surface crust may become broken and
pulverized in numerous places by the disturbance of the soil in connec-
tion with harvesting, thus offering the moths many desirable spots for
egg laying. Injury may be least expected to occur in fields in which the
surface soil is hard or crusted during the egg-laying period. In most
instances this condition can be brought about by not disturbing the
ground in any way between July 15 and September 15. If farmers in
preparing their grain fields for seeding will be governed by these prin-
ciples it is believed that Porosagroiis orthogonia injury can be greatly
reduced. Fortunately this method of handling summer-fallowed fields
does not interfere with approved farm practices, and in fact agrees very
closely with the recommendations of the agronomists.

NATURAL ENEMIES

Unlike most of our common cutworms, Porosagroiis orthogonia suffers
comparatively little from attack by natural enemies. Much difficulty
has been encountered in rearing various other species taken in the field
as larvae, particularly the army cutworm, Chorizagrotis auxiliaris, on
account of the high percentage that developed disease or parasites.
This has not been the case with the present species. Our records for
19 15 show that out of a large number of army cutworms reared indi-
vidually only 35 per cent were brought through to the moth stage,
parasites emerged from 24 per cent, 2 1 per cent died of disease, and the
remaining 20 per cent died in the pupa stage, mostly on account of in-
sect parasites. In 1919, 55 per cent of P. orthogonia larvse handled
in the same way were reared to adults. Of the 45 per cent that died,
very few seemed to die of any disease, and parasites emerged from only
two larvae.

In 1920, out of 960 Porosagroiis orthogonia larvae collected in the
field, 13.7 per cent were parasitized, 12.2 per cent by Diptera and 1.5
per cent by Hymenoptera. The parasites which emerged were 14 Bon-
netia compta Fall and i Peleteria robusta Wied.

The common wild birds of the prairie are the most beneficial natural
check that we have observed. The western grasshopper sparrow, Am-
modramus savannarum bimaculatus Swainson, particularly, has been
watched while digging out the larvae and carrying them away to its
young. In many parts of the cutworm-infested regions it has been a
common sight toward the last of June to see thousands of small excava-
tions made by the western grasshopper sparrow, horned larks Otocoris
alpestris leiicolaema Coues (PI. 30, C), and possibly other wild birds in
their search for the larvae.

Although the common ground squirrel, Citellus richardsoni Sabine, has
at times been known to seek out and devour large numbers of cutworm

314 Journal of Agricultural Research voi. xxii, no. 6

larvae, we do not believe that ground squirrels are of much importance
as a natural check.

In some instances both larvae and adults of Calosoma teptdum Lee.
have been observed to be especially numerous about cutworm-infested
fields and are, we believe, one of the lesser important predators.

DESCRIPTION OF STAGES
EGG

Spheroidal, flattened dorso-ventrally, glistening milk-white when first

laid (PI. C, i), later becoming dull gray: i mm. in diameter, 8 mm. in

height. Around the micropyle is the

■. . v.. .i. ..•,•■■•:••., .' usual rosette which lies in the cen-

'/C ^:--^r'-^'''Vi'.'''f ■■(■''''■■.•' ter of a finely reticulated area about

. •. .-• 0.3 mm. in diameter. The pattern of

■•, '• the reticulation is shown in figure i.

From the edge of the reticulated area

]!...;.... about 30 slightly raised longitudinal

■/••.'.?■• ribs radiate toward the base, extend-

fi. •' " ■ .

■.■."'^:;{.. ing approximately four-fifths the dis-

'"'/""■■/•■■■■■■} •! V '■^..•"■•. tance from the apex to the base.

"■•■■■'••■.••....• ■;'■"■ The ribs are sometimes irregularly

branched or connected by cross

^^u'Z?'"'''''"^"''", "'■'A'^^'?!?'"-" ""'^^f'"'' ridges. The shallow channels be-
about the micropyie. Highly magnified. "

tween the ribs are transversely
striated and lightly pitted. The chorion on the basal fifth of the egg
is smooth and glistening without ridges or definite reticulation.

I^ARVA ^
FIRST INSTAR

Head width, 0.4 to 0.43 mm.; average 0.41 mm.

Total length of body, 2.4 to 3 mm.; average 2.8 mm.

The head (PI. 28, A) is a very dark glistening brown, almost black;
clypeus and front same color as epicranium; adfrontals indistinct, but
apparently extending almost to deep indentations at summit; mandibles,
labrum, ring-shaped sclerite resting on mentum, and cardo dark brown;
submentum, margins of stipes next the mentum, the antennae, and labial
palpae brownish; ©cellar region blackish.

The posterior portion of the thoracic shield dark brown; anterior
margin very light brown. The thoracic legs are brownish with dark
brown transverse lines immediately anterior and partially surrounding
coxae.

■ In the description of the larva the naming of the various parts follows very largely the system of William
T. M. Forbes U).

Nov. 5, 1931 Pale Western Cutworm 315

Prolegs on segments 9, 10, and 13 (counting the head as the first seg-
ment); crotchets brown; rudimentary prolegs slightly visible as small
tubercles on segment 8.

There is a reddish brown mottling over the lateral and dorsal regions,
becoming more distinct along the posterior segments. Before the larva
has taken food this coloration appears much darker. The general color
of the newly hatched larva is brown. The spiracles dark brown, setae
single and arising from minute brownish tubercles.

SECOND INSTAR

Head width, 0.47 to 0.576 mm.; average 0.53 mm.

Total length of body, 2.8 to 3.77 mm.; average 3.3 mm.

The head (PL 28, B) remains a dark, shiny brown; mandibles and
labrum dark brown, mandibles at teeth and labrum at notch blackish;
ring-shaped sclerite resting on mentum blackened ventrally and with its
setae within the sclerite; cardo dark brown; submentum, margins of
stipes next the mentum, antennae, and labial palpse brownish; ocellar
region blackish.

Thoracic shield dark brown posteriorly, but not as dark as head;
anterior margin light brown. On each side of the light brown dorsal
stripe is a dark spot on the anterior portion of the shield with several
dark spots laterad.

Prolegs on segments 8, 9, 10, and 13; crotchets on eighth segment con-
sisting of only two or three hooks; rudimentary prolegs on segment 7
beginning to show; crotchets reddish brown.

The dorsal stripe is gray, bordered with broken lines of brown; sub-
dorsal and lateral stripes brownish; spiracles dark brown; setae single,
tubercles brownish.

THIRD INSTAR

Head width, 0.68 to 0.786 mm.; average 0.75 mm.

Total length of body, 4.19 to 5.5 mm.; average 4.29 mm.

Head brown (PI. 28, C) with upper parts of lobes of epicranium dark
brown; front brown but with its lower margin together with clypeus
dark brown; mandibles reddish brown to black at teeth; labrum dark
brown with blackened notch, ring-shaped sclerite resting on mentum
blackened ventrally and with its setae within sclerite ; cardo dark brown ;
submentum, margins of stipes next the mentum, antennae and labial
palpae brownish; ocellar region blackish.

Thoracic shield dark brown, almost black at posterior region; an-
teriorly it is a lighter brownish gray with a small black spot on either
side of the light dorsal stripe and with several dark spots laterad.

Thoracic legs tinged with brown, claws and markings anterior to and
partially surrounding basal joints reddish brown.

31 6 Journal of Agricultural Research voi. xxii, no. 6

The dorsal stripe along the body is made of broken gray which is
bordered irregularly with brown. Subdorsal and lateral stripes brownish;
general color same as dorsal stripe, or lighter, with a greenish tinge.
Spiracles dark brown; setae single, tubercles greenish brown.

FOtTRTH INSTAR

Head width, 0.84 to 1.14 mm.; average 1.02 mm.

Total length of body, 6.5 to 12.5 mm.; average 8.9 mm.

Coloration of head slightly modified from preceding instar; front not
as dark, clypeus a lighter brown, and cranial lobes considerably darker
at top (PI. 28, D) ; mandibles are black at teeth and fade to reddish
brown to dark brown near articulations; labrum dark brown with
blackened notch; ring-shaped sclerite resting on mentum blackened
ventrally and with its setae within the sclerite; cardo dark brown;
submentum, margins of stipes next the mentum, antennae and labial
palpae brownish; ocellar region blackish.

Posterior part of thoracic shield dark brown; anteriorly it is lighter,
and in this lighter area are three distinct dark spots on either side of the
dorsal line and also larger dark spots toward the lateral ends of the
shield.

Thoracic legs tinged with brown, claws and markings anterior to and
partially surrounding basal joints, reddish brown.

Prolegs on segments 8, 9, 10, and 13 and rudimentary prolegs on
segment 7; crotchets reddish brown.

The dorsal line is a greenish gray partially broken and bordered with
brown; subdorsal and lateral lines brownish; general color green to
gray; spiracles dark brown; tubercles are greenish, setae single and
ringed at base with a light color.

FIFTH INSTAR

Head width, 1.38 mm. to 1.98 mm.; average 1.83 mm.

Total length of body, 11. 5 mm. to 18.0 mm.; average 16.1 mm.

General color of head much lighter (PI. 28, E) ; ocellar region very
dark brown; ocelli i, 2, and 6 colorless, other three dark; mandibles
reddish brown to black at teeth; lower margin of labrum reddish,
blackened at notch; cardo and submentum bro^vn with the margins of
stipes next the mentum same color and with the stripe becoming wider
about the base of the palpifer; sclerite resting on mentum blackened.

The two bands of dark brown on the epicranium and bordering the
adfrontals become prominent for the first time in this instar (PI. C, 2).
The rest of the head seems to have lost color, leaving these two stripes
which run from points even with the base of the clypeus to the second
epicranial setae, above which they gradually fade out about the first
epicranial setae.

Nov. 5,1921 Pale Western Cutworm 317

The thoracic shield is brownish with a light dorsal stripe ; in the lighter
area on the anterior margin of the shield and on either side of the dorsal
stripe are distinct blackened spots, with other dark spots toward the
lateral margins of the shield.

Thoracic legs tinged with brown, claws and markings anterior to and
partially surrounding basal joints reddish brown.

Fully developed prolegs on segments 7, 8, 9, 10, and 13 are concolorous
with body; crotchets brownish. The anal plate is marked with a trans-
verse row of small brownish spots anterior to the setae.

The dorsal line is a greenish gray and bordered with brown; the sub-
dorsal and lateral stripes brownish; the general color is about the same
or perhaps a trifle lighter than the dorsal stripe.

SIXTH INSTAR

Head width, 1.98 to 2.64 mm.; average 2.41 mm.

Total length of body, 2.0 to 2.5 cm.; average 2.2 cm.

Head light brown; ocellar region very dark brown; ocelli i, 2, and 6
transparent, other three brownish; mandibles reddish brown to black
at teeth; lower margin of labrum reddish, blackened at notch; cardo and
submentum brown with the margins of stipes next the mentum same
color with the stripe broadening apically about the base of the palpifer.

The two conspicuous bands of dark brown persist on the epicranium
bordering the adfrontals (PI. 28, F); the stripes become a lighter color
at top, ending near the first epicranial setae; a denser colored portion of
each band follows the adfrontals almost to their apex.

The thoracic shield is brownish with a light dorsal stripe ; in the lighter
area on the anterior margin of the shield and on either side of the dorsal
stripe are distinct blackened spots with other dark spots toward the
lateral margins of the shield.

The thoracic legs tinged with brown, claws and markings anterior to
and partially surrounding basal joints reddish brown.

Fully developed prolegs are found on segments 7, 8, 9, 10, and 13, con-
colorous with body; crotchets brownish. The anal plate on the thirteenth
segment, a pale green in color, possesses a transverse row of brownish
spots anterior to the setae; a light-colored dorsal stripe runs through the
plate.

The dorsal stripe is a broken gray green bordered with light brown;
the subdorsal is brownish, but the lateral has become lighter in color,
brownish gray. Sets single, ringed at base with a light-colored area;
tubercles greenish brown; spiracles black.

SEVENTH INSTAR

Head width, 2.70 to 3.18 mm.; average 2.93 mm.
Total length of body, 2.9 to 3.2 cm.; average 3.02 cm.
I Head light brown; ocellar region very dark brown, ocelli 1,2, and 6
transparent, other three brownish; mandibles reddish brown to black at

3i8 Journal of Agricultural Research voi. xxn.No. 6

teeth; lower margin of labrum reddish; ringed-shaped sclerite resting on
mentum blackened ; cardo and submentum brown with margins of stipes,
next the mentum, same color with the stripe broadening about the base
of the palpifer.

The bands of dark brown bordering the adfrontals have the same
appearance as in the previous instar (PI. 29, A).

The thoracic shield is dark brown with a distinct light-colored dorsal
stripe on either side of which toward the anterior margin of the shield
is a small blackened area with other dark spots toward the lateral margins
of the shield.

The thoracic legs are tinged slightly with brown toward the apical
joints; claws reddish brown, and color markings remain the same about
the basal joints. Prolegs on segments 7, 8, 9, 10, and 13 are concolorous,
crotchets brownish; the anal plate is a gray green with dark spots in a
transverse row anterior to the setae; a light-colored dorsal stripe runs
through the plate.

The dorsal stripe is gray-green, and the brown borders in the previous
instars appear greenish in this one; the subdorsal and lateral stripes are
greenish with a slight tinge of brown ; the general color is a greenish gray ;
setae single and ringed at base with light area; spiracles black, tubercles
green; the pulsating dorsal vessel can be easily seen through the epi-
dermis.

Eighth instar

Head width, 3.18 to 3.42 mm.; average 3.36 mm.

Total length of body, 3.1 to 3.6 cm.; average 3.34 cm.

The general color of the head is a light brown with a slight yellowish
tinge; ocellar region dark brown, ocelli i, 2, and 6 colorless, other three
dark brown to black; the front is a trifle lighter in color than the clypeus;
the adfrontals, which are made very distinct by a darker brown coloration
following the frontal sutures and by the dark bands following the epi-
cranial sutures, extend to the bottom of the deep indentation separating
the epicranial lobes at the summit; the mandibles are black at teeth and
at points of articulation, and between lies an area which is a very pale
brown in color; submentum and cardo brown; chitinized brownish stripe
on margin of stipes, next the mentum, broadened about base of palpifer;
antennae, labial and maxillary palpse brownish.

The two bands of dark brown which border the adfrontals and which
are very conspicuous in the fifth, sixth, and seventh instars, are some-
what reduced, especially the upper parts of the bands in the regions of
the first and second setae, in the advanced stage of the last instar (PL
29, B) ; during the first days of the instar the bands extend from points
even with the base of the clypeus to the second epicranial setae, running
between the setae and the adfrontals; here they become slightly less
dense in color and divided, parts of the stripes continuing along the

Nov. s, 192 1 Pale Western Cutworm 319

adfrontals and the other, lighter but wider parts, extending back to the
regions of the first epicranial setae.

The thoracic shield is pale brown with a prominent whitish dorsal
stripe; there are small spotted dark brown areas toward the lateral
margins of the shield and several smaller brown spots on either side of
the dorsal stripe.

The thoracic legs are tinged with brown, especially laterad; about the
base of the coxae and femora anteriorly are reddish brown stripes.

The prolegs on segments 7, 8, 9, 10, and 13 are concolorous with body
and possess reddish brown crotchets.

In the first part of this instar (PI. C, 3) there are distinct dorsal and
subdorsal stripes, the dorsal appearing as dark green and produced by
the pulsating dorsal vessel beneath the epidermis, and subdorsal as
brownish ; a broken whitish lateral stripe is quite distinct; in the advanced
stage of the instar with the exception of the tubercles there are no mark-
ings on the body, which becomes a bleached out yellowish color.

The spiracles are black; setae, which are a reddish brown especially
in the head region, are single; tubercles a greenish brown but imme-
diately about the base of each seta there is a ring of lighter color; the
anal plate is marked by a transverse row of dark brown spots anterior
to the setae.

PUPA

Length 17.5 mm., width 5.7 mm.

Typical noctuid pupa; labial palpa? exposed for entire length; maxillae,
mesothoracic legs and antennae of practically same length and extend-
ing almost to caudal margin of wings ; prothoracic femora exposed ; tips
of metathoracic legs visible and mesothoracic legs not extending to eye
pieces; dorsal cephalic margins of abdominal segments 5, 6, and 7 marked
with many small chitinized circular pits which extend to ventral surfaces
of segments but where they are fewer in number and less prominent.
The slightly bifurcate, blackish, rough cremaster ends in two stout often
incurved spines set far apart. The color of pupa varies from a light
straw color to a dark brown, according to age (PI. C, 4).

ADULT '
"Agrotis orthogonia" nov. sp.

All the tibiae spinose. Antennae of the male strongly serrate. Middle of the
second joint of palpi black, its outer edge and tip, as well as the third joint, light.
Head and thorax gray. Anterior wings dark gray; all the markings well expressed;
half-line followed by a white shade line; basal space lighter than the other portions
of the wing; interior line forming a very long outward projection below the submedian
vein, and another shorter one on the costa, the line is white and distinct, bordered
with black on each side, between the submedian and subcostal veins it is straight,
except one lobe below the median vein, to which the concolorous, black edged clavi-
form spot is attached; subcostal median and submedian veins white, and contrasting

1 The description of the adtilt is quoted from Morrison (z^).

320 Journal of Agricultural Research voi. xxn. no. 6

(PI. C, 5); orbicular spot elliptical, with an outer black ring, within which appears a
white annulus, inclosing the gray center; reniform spot large and of the usual shape,
the portion of its black annulus, beneath the median vein, separated and very dis-
tinct; exterior line rounded, formed of interspaced luniform marks, followed by a white
shade line; subterminal space rather lighter than the median space, terminal space
again dark; a series of partially effaced cuneiform marks, before the white subterminal
line, which forms two short teeth on the second and third median branches. Pos-
terior wings whitish at the base, with a black terminal band and contrasting white
fringes. Beneath whitish, the center of the median space dark, and the neighbor-
hood of the median vein, on the anterior wings, clothed with long soft hair.

Expanse, 34 mm.

Hab. Glencoe, Nebraska. Received from Mr. G. M. Dodge. (No. 66).

The nearest ally of this fine species is the European Agrotis vestigiales Rett.

LITERATURE CITED

(1) COOLEY, R. A.

1919. SEVENTEENTH ANNUAL REPORT OF THE STATE ENTOMOLOGIST OF MONTANA.

Mont. Agr. Exp. Sta. Bui. 133, 15 p.

(2) DoD, F. H. Wolley.

1901-06, PRELIMINARY LIST OP THE MACRO-LEPIDOPTERA OF ALBERTA, N.-W. T.

In Canad. Ent., v. 33, p. 40-42, 157-172, 1901; v. 36, p. 345-355.
1904; V. 37, p. 17-28, 49-60, 145-156, 173-184, 221-230, 241-252,
1905; V. 38, p. 45-54. 89-94, 263-267, 1906.

(3) Dyar, Harrison G.

1902. A LIST OP NORTH AMERICAN LEPIDOPTERA... U. S. Nat. Mus. Bill. 52, xix,

723 p. List of works quoted, p. ix-xix.

(4) Forbes, William T. M.

1910. A structural study op some caterpillars. In Ann. Ent. Soc. Amer-
ica, V. 3, no. 2, p. 94-132, pi. 10-20. Bibliography, p. 125-127.

(5) Gibson, Arthur.

1912. THE entomological RECORD, 1911. In 42d Ann. Rpt. Ent. Soc. On-
tario, 1911, p. 89-112.
(6)

(7)

1912. cutworms and army-worms. Canada Dept. Agr. Div. Ent. Bui. 3 (70,
Exp. Farm ser.), 29 p., 10 fig.

1914. A NEW destructive CUTWORM OP THE GENUS POROSAGROTIS, OCCURRING

IN WESTERN CANADA. In Jour. Econ. Ent., v. 7, no. 2, p. 201-203.

(8)

191 5. CUTWORMS AND THEIR CONTROL. Canada Dept. Agr. Div. Ent. Bui. 10.

31 p., 20 fig.

(9) Hampson, G. F.

1908. notes on noctuidaE... In Canad. Ent., v. 40, no. 3, p. 102-107.

(10) Hewitt, C. Gordon.

1912. report op the dominion entomologist, /w Canada Exp. Farms Rpts.
[i9ii]/i2, p. 173-189, pi. 5.

(11)

1914. report prom the division of entomology... [i9i2]/i3. In Canada

Exp. Farms Rpts. [i9i2]/i3, p. 499-518, pi. iq.

(12)

1915. REPORT FROM THE DIVISION OP ENTOMOLOGY... [i9i3]/i4. In Canada

Exp. Farms Rpts. [i9i3]/i4, p. 851-876.

Nov. s. 192 1 Pale Western Cutworm 321

(13) Canada Department op Agriculture.

1917. REPORT OF THE DOMINION ENTOMOLOGIST, [i9i5]/i6. 73 p., 4 pi. Ottawa.

(14) Maxson, Asa C.

1920. PRINCIPAL INSECT ENEMIES OK THE SUGAR BEET IN THE TERRITORIES
SERVED BY THE GREAT WESTERN SUGAR COMPANY. vii, 157 p., 30 fig.

9 col. pi. Denver, Colo. Bibliography, p. 147-148.

(15) Morrison, H. K.

1877. descriptions OP NEW north AMERICAN NOCTUIDAE. In PfOC. BoStOH

Soc. Nat. Hist., v. 18, 1875/76, p. 237-241.

(16) Smith, John B.

1890. contribution toward a MONOGRAPH OF THE INSECTS OF THE LEPI-
DOPTEROUS FAMILY NOCTUIDAE OF TEMPERATE NORTH AMERICA. RE-
VISION OF THE SPECIES OF THE GENUS AGROTIS. U. S. Nat. MuS. Bul.
38, 237 p., 5 pi.

(17) Strickland, E. H.

1915. POISONED BAIT FOR CUTWORMS. In Canad. Ent., V. 47, no. 7, p. 201-204.

(18)

1916. THE CONTROL OF CUTWORMS IN THE PRAIRIE PROVINCES. Canada Dept.

Agr. Ent. Branch Circ. 6, 8 p., 5 fig.

65769°— 21 3

PLATE C
Porosagroiis orthogonia:

—Eggs.

— Cast head of fifth-instar larva.

— Eighth-instar larva.

— Pupa in earthen cell.

— Adult, male.

(322)

Pale Western Cutworm

Plate C

1 ^^i3^

Hit- i^i/^'<MA i\ I

Journal of Agricultural Research

Vol. XXII, No. 6

PLATE 28
PoTOsagrotis orthogonia:
A, B, C, D, E, F. — Cast heads of first- to sixth-instar larva?.

Pale Western Cutworm

Plate 28

Journal of Agricultural Research

Vol. XXII, No. 6

Pale Western Cutworm

Plate 29

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Journal of Agricultural Research

Vol. XXII, No. 6

PLATE 29

Porosagrotis orthogonia:

A. — Cast head of seventh-instar larva.

B. — Cast head of eighth-instar larva with setae numbered. Adf., adfrontal sclerite,
adf. i, adf. it, its setae; fr., frontal sclerite; fr. i, frontal setae; fr. 0, frontal puncture;
cL, clypeus; cl. i, cl. ii, its setse; Ihr., labrum; ant., antennae; md., mandible; md. i,
md. ii, its setae; i to xi, setae of epicranium, / to IV , first four ocelli.

PLATE 30

Porosagroiis orthogonia:

A. — Moth feeding on clover blossom.

B. — Light trap.

C. — Excavation made by horned lark in digging out cutworm.

D. — ^Wheat field attacked by the larv'se.

Pale Western Cutworm

Plate 30

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Journal of Agriculturjil Researcli

Vol. XXII, No. 6

BIOIvOGY OF EMBAPHION MURICATUM

By J. S. Wade, Scientific Assistant, Cereal and Forage Insect Investigations, Bureau
of Entomology; systematic description of the larva by Adam G. Boving, Bureau
of Entomology, United States Department of Agriculture

INTRODUCTION

Considerable damage has been wrought during the past six or seven
years by the larvae of Embaphion fmiricatum Say and related species of
false wireworms to growing wheat and other field crops throughout the
semiarid and middle western United States. The area of greatest
injury embraces approximately the western half of Nebraska, Kansas,
and Oklahoma, and the eastern third of Colorado and New Mexico,
although losses of varying magnitude have been reported in various
localities over the greater part of all these States. In view of the obscure
character of the injury, it seems quite probable that much crop damage
commonly charged to other causes in reality has been brought about by
this pest. The steady transformation in recent years of grassy prairies
into cultivated fields has been an important factor, because the removal of
native food plants causes this and related species to feed more and more
upon cultivated grains. The hardiness of the insect and the rapidity of
its adaptation to changed conditions and to new host plants indicate
that this species is potentially a serious menace to grain production
within the infested region.

EARLY RECORDS

The species under discussion was originally described in 1824 as Akis
muricata by Thomas Say (/) ,^ who stated that it —
inhabits Arkansa at the Rocky Mountains

and that —

as it does not entirely agree witli any genus the characters of which Latreille has
noted, it may be proper to remove it to the Blapsidae, under a separate genus, which
may be named Embaphion.

This description was reprinted in 1859 in the LeConte edition of
Say's (x) works, with a brief supplementary editorial note indicating
relationship of the genus Embaphion to the genus Eleodes. A single
specimen from Texas was described as Eleodes contusum by LeConte (2)
in 1853, who stated that it —

resembles E. muricatum Say but is longer and narrower with the broad margin of
the elytra more suddenly reflexed and almost perpendicular. Although so different
in form, this genus is only distinguished from Eleodes by the inferior plane of the

' Reference is made by number (italic) to "I<iterature cited," p. 334.

Journal of Agriculture Research, Vol. XXII, No. 6

Washington, D. C. Nov. s. 192 1

aaj , Key No. K-103

(323)

324 Journal of Agricultural Research voi. xxii. no. 6

mentum being more rounded and more deeply impressed; its anterior margin is
slightly incised; the lateral angles are so much reflexed as to be invisible; the tarsi
are silicate beneath and fringed at the apex and sides with short spines; the middle
joints of the posterior tarsi appear more elongated than in Eleodes. I have grave
doubt of the generic value of any of these differences, and several nondescript species
from New Mexico seem to be intermediate both by the form of the body and by the
dififerences in the mentum.

The characters distinguishing Embaphion contusum from Embaphion
muricatum were discussed by LeConte (j) in 1859. A brief resume of the
previous history of the genus was made by Lacordaire {4, v. 5, p. 152;
atlas, pi. 50, fig. 2) in 1859, in which attention was called to Say's inade-
quate designation of the genus Embaphion and to variation of the species
with its geographical distribution. Horn (5, p. 320-322) in 1870, in his
discussion of the genus, indicated the feeble taxonomic characters which
separate this genus from Eleodes. In referring to the species Embaphion
muricatum he states :

This species may be readily distinguished from the others of the genus by the very
broad foliaceous margin of the thorax and elytra, very strongly reflexed. The elytral
margin extends beyond the apex and the two meet on a line with the suture. The
thoracic margin is broad and widens behind, so that the hind angles are prominent,
sub-acute, and project backwards over the basal angles of the elytra. The thorax
itself (less the margins) is narrow, longer than broad, and about equal to half the width
of the elytra (without margin). The disc of elytra (without margin) is elongate oval,
the humeral angles not prominent and are rounded. The angles formed by the margin
are nearly right. The base of the thorax is strongly trisinuate; the base of the thorax
proper being rounded, that of the margin on each side emarginate. The base of elytra
is emarginate at middle, and on each side broadly rounded.

He stated further that Embaphion concavum, described by LeSonte
(2) in 1853, is

merely a large form with more strongly reflexed margins. The elytra of both forms are
sculptured with approximate series of fine punctures, each bearing a short hair.

Blaisdell's (//, p. 473-477) very full discussion (1909) of the adult
forms of the species and their taxonomic relationships leaves little to be
desired. He especially emphasizes the salient type characters.

Margins of the thorax and elytra broadly foliaceous and strongly reflexed, basal
angles of the prothoracic margins projecting strongly backward over the basal angles
of the elytra.

DISTRIBUTION

Nebraska: Alliance, altitude 3,971 feet, August, H. F. Wickham; Beaver City, alti-
tude 2,150 feet, M. H. Swenk {12), September, J. S. Wade; "Nebraska," May to
August, H. F. Wickham (<S).

New Mexico: Chico, altitude 6,882 feet, September, D. J. Caffrey; Clovis, August,
H. F. Wickham; Koehler, altitude 5,500 feet, June, V. L. Wildemiuth, August,
W. R. Walton; Vaughn, September, H. F. Wickham; Maxwell, altitude 5,894 feet,
D. J. Caffrey; Willard, altitude 6,091 feet, H. F. Wickham.

Kansas: Clark County, altitude 1,962 feet, June, F. H. Snow {10); Colby, altitude
3,150 feet, August, J. S. Wade; Dodge City, altitude 2,509 feet, August, J. S. Wade;
Hamilton County, altitude 3,000 feet, F. H. Snow {10); Liberal, altitude 2,839 feet,

Nov. 5, 1921 Biology of Embaphion muricatum 325

July, J. S. Wade; Meade, altitude 2,503 feet, July, J. S. Wade; Morton County, alti-
tude 3,000 feet, F. H. Snow {10); Norton, altitude 2,284 feet, August, J. S. Wade;
Rice Coimty, June, H. F, Wickham; Scott City, altitude 2,971 feet, August, J. S.
Wade; Wallace County, altitude 3,000 feet, F. H. Snow (7); Wellington, altitude
1,205 feet, July, J. S- Wade; "Kansas to Texas," G. H. Horn (5), "Western Kansas:
In Arkansas and Smoky Hill Valleys," E. A. Popenoe ((5).

North Dakota: Dickinson, altitude 2,411 feet, August, H. F. Wickham; "Dakota,"
W. G. Dietz; "Dakota," May to August, H. F. Wickham {8).

Colorado: Bellevue, altitude 8,993, H. F. Wickham (p); Colorado Springs, altitude
6,072 feet, H. F. Wickham {g) ; Denver, altitude 5,279 feet, April, H. Soltau; Greeley,
altitude 4,652 feet, June, H. F. Wickham; Fort Collins, altitude 4,994 feet, H. F.
Wickham (p); LaSalle, altitude 4,676 feet, September, H. F. Wickham; Pueblo,
altitude 4,685 feet, October, H. Soltau; West Las Animas, H. F. Wickham (p);
"Colorado," May to August, H. F. Wickham (8).

Texas: Amarillo, altitude 3,676 feet, August, H. F. Wickham; Canadian, altitude
2,340 feet, August, H. F. Wickham ; Mobeete, July, H. S. Barber; Texline, altitude
4,694 feet, September, I. R. Crawford.

Montana: Assinniboine Mountains, Hubbard and Schwarz; "Montana," May to
August, H. F. Wickham (8).

South Dakota: Alexandria, altitude 1,354 feet.

Mexico: Nuevo Laredo, Tamaulipas, Hoge.

INJURY

The principal damage caused by these insects is that wrought by the
larvae during the fall in devouring recently sown or newly sprouted wheat
grains shortly after the seed wheat has been drilled. These larvee often
may be found in large numbers in infested fields at such periods working
steadily along through the soft soil of the drill rows, either wholly devouring
or destroying for germination purposes every wheat grain within a drill row
for many yards. Within the region of greatest infestation the principal
injury is done between September 20 and October 15. The injury to the
grain is characteristic of this family. Sometimes the entire contents of
the grain are removed, lea\'ing all or part of the shriveled outer husk; in
some cases the ends of the grain are nibbled away or portions of the ven-
tral crease are neatly fmrowed out. The adults also are known to feed
upon wheat grains and other seeds, being present around the bases of
wheat stacks in July, where they may be found tearing away the spikelets
of grain in newly cut wheat heads to devour the kernel within, or they
may be found feeding upon the scattered grains. The extent of the injury
varies annually in accordance with seasonal conditions, little or no damage
being done in localities where an abundance of rainfall occurs, and where
temperature and other factors are favorable to growing crops, whereas
at the same time considerable loss may be experienced in other localities,
varying from 10 to 50 per cent or more of the wheat of an entire neighbor-
hood, where weather and other conditions render normal development
of this crop impossible. In view of the fact that the larvae of this pest
usually may be found working with those of other nearly related species
of true and false wireworms, it becomes increasingly difficult to isolate and
estimate singly the exact amount of injury wrought by this particular pest.

326 Journal of Agricultural Research voi. xxu, no. 6

HABITS

The larvae are exceedingly active and quick and, if exposed to light by
the plow or otherwise disturbed, have the power of ^vriggling very
quickly down out of sight into the soil. They are also occasionally
found upon the surface of the ground feeding upon seeds of weeds and of
other plants, in spots where the soil may be slightly moist and where
they are covered by wheat shocks or by matted masses of dried Russian
thistles or other weeds. While they appear to prefer habitats where
there may be a slight degree of moisture, such as moist, poorly drained
spots in fields, and cool, damp cellars, yet they do not live long in thor-
oughly wet soil. Both larvae and adults often may be found in numbers
beneath dried weeds along irrigation canals. The larvae habitually feed
during warm weather at a depth varying from 2 to 5 inches, according to
condition of the soil. As they burrow from place to place, they feed upon
the roots and seeds of plants, and possibly to a certain extent upon
organic matter where this is sufficiently decayed. When placed under
artificial conditions the larvae feed readily not only upon germinating
wheat, but upon com and roots of grasses. They are cannibalistic in that
they feed upon other larvae of the same species which die or become
weakened because of injury or disease. They also feed upon their own
exuviae.

The adults, in common with those of other nearly related species, are
very hardy and active and appear to be able to withstand considerable
variations of temperature. While they, like the larvae, appear to prefer
cool, moist spots, they do not survive temperatures as low as —9° F.
They have been collected in August beneath wheat shocks in fields where
the temperature was as high as 100°. The adults easily climb all over
wheat where standing or in the stack or shock, and they burrow with
apparent ease far into the piles of unthrashed grain. They are also fre-
quently found in the burrows of small mammals. During periods of pro-
longed drought the beetles may seem to have entirely disappeared, yet
immediately following a shower or rainstorm, curiously enough they
reappear in large numbers, where previously none could be found.

DESCRIPTIONS

EGG

Size slightly variable, being i.i to 1.3 mm. in length and 0.60 to 0.62 mm. in width;
shape circular in cross section and oval in longitudinal section; without sculpturing;
color pure white when first deposited, changing to yellowing brown before hatching.

MATURE LARVA *

Length 27 mm.; color testaceous with head and legs somewhat darker; anterior
and posterior margins of prothorax and posterior margins of the following segments
castaneous-testaceous. Surface corneous. Form elongately cylindrical, more than

1 Description and Plates 31 and 32 by Adam G. Bdving.

Nov. 5. 1921 Biology of Embaphion muricatum 327

10 times longer than wide; dorsally verj'^ convex, ventrally flattened; pygidium
movable in the directions up and down, subconical, obtusely pointed. Head, ven-
tral sides of the thoracic segments and of the first abdominal segment, legs, and
pygidium (PI. 32, C) clothed with rigid or soft setae; rest of body glabrous with very
few and small ventral hairs.

Cranium (PI. 31, B) rounded, nutant, exserted, one-third broader than long (from
epistomal margin (epi) to foramen occipitale), broadest medianly, dorsally somewhat
convex. Anterior frontal angle (fa) low and rounded. Frons (/) three-fourths the
length of cranium, about as long as wide with extreme width anteriorly, side margin
convex. Epicranial halves (epc) meeting dorsally; epicranial sutiue one-fourth the
length of cranium; ventrally (PI. 31, E) the halves are separated by gula (gu); dor-
sally with a few, laterally and ventrally with many hairs. Gula and submentum
(sm) both distinct, coriaceous. Gula almost square, with tentorial pits (ip) at the
middle of the side margins. Submentum trapezoidal, broadest posteriorly; side
margins slightly concave and adjacent to maxillary articulating area. Clypeus
(cl, PI. 31, B) trapezoidal, widest behind, length to extreme width as one to four,
medianly with slight transverse deepening, set on each side with one minute seta
near the middle line and two well-developed setae near the lateral margin. Labrum
well-developed, movable, transversely rectangular, length to width as one to three,
anterior margin almost straight, anterior comers rounded; disk on each half with a
median transverse series of five large setae, and an anterior series of three long, thin,
and straight setae; right behind those but on the ventral side of labrum another series
of four shorter, stronger, and ciurved setae. Epipharynx (eph, PI. 31, A) forming the
buccal surface of labrum, soft-skinned with posterior transverse, broad, sinuous, chitin-
ous band, that carries one pair of stublike sharp teeth; on the soft-skinned part
anteriorly to these teeth a pair of tiny hooks; near anterior margin scattered minute
setae and ring-shaped punctitfes. Just behind antenna two ophthalmic spots, both
transverse, slightly posteriorly convex, the anterior a little more external and about
three times longer than the posterior; immediately in front of the anterior are numer-
ous setae; the ophthalmic spots are likely to disappear in full-grown larvae. Antenna
(PI. 31, B) closely behind the mandible, attached in articular cavity with distinct
border; basal antennal membrane well developed; basal article cylindrical, about as
long as epicranial suture, second article as long as basal article, more clavate, apical
article very small, conical, papilliform, carrying one seta; no supplementary appendix
besides the apical article. Mandibles (Pi. 31, F) of right and left side differing in
shape; both apically bifid (a^, a^); both with one tooth (/) between apex and molar
part (m); tooth of right mandible, however, prominent and placed near apex, that of
left mandible less developed and placed closer to molar part; molar part of right mandi-
ble with bituberculate crown, that of left mandible with hollow crown; ventrally (PI.
31, D) with cutting part deeply excavated; exterior stirface ("the back of the mandi-
ble") distally with a slightly carinate margin (PI. 31, F, c), proximally with a soft-
skinned, whitish swelling (s) from an excavation (e) opposite the molar part; three
to four strong setae from the anterior portion of the swelling, two from the posterior,
several small, soft setae near dorsal mandibular articulation. Maxilla dorsally com-
pletely covered by mandible; palp (PI. 31, E) siu-mouiiting mala (ma) (maxillary
lobe) with one-third of its own length; palpiger (pag) small, ring-shaped; basal article
about as wide as long, second article cylindrical, somewhat narrower and more than
twice as long as basal article, apical article two-thirds as long and half as thick as the
second, conical, with soft tip; each article with one or two thin setae; mala (ma) on
buccal stuface (PI. 32, F) with two series of well-developed, somewhat curved setae;
base of stipes (PI. 31, E, bs) (that is, region where stipes and cardo meet) rather short;
proximal half of inner margin of stipes (is ,) connected with exterior half of maxillary
articulating area (ar^), distal half (js- 2) right behind mala, free; ventral stipital
surface with several strong setae; other setae on the exterior surface; cardo as long as

328 Journal of Agricultural Research voi. xxii, no. e

exterior margin of stipes, adjacent to slightly curved hypostomal thickening {hyp)
between fossa for ventral mandibular condyle {fm) and fossa for tip of cardo (/c); inner
margin of cardo connected like stipes with exterior half {ar ^) of maxillary articulating
area. Maxillary articulating area protuberant, soft, divided into two halves; exterior
half (ar I) connected with maxilla, subdivided into an upper and low^er portion;
interior half (ar^) connected with submentum, entire; no seta. Mentum {me)
almost square, side margins free; on each side about five setse of different length.
The two stipites labii {stla) fused into a slightly chitinized unit, carrying on each
side two setae; labial palp about half as long and half as thick as maxillary palp;
basal and apical articles slightly different in length, basal article somewhat clavate,
apical article conical and half as thick as basal article; ligula {li) small, narrow conical,
with one terminal pair of setae. Hypopharyngeal sclcrite (PI. 32, A, G, H, hsc)
elongate rectangular, projecting, strong; anteriorly tricuspidate with median cusp
largest; disk somewhat excavate with a posterior semiglobular tubercle; molar part
of mandible and hypopharyngeal sclerite grinding together (PI. 31, D, F, G). The
hypophar>'ngeal bracon (PI. 32, A, G, H, hbr) is well developed as a chitinous rod in
the buccal membrane between the ventral mandibular articulation and the hypo-
pharyngeal region. Prothoracic legs (PI. 31, C, H, I; 32, B) considerably stronger
than the mesothoracic and metathoracic ones and with coxae attached so closely
together that they almost touch each other at base. Coxa of first pair about as long
as wide ; many fine , scattered hairs on exterior and interior sm"f aces ; trochanter about
as long as coxa, on the inner side (PI. 31, H) distally with two spinelike setae and also
with a few other thin hairs; femur {fe) about as long and wide as trochanter, armed
with five spinelike setae, also with many thin, scattered hairs; tibia {ti) about twice as
long as thick, almost same length as femur but not fully as wide, armed with five
spinelike setse and also with fine, scattered hairs; tarsus {ta) of almost same length
as tibia, claw-shaped, strong, but rather slender, with backward-facing surface distally
excavate and proximally carrying a round soft-skinned region around a short but strong
seta; another and similar seta set close to it at the end of the excavation; otherwise
no setae or hairs on tarsus. Second and third pairs of legs inserted farther apart than
the first pair; the arrangement of their setae very similar to that of the first pair, but
the proportional sizes between the articles somewhat different from those of the
first pair. Prothoracic eusternum (PI. 32, B, eu) large, rhomboidal, anteriorly almost
reaching the front margin of the segment, only separated from this margin by a small
presternal area (y); the hypopletu-al chitinization {hi and h-^), and especially its
prehypopleural part (A i), large and strong; prothoracic tergal shield siibquadrate,
slightly wider than long, with anterior and posterior margins, as mentioned above,
darker than the rest of the shield and finely longitudinally striated. Mesothorax
and metathorax with transverse, sub triangular, narrow presternum {y), laterally
adjacent to poststernellura (2) of the preceding segment; hypopleiiral chitinizations
{h I and h n) well developed, but considerably smaller than those of prothorax;
poststemellum of metathorax not present, preepipleurum of mesothorax {e i)
subtriangular, carrying first thoracic spiracle; preepipleurum of metathorax not
distinctly limited, carry'ing the rudimentary second thoracic spiracle; postepipleiuiim
(e u) of both segments well developed, more or less fused with the corresponding
preepipleura ; mesothoracic and metathoracic tergal shields transversal, subrectangu-
lar, about twice as wide as long, right behind anterior margin with a dark transverse
line; posterior margin darker than rest of segment, finely longitudinally striated.
Typical abdominal segment (that is, one of the eight anterior abdominal segments)
with fused sternal and hypopleural areas {ster), covered by a single, longitudinally rec-
tangular shield, which posteriorly has a rather dark, transverse, longitudinally finely
striated margin; one seta present near the anterior and one seta near the posterior
margin; additionally the sternum of first abdominal segment is anteriorly densely

Nov. 5, 1921

Biology of Embaphion muricatum

329

set with small, soft sets; similar outfit lacking on the other abdominal segments.
Epipleural region narrow. Tergal region with a dark line above the spiracle. Tergal
shield (ter) single, posteriorly with a dark, longitudinally striated margin. Anterior
abdominal segments transverse, slightly wider than long; sixth, seventh, and eighth
abdominal segments subquadrate. Ninth abdominal segment smaller than the
preceding segment ; dorsal part or pygidium pointing upwards, subconically produced,
above somewhat flattened, below broadly convex, apex obtuse, laterally with margin
set with a single series of strong, short setse, whole siuface with scattered, fine setae;
ventral part of ninth segment small, transverse, soft. Tenth abdominal segment
(or "anal segment") small, with trilobate upper transverse anal lip, with a pair of
conical and, except at the tip, setose ambulatory warts, laterally to anus a small
triangular lower lip. Spiracles (Pi. 32, I) annular, shortly oval, transversely placed;
opening at the bottom of cup-shaped peritrema, linear, unprotected by hairs. The
number and development of setae on the first pair of legs vary according to species
and do not offer any generic character. The same is the case with the setal arrange-
ment of pygidium.

Fig. I. — Pupa of Embaphion mu-
ricatum, dorsal view.

Fig. 2. — Pupa of Embaphion mw
ricaium., ventral view.

PUPA (FIG. I, 2)

Length 11 mm. Width 5.6 mm. Free. Arcuate. Color pinkish white, with
ferruginous tinge on pronotiun. Femora and tarsi fuscate, other appendages partly
translucent. Pupa becomes more strongly colored immediately before issuance of
adult. Head pressed to prostemum. Pronotum broad and projecting somewhat
anterior to head, making the head nearly invisible from above. Frons impressed.
Vertex prominent. Antennae placed backward near sides of prothorax. Mesonotum
narrow. Legs not pressed against body. Tips of wing cases extending to the an-
terior margin of metanotum. Second to fifth abdominal segments bearing on each
side of tergites flat, lacerated protuberances, obtuse, pointed and directed posteriorly.
Eighth segment ending in two slightly divergent, acute processes.

330

Journal of Agricultural Research voi. xxn.N>. e

ADULT (fig. 3) *

Oval to oblong-oval, brownish to piceous black, thoracic and elytral margins very-
broad and foliaceous, strongly reflexed.

Head small, less than twice as wide as long, plane, sides of the frons slightly promi-
nent, punctate, punctures very feebly subasperate, fine, not dense, each with a small
curved and short seta, frontal suture usually not visible. Antennce rather long, quite
slender, outer four joints very slightly compressed and scarcely widened, tliird joint
shorter than the next two taken together, fourth scarcely longer than the fiftli, the
latter and sixth subequal, seventh shorter, eighth feebly shorter than the seventh
and slightly triangular, ninth and tenth suborbicular, eleventh subovate.

Pronotum with margins very broadly foliaceous, the margin more than one-half
wider than the disc, the latter comparatively narrow, longer than wide at middle, very

feebly convex, usually with irregular de-
pressed areas; finely, more or less subas-
perately and sparsely punctate; reflexed
margins wider posteriorly and more or less
concave, a little more distinctly punc-
tate, punctures less sparse, each with a
short curved seta; o/>ex deeply and feebly
subquadrately emarginate, the emargina-
tion about one-half wider than deep, sides
almost parallel, and scarcely margined;
sides evenly but not strongly arcuate,
moderately converging from base to apex;
base proper feebly arcuate, not margined
and about equal to the length, 1^.' ,rally
sinuate; apical angles rather narrowly
rounded and formed by the advanced
foliaceous margins and nearly as long as
the head; basal angles are posteriorly
prominent, subacute, and projecting back-
ward over the basal angles of the elytra.
Propleurcc smooth and impunctate; in-
ferior surface of the foliaceous margins
obsoletely punctate.

Elytra oval to elongate oval; margins
broad andreflexed, angles at humeri nearly
rectangular and more or less truncate at base, posteriorly extending beyond the apex,
the two meeting on a line with the suture above the true elytral apex, and defined
from the same by a slight groove, borders evenly arcuate from base to apex or more
or less parallel basally ; base evenly but not strongly emarginate; humeri proper broadly
rounded and not prominent ; sides proper more or less evenly arcuate ; apex proper not
produced and narrowly rounded; disc plane, feebly convex, at times slightly concave,
the inflexed sides nearly straight and oblique, gradually and not strongly arcuately
declivous behind; surface sculptured with approximate series of fine asperate punc-
tures, which become more irregular and slightly denser laterally. Each puncture
bears a short and ratlier robust cur\'ed seta.

Epipleurce narrow, not attaining the humeral margin and not dilated, but gradually
narrowing to apex, not defined from the inflexed sides of the elytra, and on the same
plane; superior margin obsolete, except near apex; elsewhere represented by a line
of punctures or a faint groove.

Fig. 3. — Adult of Embapkion tnurkalum, dorsal view.

^Reprinted from F. E. Blaisdell (//, p. 473-476).

Nov. 5, 1931 Biology of Embaphion muricatum 331

Sterna more or less dull, finely and not distinctly sculptured.

Parapletirse smooth, rather sparsely but not very distinctly punctate.

Abdomen horizontal, very finely and sparsely punctulate, obsoletely rugulose and
quite evenly convex.

Legs rather slender, moderate in length. Anterior femora mutic, pro tibial spurs
similar in the sexes, the anterior slightly longer than the posterior. Protarsi simple.

LIFE HISTORY AND DEVELOPMENT

The principal observations on the hfe history and development of
Embaphion muricatum, as given below, were made under laboratory and
field conditions in south-central Kansas, at an altitude of approximately
1,200 feet. Under different conditions of latitude, altitude, and humidity
there would doubtless be found more or less marked variations. The rec-
ords are unfortunately based on incomplete studies for, owing to working
conditions and to pressure of other duties, there was no opportunity to
conduct a sufficiently extensive series of experiments to render all obser-
vations conclusive.

The eggs are deposited in loose, dry, or slightly moist soil at a depth
of ^ to I inch, sometimes singly, but more often in clusters of two or
three to a dozen or more eggs at one place. At temperatures ranging
from 80° to 90° F. the average period of incubation is approximately
10 days, whereas at temperature's of 68° to 70° F. the egg stage is approxi-
mately 13 days. Undoubtedly weather conditions and the time of year
have a direct bearing on the duration of the egg period.

During the later stages of development and shortly before hatching,
the surface of the egg becomes light brown in color, and the shell appears
to expand slightly and to become more flexible, while the movements of
the young larva can be noted within. During the process of hatching,
the struggles and the lifting pressure of the young larva burst the shell
and the larva emerges by rather slow periodic movements, as its integu-
ment is very soft and fragile. Though the young larva often remains
for some time near the place of hatching, yet it is capable of locomotion
soon after emergence. All normal eggs of the same egg cluster usually
hatch within a short period, generally a few hom-s. While abnormal
weather conditions may prolong the period of hatching, no injurious
effects of such retardation are noted in the eggs. No infertile eggs were
ever collected under field conditions. Soon after the emergence of the
larvae the empty eggshells become more and more contracted and dried
up, until eventually only tiny, shrivelled fragments remain.

Upon hatching the larva averages 3.5 to 3.75 mm. in length and is
yellowish white. The color changes sHghtly after each molt until at
maturity the larva becomes a deep yellow.

The length of the larva stage, according to an experiment consisting of
3 1 larvae hatched in June and kept in a cool cellar at an average tempera-
ture of 68° F., averaged for the siu-vivors 79 days, while in an experiment
consisting of 49 larvse, under similar conditions, it varied from 76 to

332 Journal of Agricultural Research voi. xxii, no. 6

96 days, though the average duration was 85 days. The larvae as hatched
were placed in small tin salve boxes containing about X inch of slightly
moist soil and split wheat grains. As the larA^se became large, whole
wheat grains were used as food. Under field conditions many of the
larvse appear to become nearly mature during late fall and overwinter
in this condition. From about November i to March 15 in the latitude
of southern Kansas they are exceedingly inactive and feed but little.
The rapidity of growth of the larvffi undoubtedly depends to a large
degree upon weather and seasonal conditions and the quantity and
quality of food available. Shortly before the period of pupation the
larva does not feed and assumes a semidormant stage of approximately
7 to 9 days' duration.

The pupa stage, when rearings were conducted under laboratory
conditions, comprised 18 to 20 days. The pupse are pinkish white im-
mediately after transformation, and as development proceeds the color
changes to light yellow. Shortly before the adults emerge the append-
ages take on a yellowish brown tint.

The newly issued adults are of a brighter color, and the chitinous
portions of the body are soft. Within a few days, however, the color
darkens and the integument hardens so that the newly emerged adults
are not distinguishable. Under artificial conditions mating does not
become general for a week or more after emergence. Oviposition and
feeding appear to occur usually at night. The adults are crepuscular.
They may be found abroad in greatest numbers on cloudy days or in
early morning or late evening. On clear days, during the middle or
warmer portion of the day, they remain under shelter. While usually
inactive at such periods, if disturbed they will run with great rapidity.
The insect may overwinter both in the adult and in the larva stages. In
the latitude of southern Kansas, however, the mortality of such over-
wintering adults is great.

ENEMIES

While the incomplete character of the life-history work performed with
Embaphion muricahtm afforded comparatively little opportunity for
obtaining parasites under artificial conditions, or for obtaining data on
other enemies for possible use in control work, yet some noteworthy
information was obtained. From adults of E. imiricaium collected by
the MTiter from barley at Colby, Kans., on August 25 there were reared
on October 23 adults of a parasite determined by A. B. Gahan of the
Bureau of Entomology as Perilihis eleodis Viereck {13). No life-history
work on these parasites was attempted.

Considerable difficulty was experienced in rearing larvae owing to the
presence in the cages of a fungus, Metarrhizium anisoplae Metsch. Soc. ;
and though the apparatus and soil were sterilized, yet the mortality was
sufficient at times to interfere to a marked extent with the rearing. A

Nov. 5, 1921 Biology of Embaphion muricatum 333

number of the larvae in the cages were also attacked by an obscure bac-
terial disease. This appeared to be identical with that described by
Prof. Swenk {12). There would appear somewhere upon the body
sutures small circular or irregularly shaped dark brown spots, and these,
after a few days, would become larger, until in some instances they
would cover one-third to one-half of the body surface. This disease
usually caused the death of the larvae within varying periods of time.
Larvas found under normal field conditions are sometimes found to be
affected both with Metarrhizium and with the disease.

CONTROL

While no extended series of experiments relative to control of the
insect thus far has been found possible, yet the information secured on
the subject has been sufficient to assure the practical value of the measures
here recommended in reducing or preventing damage.

A systematic rotation of crops is one of the most effective procedures
in cutting down dam.age. The maximum injury always may be found
upon those areas where the ground has been cropped to wheat continu-
ously for several years, whereas the minimum injury is found where
com, kafir, milo, and other crops are grown which require some degree
of cultivation during the growing season. An important factor in migra-
tion and infestation lies in the fact that the beetles are wingless and
therefore become dispersed much more slowly than do winged forms.

A number of fields within infested areas, which were also infested by
the corn earworm {Chloridea obsoleta Fab.) and other insects of somewhat
similar habits, were plowed by farmers during late fall or early spring to
destroy the pupae, and it was found that such measures were of consid-
erable value in control of the false wireworms in the soil. The pupal
cells were crushed and the pupae buried or thrown out upon the surface,
where they were exposed to the elements and to predatory enemies.

It is not only good farm practice but also advantageous as a control
measure to destroy and remove from infested fields and adjacent fence
rows all clumps of Russian thistles and other weeds or heavy growths of
grasses likely to shelter these beetles.

While adults in small numbers are known to feed upon poisoned bran
mash and similar preparations, experiments in poisoning the larvae were
not satisfactory. It appears doubtful that such poisoning will ever
prove of practical value in dealing with this pest.

Late sowing of wheat in the fall also has been tried as a possible control
measure, but does not appear to be successful unless the season is a very
dry one, and even then if the seed has to lie in the ground any appreciable
length of time before rain and germination much injury is likely to result,
for the larvae are most active in the dry, loose soil under such conditions.
65769°— 21 4

PLATE 31
Embaphion muricatum:

A. — Epipharynx (eph) and anterior margin of labrum.

B. — Head: cl., clypeus; fa, anterior angle of front; epi, epistoma; /, frons; epc,
epicranium.

C. — Lateral view of larva.

D. — ^Mandibles and hypopharyngeal sclerite from below. Concavity of molar part
of left mandible grinding against the sclerite.

E. — Gula, labiiun, and right maxilla from ventral side: gu, gula; tp, tentorial pit;
sm, submentum; me, mentum; stla, stipes labii; li, ligula; hyp, hypostoma; fm,
fossa for mandible; fc, fossa for cardo; ar, maxillary articulating area; ca, cardo; sti,
stipes maxillaris; &.y, basis of stipes; z>i and Uj' i^mer margin of stipes; jmo, mala maxil-
laris (probably lacinia); pag, basal membrane of maxillary palp.

F. — Dorsal side of right and left mandible, hypophar>Tigeal sclerite between them:
a^ and a^, the bicuspidate mandibular apex; t, tooth of cutting edge; m, molar part; c,
carinate edge on exterior side of cutting part of mandible ; s, soft-skinned, seta-bearing
elevation below the carinate edge; e, margin of chitin framing the soft elevation.

G. — Mandibles and hypopharyngeal sclerite from below; no grinding in this position.

H. — Left anterior leg showing the anterior face of the leg hanging perpendicularly
down from a horizontally placed larva.

I. — Left anterior leg, exliibiting its posterior face: cox, coxa; ir., trochanter; /e,
femur; ti, tibia; ia, claw-shaped tarsus, shortly but not correctly designated as "claw."

Biology of Embapliion muricatum

Plate 31

Journal of Agricultural Research

Vol. XXII, No. 6

Biology of Embaphicn muricatum

Plate 32

/^c^^^

Journal of Agricultural Research

Vol. XXII, No. 6

PLATE 32
Embaphion muricatum:

A. — Lateral view of buccal cavity with mouthparts removed: eph, epipharynx;
hsc, hypopharyngeal sclerite; hbr, hypopharyngeal bracon;/jw, fossa of ventral con-
dyle of mandible; oes, oesophagus (note the distance between sclerite and entrance
to oesophagus).

B. — Ventral view of head, the thoracic segments, and the anterior portion of first
abdominal segment: y, presternum; eu, eustemum (Snodgrass) or basistemum
(Crampton); hi, prehypopleurum ; hn, posthypopleurum ; 2, poststernellum (prester-
num and poststernellum constitute together the ventral intersegmental region); ej,
preepipleurum ; %, postepipleiunim ; te, tergite; ster, sternal shield of abdominal
segments; ep, abdominal epipleimim ; ter, abdominal tergite.

C. — Pygidium, lateral view.

D. — Pygidium, dorsal view.

E. — Pygidium, ventral view; IX, ninth abdominal ("pygidial") segment; X,
tenth abdominal ("anal") segment.

F. — Maxillse, ligula, labial palpi seen from the buccal cavity. (Hypopharyngeal
region removed.)

G. — Hypopharyngeal region, oesophagus, and hypopharyngeal bracon which all
were removed from figure F: hsc, hypopharyngeal sclerite; hbr, hypopharyngeal
bracon; fm, mandibular ventral xossa; oes, oesophagus.

H. — Hypopharyngeal region, same piece as figure G, reversed: hsc, base from which
hypopharyngeal sclerite originates; hbr, hypopharyngeal bracon; /w, mandibular
ventral fossa; oes, oesophagus.

I. — First thoracic spiracle.

ADDITIONAL COPIES

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iToL. XXII NOVEMBER 12, 1921 No. 7

JOURNAL OF

AGRICULTURAL
RESEARCH

CONTENTS

Page

Genetic Behavior of the Spelt Form in Crosses between
Triticum spelta and Triticum sativum- - - - 335

CLYDE E. LEIGHTY and SARKIS BOSHNAKIAN

( Contribution from Bureau of Plant Industry )

Plum Blotch, a Disease of the Japanese Plum Caused
by Phyllosticta congesta Heald and Wolf - - - 365

JOHN W. ROBERTS

( Contribution from Bureau of Plant Industry )

A Comparison of the Pectinase Produced by Different
Species of Rhizopus - - "" ~ " -371
L. L. HARTER and J. L. WEIMER

(Contribution from Bureau of Plant Industry)

PUBUSHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE,

WITH THE COOPERATION OF THE ASSOCIATION OF

LAND-GRANT COLLEGES

WASHINGTON, D. C.

GOVERNMENT PRINTING OFFICE

1921

EDITORIAL COMMITTEE OF THE

UNITED STATES DEPARTMENT OF AGRICULTURE AND

THE ASSOCIATION OF LAND-GRANT COLLEGES

FOR THE DEPARTMENT

KARL F. KELLERMAN, Chairman

Physiologist and Associate Chief, Bureau
of Plant Industry

EDWIN W. ALLEN

Chief, Office of Experiment Stations

CHARLES L. MARLATT

Entomologist and A ssisfant Chief, Bureau
of Entomology

FOR THE ASSOCIATION
J. G. LIPMAN

Dean, State College of Agriculture, and
Director, New Jersey Agricultural Experi-
ment Station, Rutgers College

W. A. RILEY

EntoTnologist and Chief, Dnision of Ento-
mology and Economic Zoology, Agricul-
tural Experiment Station of the University
of Minnesota

R. L. WATTS

Dean, School of Agriculture, and Director,
Agricultural Experiment Station, The
Pennsylvania State College

All correspondence regarding articles from the Department of Agriculture should be
addressed to Karl F. Kellerman, Journal of Agricultural Research, Washington, D. C.

All correspondence regarding articles from State Experiment Stations should be
addressed to J. G. Lipman, New Jersey Agricultural Experiment Station, New
Brunswick, N. J.

JOlNiaOFAGiaaiTiAllSEMCH

Vol.. XXII Washington, D. C, November 12, 1921 ,^ No. 7

GENETIC BEHAVIOR OF THE SPELT FORM IN CROSSES
BETWEEN TRITICUM SPELTA AND TRITICUM SATI-
VUM '

By Clyde E. LEighTY, Agronomist, Office of Cereal Investigations, Bureau of Plant
Industry, United States Department of Agriculture, and Sarkis Boshnakian, Depart-
ment of Plant Breeding, College of Agriculture, Cornell University

INTRODUCTION

In connection with genetic studies of density in the wheat spike, it
was noted that the appearance of dififerent specific forms in certain wheat
crosses introduced marked irregularities in density curves of the second
and following generations, and that the densities of Triticum sativum
Lam., T. polonicum Linn., T. spelta Linn., etc., were affected in different
degrees when a certain known density factor was introduced through
hybridization. In some instances there were partial and sometimes
total inhibitory effects in regard to density, depending upon the sub-
species and also the kind of density factor involved in the cross.

The occurrence of these irregularities which appeared to be caused
by the spelt character in some crosses led to the study of the nature and
genetics of the species T. spelta. Although studies have been made of
the mode of inheritance of the spelt form in a large number of inter-
specific crosses, only the different modes of inheritance in crosses where
the parents are spelta and sativum are presented in this paper. In other
crosses, such as turgidum Linn. X sativum, durum. X sativum, dicoccum
Schr. X .yafwwm, etc., spelts invariably appear in the F2 generation. The
mode of inheritance of these spelt forms is complex and variable, so their
discussion here has been omitted.

The plants on which these studies were made were grown, with a few
exceptions, on Arlington Farm, near Washington, D. C, or on the Plant
Introduction Station, Chico, Calif., both operated by the United States
Department of Agriculture. The crosses were made at the former place
in 1913.

> The specific name T. sativum as used in this paper refers only to the forms T. vulgare Vill., T. compactum
Host., and T. capitatum Schlz. These three forms are essentially the same species, their differences being
merely a question of intemode length. The word wheat is frequently used as an English designation for
these forms, and when so used does not include such other forms as T. durum Desf . , T. polonicum, etc.

Journal of Agriailtural Research, Vol. XXII, No. 7

Washington, D. C. Nov. 12, 1921

aak Key No. G-isa

336

Journal of Agricultural Research voi. xxn, no. 7

SPECIFIC DIFFERENCES BETWEEN TRITICUM SATIVUM AND TRITICUM

SPELTA

The shape of the outer or sterile glume is an important character in the
dififerentiation of wheat species. The glume of the true T. sativum form
(fig. I , B) is, as a rule, soft, with a somewhat pointed apex. It is rarely
and very weakly keeled along the entire length. About 0.5 to i mm.

Fig. I. — Characteristic spikelets of Triticum spelta (A) and Triticum sativum (B). Note upright and
tight position of the glumes of spelta and the loose, spreading habit of satiiiim. Spelta has a flat shoulder
(a) with two or three indentations; the shoulder of sativum (d), though varying widely, is generally not so
prominent. It is often rather tapering with no indentations. The base of the sterile glume of spelta (b) is
broad, showing firm attachment to the rachis. The glumes can not be opened without breaking them at the
base. In sativum the base (/) is narrow and is weakly attached to the rachis, and the glumes can be opened
easily. There are one or more depressions («) at the base of the glume in sativum which are not present in
spelta. The glume of spelta is more or less uniform in width. It is stiff and has prominent corrugations
(c); that of jaijVwwj is narrow at base, widens, then tapers again gradually. It is very thin and soft, and the
veins on the glumes are not so prominent. Sativum has a weak keel while spelta has a very strong one .
The Spelta spikelet usually develops only two kernels; sativum, often develops three or more.

above the point of attachment there is a wrinkle or depression. The
base is rather narrow and is very weakly attached to the rachis. The
glume characters of the typical spelt, on the other hand, are quite
different (fig. i, A). The glume is stiff and thick, with a very blunt
apex. It is strongly keeled and has no depression above the base,
which is wide and firmly attached to the rachis.

Nov. 12, 1921 Genetic Behavior of Spelt Form in Crosses 337

Some wheats exhibit some of the spelt characters in a very weak form.
For instance, some are more or less strongly keeled or have a flat shoulder
at the distal part of the glume. While such wheats are classified as
T. sativum, they are not true sativum in the sense used here. They do
not represent the type.

Aside from the glumes, these species have other distinguishing char-
acters. Of these, the brittleness of the rachis and nonshattering qualities
of the spikelet in spelt may be mentioned as contrasted with the sativum
types, the latter being nonbrittle and shattering easily.

Although the heads of the spelt varieties commonly grown at experi-
ment stations in this country usually are lax, laxness of the head is not
necessarily a characteristic of the spelt. Compactness may easily be
introduced into spelts when crossed with T. compactum, which is really
only a wheat carrying a genetic factor or factors for compactness.

The characters differentiating these two species are recapitulated
below. This list includes also some other minor characters.

T. sativum. T. spclta.

Outer glumes — Outer glumes —

Weakly attached by narrow base. Firmly attached by wide base.

Weakly keeled. Strongly keeled.

Apex tapering. Apex blunt.

Depression or wrinkle near point of at- No wrinkle near base.

tachment.

Veins not prominent. Prominent parallel longitudinal

veins.

Shoulder narrow to broad, with no Shoulder broad, with two or three

dentation. dentations.

Glume soft. Glume, lemma, and palea firm and

thick.

Spikelets — Spikelets —

Kernel loosely held between glumes Kernel tightly held (nonshattering).

(shattering).

Spikelets spreading somewhat from Spikelets oppressed tightly against

rachis. the rachis.

Usually three or more kernels per Usually two and rarely three kernels

spikelet. per spikelet.

Rachis — Rachis —

Tenacious. Fragile.

There also are differences in the shape of the kernels of these two
species (i).^

LINKAGE OF SPECIFIC CHARACTERS

In order to understand the manner in which these two forms are here
classified, it is necessary to refer to the linkage of their specific characters,
without discussing the details.

Most of the characters tabulated above show two limits of contrasts.
When a wheat is crossed with a spelt, a gradation of forms naturally

' Reference is made by rmmber (italic) to " Literature cited," p. 363-364.

338 Journal of Agricultural Research voi. xxii.no. 7

appears in the Fj generation, ranging from the true spelt to the typical
wheat form. In the segregates, a plant which has the glume form of
the spelt invariably has a brittle rachis and a nonshattering habit. If
the glume form is intermediate, the brittleness and shattering qualities
also are intermediate These three characters do not segregate inde-
pendently. A plant with sativum glumes, for instance, has not been
found whose rachis is brittle (a characteristic of the spelt only), nor
have we obtained a spelt-glumed plant which has as tenacious a rachis
as that of T. sativum. These species may present other characters,
such as pubescence, beardedness, glume color, etc. These are common
to both and segregate independently irrespective of the species. The
presence of correlation between some of these specific characters in
sativum X spelta crosses also has been noted by von Tschermak (12).

Unless the linkage is absolute it does not exclude the possibility of
the occurrence of crossovers, but if crossing over ever occurs with respect
to these characters it must be very rare.

The linkage of specific characters of spelt is very similar to the linkage
of a number of glume characters of the wild oats in crosses between wild
and cultivated forms described by Surface (u) and by Love and Craig (5),

It is absolutely necessary to bear in mind that the determination of the
species is based only upon the presence, intensity of development, or
absence of the specific characters (glume characters, brittleness of rachis,
and seed-holding habit) , all of which are linked to a very large extent.
These characters show no independent segregation. It is obvious,
therefore, that they do not mendelize independently. The characters
which do segregate independently, such as pubescence, beardedness,
glume color, etc., are not taken into consideration.^

METHOD OF CLASSIFYING THE FORMS

The second and subsequent filial generations of spelta X sativum crosses
show numerous gradations between the two parental forms. Some of
the F2 plants produced a progeny consisting of individuals which were
decidedly spelts, while others produced a progeny which, while all spelts,
yet resembled T. sativum to some extent and were distinctly different
from the former group of plants where all the individuals were markedly
spelt-like.

Ten arbitrary classes were made in order to record the degree of in-
heritance of spelt characters. The typical or intense spelt which ex-
hibited all the specific spelt characters in their extreme forms was graded
I. As the exhibition of the intensity of these characters diminished —
that is, as they tended to approach those of the wheat — ^the heads were

' Even these independently segregating characters vary in intensity and quality in these two species
when segregating in crosses.

Nov. 13, 1921 Genetic Behavior of Spelt Form in Crosses 339

classed 2, 3, and so on to 9. Class 10 includes only true wheats, the
forms which show absolutely no trace of the spelt characters. It must
be emphasized that these 10 classes are purely arbitrary, based upon
the appearances of the heads, and are not intended to represent any geno-
typic classes. The types representing these 10 classes are presented in
Plate 33, A.

When the degree of inheritance of the spelt characters by a heterozy-
gous population is recorded on two different occasions, a variation in
the class frequencies naturally may be expected, but the general form
of the curve remains practically the same. The error which may affect
the conclusions rests in the decision as to whether a particular plant
belongs to class 9 or 10. The degree of error depends upon the cross
examined.

Of the plants discussed herein, which were tested in the Fg generation,
there were five cases where individuals belonging in class 9 had been
placed in class 10 and only one case where a class 10 individual was
erroneously recorded as belonging to class 9. In the tables these six
corrections have been made. As the conclusions are based not merely
on the F2 individuals but upon the progeny of these, it does not seem
likely that this source of error could have affected the results to any
extent.

The spelt parents used for making the crosses graded from i to 4 ; the
variety of wheat known as Gatineau ^ and herein considered as speltoid
in form graded from 4 to 7; and all the T. sativum parents of course
graded 10. As the discussions in this paper are confined only to the
spelt character it does not seem necessary to describe further the agrono-
mic and botanical characteristics of the plants used in making the crosses,
as these have no direct bearing on the subject.

FAMILIES SHOWING THE PRESENCE OF ONLY ONE FACTOR FOR

SPELTING 2

The hybrids of the first filial generation (FJ of a spelta X sativum cross
are slightly intermediate in form, resembling the spelt more than the
sativum parent. They grade from 4 to 7, depending upon the cross.
They possess all the characteristics of a spelt, but the spikelets may be
somewhat more open and the grains may not be so firmly held within
the glumes. The spelt characters are so nearly completely dominant
that they inhibit all wheat characters.

In the second generation a segregation is obtained where the indi-
viduals vary, producing forms ranging from the typical wheat form to

' This variety originated as a cross of Red Fife 9 (T'. sativum) and Goose S {T. durum) (lo, p. 239). It
is a striking illustration of the spelt-like segregates which appear when these two wheat species are crossed.
The heads are sc much like spelt that at blooming time especially it is easily mistaken for spelt, but it
thrashes free and is otherwise like the common wheats.

' For brevity, "spelting" is used throughout this paper in place of "inheritance of the spelt characters."

340

Journal of Agricultural Research vou xxii, no. ?

the spelt. The curve of these gradations is not a binomial frequency
curve, but, on the contrary, more individuals are found, as a rule, at the
extremities of the range than near the center. This fact may be ob-
served in Table I, which shows the forms of the Fj segregates.

Table I. — Number of plants of the F^ generation falling into each of lo classes, based on
presence or absence of spelt characters, with total number of spelts arid wheats actually
obtained, calculated numbers on basis of j to i ratio, deviation, probable error, and ratio
between deviation and probable error

Series.

Crosses.

Number falling in class —

To-
tal.

13255a

13260a

13263a
3094a

3085a

13124a

13125a

Spelt sel. i344oXDale Gloria set.

13401

Spelt sel. 13438 X Turkey (C. I.
3375) sel. 13389

Dale Gloria sel. i340iXspelt

Black Bearded spelt X Early Red
Chief

Black Bearded spelt X Jones Long-
berry

Vulgare (C. I. 3338)Xspelt sel.

13437

Crimean (C. I. 3340) sel. 13351X
spelt sel. 13437

50
76

5°

Total of crosses .

418

Series.

13255a
13260a
13263a
3049a
3085a
13124a
13125a

Crosses.

Spelt sel. i344oXDale

Gloria sel. 13401

Spelt sel. 13438 XTurkey

(C.I- 3375) sel. 13389-
Dale Gloria sel. 13401X

spelt

Black Bearded spelt X

Early Red Chief

Black Bearded spelt X

Jones Longbcrry

Vulgare (C. I. 3338) X

spelt sel. 13437

Crimean (C. I. 3340) sel.

i335iXspeltsel. 13437

Total of crosses . . .

Number of plants.

Obtained.

Spelts. Wheats.

52
49
18

52
37
56
41

305

113

Calctilated.

Spelts. Wheats.

54-8

47-3
18.8
60.8

37-5
57- o
37-5

313-5

IS- 7
6.2
20.3
12.5
19. o
12.5

104.5

Devia-
tion.

8.5

Prob-
able
error.

2-5
2-3

1-4
2.6
2. I

2.5
2. I

6.0

Ratio

Dev.
P.B.

•74
•57

3-38
.24
.40

I. 67

I. 42

As it is impossible to distinguish the homozygous spelts from the
heterozygous forms, in determining the ratios all the spelt and speltoid
forms (classes i to 9) have been grouped together and compared with

Nov. 13, 1921 Genetic Behavior of Spelt Form in Crosses

341

the wheat forms (class 10), which show no trace of spelt characters.
The proportions between spelta and sativum forms of each of the crosses
taken separately and that of the totals of these two groups approximated
the monohybrid ratio of 3 to i. The obtained ratio of the totals of the
crosses was 305 speltoid and spelt forms to 113 wheats, the expections
being 313.5 to 104.5, respectively, showing a deviation of 8.5 with a
probable error of ± 6.0.

Two of the Fj families, 13260a and 13255a, gave the results shown in
Table II when the F3 generation was grown.

Table II shows that the Fj population of the families tested consisted
of individuals in the proportion of i homozygous spelt to 2 heterozygous
forms and i homozygous wheat.

Table II. — Number of F2 plants from series ij26oa and Ij2^ja which proved to be homo-
zygous spelts, heterozygous forms, and homozygous wheats {i : 2 : i) when tested in the
/• 3 generation

13260a, Spelt X Turkey.

132553, Dale Gloria X Turkey.

Nature of data.

Homozy-
gous
spelts.

Heterozy-
gous
forms.

Homozy-
gous
wheats.

Total.

Homozy-
gous
spelts.

Heterozy-
gous
forms.

Homozy-
gous
wheats.

Total

Obtained

Calculated

Deviation

7-5
•5

13
15.0

2. 0

7-5
•5

8.2

1.2

16.5
2-5

12
8.3

3-8

The ratios of the totals of the forms produced in the F3 generation by
the heterozygous Fg individuals of these two families (Table III) seem
to conform to the foregoing assumption, although the ratios of the
forms produced by each of the F2 individuals sometimes are not so
close to the 3 to i expectation. Of the total individuals produced by
the Fj heterozygous plants of series 13260a (spelt X Turkey) , 212 were
spelts and 71 wheats. These results were surprisingly close to the
expectation, the deviation from the calculated numbers being but
0.3 with a probable error of ± 4.9. In series 13255a (Dale Gloria X spelt),
the numbers obtained from the heterozygous individuals were 365
spelts and speltoids and 156 wheats; the deviation here was 25.8 with a
probable error of ±6.7. This apparent dominance of the spelt character
over that of the wheat and its segregation into the 3 to i ratio are in
accord with the observations of Pitsch, as cited by von Tschermak
{12, p. ijg) and of Kajanus (j).

342

Journal of Agricultural Research voi. xxii. no.

Table III. — Numbers of spelts and wheats produced in the F3 generation from the F^
heterozygous plants, and comparison of these with theoretical expectations, calculated
on the J to I basis

Total F3
plants.

Number of plants.

Devia-
tion.

Probable
error.

Ratio

Pedigree No.

Obtained.

Calculated.

Dev.

Spelts.

Wheats.

Spelts.

Wheats.

P.E.

132603-3 . . . .

7. ...

9. ...

10. . .
13- ••

14. ..

15. ..
19. ..
25. ..

28. ..

29. . .

30. ..

23
20
28
17
23
24
17
20
29
17
17
18

13
16
20
10
18
17
13
16
26
13

12
16
22

4
8

7
5
7
4
4
3
4
5
2
8

17-3
15.0

21. 0
12.8

17-3
18.0
12.8
15.0
21.8
12.8
12.8

13-5

22. 5

5-7
5-0
7.0
4. 2

5-7
6.0
4.2

5-0
7.2
4.2
4.2
4-5
7-5

4-3
I. 0
I. 0
2.8

•7

I. 0

. 2

I.O

4.2
. 2
.8

2-5

•5

1.4
1-3

I- 5
I. 2
1.4
1.4
1.2

1-3
1.6
I. 2
I. 2
1.2
1.6

3-1
.8

•7

2-3

•5
•7
. 2
.8
2.6
. 2

•7
2. 1

•3

Total..

283

212

212.3

70.7

•3

4.9

. I

i325Sa-7 •• • ■
8. ...
10. . .
II . .

15. ..

16. ..

17. ..
21 . . .

23 •• ■

24. . .

25. . .

35
59
33
13
52
49
35
44
37
50
35

34
15
20

29
18

33
25
33
32

2
4
4
8

15
10

9
24
20

17
II
12

17
3

27. 0

14-3
18.0
26.3

44-3

24.8

9.8

39- 0
36.8
26.3

33- 0
27.8

37-5
26.3

9.0

4-7
6.0
8.7

14.7
8.2
3-2

13.0

12. 2
8.7

II. 0
9.2

12.5
8.7

7.0

•7
2. 0

•7

•3

1.8

5-8

II. 0

7.8

S.3

2.8

4-5
5-7

1-7
1-3
1.4

1-7
2. 2

1-7

1. 0

2. I
2. 0
1-7

4.1

•5
1.4
4.1

. I
1. 1
5-8
5-2

3-9
4.9

26. ..
28. ..
3°- ••

1.8
2. 1

1-7

1.6
2. I
3-4

Total..

521

365

156

390.8

130. 2

25.8

6.7

3-9

FAMILIES SHOWING THE PRESENCE OF TWO SPELT FACTORS

Of the crosses studied, two families, 13126a (Giant Squarehead X spelt) and 3019a
(spelt X Salt Lake Club) produced a very low proportion of wheat types. Not much
importance would have been attached to the irregular behavior of these families if
an apparently similar behavior had not been observed in another sativum X spelta
cross. The manner of segregation of the progeny of these two crosses is given in
Table IV.

Table IV. — Degree of spelling and proportions of spelts and wheats obtained in the
F2 generations of spelta X sativum crosses which did not segregate in the 3 to T ratio

Degree of spelting in class—

Total.

Niunber of plants.

Devi-
ation.

Series.

7
15

7
9

9
3

3
2

12
8

4
2

Obtained.

Calculated.

bable
error.

Spelt.

Wheat.

Spelt.

Wheat.

13126a '. . . .
3019a ^

74
83

70
81

4
2

69.4

77.8

4.6
5-2

0.6
3-2

I. 40
1.49

1 Series 13126a, Giant Squarehead (C. I. No. 3351, selection 13366) X spelt (selection 13437).
' Series 3019a, white spelt X Salt ijake Club.

Nov. 12. 1921 Genetic Behavior of Spelt Form in Crosses 343

In series 13126a (Table IV), only 4 wheats were produced in a popula-
tion of 74 F2 plants, while in series 3019a, 2 wheats were produced in an
F2 population of 83 individuals. The deviation from the 3 to i ratio is
so great that even by grouping class 9 with class 10 — that is, by making
generous allowances for observational error — the proportion approached
more nearly the 15 to i ratio. On the basis of the 15 to i ratio, the
expectation in series 13126a, is 69.4 to 4.6, in series 3019a, 77.8 to 5.2.
The deviations are 0.6 and 3.2, and the probable errors ±1.40 and
± 1.49, respectively.

On examining the Fg generation produced from 27 plants of series
13126a, it was found that 12 of these had produced only spelts (Table V),
two plants yielded only sativum types, and the remaining 13 F2 plants
yielded progeny of mixed forms. Assuming that the spelt parent in
this particular cross carried two spelt factors, Sj and Sj,^ the first two
generations will consist of the following genotypic forms:

(Giant Squarehead)

S1S1S2S2 X wibib2>-'2

(Winter spelt)

T. sativum

T. spelta

(Speltoid)

I SjSjS2S2

I ^1^1^2^2 4 W1S1O2S2

2 S1S1S2S2

2 Oj^Oj02S2

2 SiSiS2S2

I D]^OiS2S2

2 OjSj02W2

I SjSj^0202

Tot

al I T. sativum.

I q spelt.

If these genotypes were carried through the F3 generation the theo.
retical behavior of each of the Fg plants would be as follows:

GROUPS. F2 GENOTYPES. PHENOTYPES OF THE Fs AND THEIR BEHAVIOR IN THE F3 GENERATION.

A I SjSjSjSj I wheat will yield wheat only.

B I SjSiS2S2

2 t3iOi02S2

1 S1S1S2S2

2 DiSib202
I SiSjv!)2v32

C 4 SiSiS2S2 4 Spelts will segregate 15:1

15 2 S1S1S2S2 1 ^ cr.«ife will oo^.-o„o+» ... \^ unstable forms.

>7 spelts will yield spelts only.

4 SiSiS2S2 4 spelts will segregate 15:1 |

2SisaS }4 spelts will segregate 3:1 f

Total 16

When the performances of the Fj plants were examined, a close
approximation was found to the above-mentioned theoretical ratios.
The numbers of constant wheats, constant spelts, and unstable spelt
forms obtained are shown in Table V, together with the theoretical
expectations.

' In the factorial explanations given in this paper the spelt factors are assumed to be Si and S2. Although
the assumption of the factors si and sz to stand for the wheat (T. sativum) character will fully agree with the
results obtained, so far as the ratios go, there is no evidence as yet to warrant the assumption that the wheat
and spelt characters are allelomorphic to each other. In fact, results with other specific crosses show the
possibility that these are caused by two sets of independent factors. The behavior of the sativum X spelta
crosses may be compared with the behavior of a maize cross where one parent has yellow endosperm and
purple aleurone color (YYCCPP), and the other differs from this by its lack of purple color (YYCCpp).
In such a cross, where the F2 shows segregation into 3 purple to i yellow, the assumption that yellow and
purple are allelomorphic may be used as a working hypothesis for crosses of this type, although it is not the
correct explanation, as endosperm and aleurone color are two different characters altogether.

344

Journal of Agricultural Research voi. xxii.no. 7

Table V. — Frequencies of spelt and wheal classes in F3 progeny of F2 individuals of
family Iji26a, Giant Squarehead X spelt

FAMILIES CONSISTING OP WHEATS ONLY

Class
of F2
parent
plant.

Classes of spelt inheritance Fa —

Number of plants.

Pedigree numbers of Fs.

4
10

Spelts
and
spel-

toids.

Wheats.

Total.

10
10

4
10

FAMILIES CONSISTING

OF SPELTS ONLY

5
3
9
7
3
6

4
5
6

3
I
2
3

2
2

I
2

3
4

I
2
3
3
I
2
2

I
I

2
2

I
I

I
3

I
I

3
2

18
10
13
13
13
5
18

14
5

14
10

13
13
13
5
18

14
5

14
10

24.

21;

Total

138

FAMILIES CONSISTING OF

SPELTS

AND

WHEATS

(heterozygous f

2 plants)

\
I

5
6
8

8
8

7
5
8
I
I
9
5

2
I
I
2

3
2

5
2

I
3

5
I

I
2

I
I

I
I
I

3
I

I
2
2

I
I
I

4
I
I

4
2
I

16
9

5
3
7

14
7

14
16

5
18

2
2

I
I
1

I
I
I

4
2
I

17
II

7
4
8

i-j

15
II

14.

Total progeny of he-
terozygous F2 plants. .
Expectations

123
108. 75

22
36.25

145

SUMMARY AND GROUPING

Types.

Number of plants.

Groups.

Obtained.

Calculated

7:8:1.

Deviation.

Wheats producing wheats only

2
12
13

1-7
II. 8

13-5

0.3
. 2

Spelts producing only spelts

Cand D...

Spelts producing both spelts and wheats. .

•5

Nov. 12. 1921 Genetic Behavior of Spelt Form in Crosses

345

The agreement between the proportions expected and those obtained
is very close indeed to the 7 to 8 to i ratio, and perhaps too close to be
ordinarily expected from such a small population.

The analysis may be carried a step further. As shown above, the
plants which would show unstability in the F3 (groups C and D), were
expected to be of two different genotypes. One of them, containing the
S1S1S2S2 forms (group C), was expected to segregate in the 15 to i ratio,
while the other (group D), containing the S1S1S2S2 and S1S1S2S2 genotypes,
should segregate in the simple 3 to i monohybrid ratio. Apparently
the individuals belonging to each of these two groups are those analyzed
in Table VI.

Table VI. — Analyses of unstable spelts of the F2 generation, series 12126a. Number of
individuals of the F^ generation produced from Fn plants of groups C and D, compared
with the theoretical expectation

■p2 INDIVIDUALS APPARENTLY SEGREGATING IN THE 1 5 TO I RATIO (GROUP C, 81818252)

Total.

Number of plants.

Devia-
tion.

Probable
error.

Ratio

Dev.
P.E.

Pedigree.

F3 obtained.

F3 calculated.

Spelts.

Wheats.

Spelts.

Wheats.

I3i26a-i . . . .
II . . .

13. . .

17. . .

18. . .

17
8

IS
15
17

16
7
14
14
16

IS- 9
7-S
14. I
14. I
IS- 9

I. I

■ s

-9

I. I

0. I

• s

. I
. I
. I

0.68
.46
.62
.62
.68

0. I

1. I
. I
. I

. I

Total..

67- S

4-5

• s

1.38

•3

F2 INDIVIDUALS APPARENTLY SEGREGATING IN THE 3 TO I RATIO (GROUP D, S1S1S2S2

AND S1S182S2)

i3i26a-6. . . .

8.3

2.7

0.7

0. 96

0.7

5-3

1-7

•3

•77

•4

4
II

3-0
8.3

I. 0

14. . .

2.7

1-3

.96

1.4

19. . .

4-S

i-S

•S

.72

•7

21 . . .

16.5

s-s

I- 5

1-37

. I

26. . .

4-S

1-5

•5

.72

•7

27. . .

4-S

I- 5

•S

.72

•7

Total..

S4-8

18.2

I. 2

2. 50

-5

Total Fa plants segregating in 15 to i ratio, obtained, 5.
Total F2 plants segregating in 15 to i ratio, calculated, 6.7.
Deviation, 1.7.

Total F2 plants segregating in 3 to i ratio, obtained, 8.
Total F2 plants segregating in 3 to i ratio, calculated, 6.7.
Deviation, 1.3.

The data in Table VI show that forms were obtained in the F2 some
of which segregated in the 15 to i and others in the 3 to i ratio as ex-
pected. The agreement to the theoretical numbers of the progeny of
each F2 plant is as close as can be expected with such small numbers, even

34^ Journal of Agricultural Research voi. xxn, no. 7

though it is borne in mind that the values of the probable error are likely
to be too high in data of this kind.

Summing up the types of groups C and D, the former yielded 67 spelts
to 5 wheats, and the latter group yielded 56 spelts to 17 wheats, the
deviation being 0.5 and 1.2 and the probable errors ±1.38 and ±2.50,
respectively.

Of the 2 7 plants tested (Tables V and VI) , 5 (group C) showed an ap-
proximation to the 15 to I ratio and 8 (group D) to the 3 to i ratio. The
theoretical number of plants belonging to each of the two unstable groups
was 6.7 — that is, one-fourth of the total Fj plants tested. Comparing the
results obtained with those expected, it will be noted that there were in
the F, generation two (1.7 actual) SjSiSaSg individuals less and one (1.3
actual) SjSiSjS, or s^SiSaS, more than expected (Table VI) .

In considering the 15 spelt to i wheat segregation, it should be borne
in mind that if the progeny of a heterozygous Fj plant is less than about
10 individuals, the chances are that the wheat form, which is expected
to appear but once in a population of 16 individuals, will not be obtained.
Such heterozygous plants producing only spelts and no wheats, on account
of their small F3 population, would be classified under group B. Had Fj
plants 13126a — 12, — 20, and — 25 produced more than 4 or 5 individuals,
I or 2 of them might have produced a wheat form which would have placed
them in group C. The experimental ratios then would almost coincide
with the theoretical.

Considering the closeness of agreement even in the details of the
analysis, with such small numbers, there seems to be no question that we
are dealing here with two spelt factors and that the ratio observed is the
ratio of 15 to I.

In the absence of more experimental evidence, the simplest hypotheses
were given to account for the 3 to i and 15 to i segregations. Notwith-
standing the surprisingly close agreements between the experimental and
theoretical ratios, however, the real explanation concerning the produc-
tion of the spelt character is still a matter of speculation.

The same spelt parent plant was used in crosses 13124a, 13125a, and
13126a. The wheat parents were of different varieties. No satisfactory
explanation can be offered as to why the same spelt parent should pro-
duce a 15 to I ratio in cross 13126a and a ratio of 3 to i in crosses 13124a
and 13125a. Three possibilities, however, may be mentioned.

1. The spelt plant used in the above-mentioned three crosses might
have been heterozygous for one of the spelt factors. Such a heterozygous
SjSiSjSj plant bears gametes producing 3 to i and 15 to i ratios in the
F2 generation when crossed with a double recessive (SjSjSjSj) wheat.

2. The spelt parent may be assumed to have carried two spelt factors
Sj and S2 and some of the wheats might have carried an inhibiting factor
I. If the wheat carried the I factor the ratio of spelt to nonspelt would
be about 3 to i and if it did not the ratio would be 15 to i.

Nov. ij. I93I Genetic Behavior of Spelt Form in Crosses

347

3. The spelt might have carried a spelt factor Sj and in addition
another factor 83, which would produce the spelt character if there were
present its complementary factor C, which might have been supplied by
the wheat parent. In this case if C were present the ratio would be
about 8 spelts to i wheat; and if C were not present, it would be 3 to i.
The ratios to be expected on the basis of this last possibility, however,
are not in accord with the experimental results.

The second explanation seems to be the most plausible of the three.
The assumption that some wheats carry the factor for the inhibition of
the spelt character is not a mere speculation but a fact, as will be seen
later when the question of the production of synthetic spelts by crossing
two wheats is taken up. As commercial strains of T. sativum are not
purified with respect to inhibitors, there are undoubtedly some strains
which contain individuals heterozygous for this factor. If such a plant
is used, for instance, as the female parent and is crossed with a spelt
carrying the factor for spelting, according to the hypothesis some of the
seeds will produce Fj progeny where in some instances the ratio will sug-
gest a 15 to I, and in others a 3 to i segregation. As will be seen later,
other modifications of these ratios may also be expected to arise.

PROGENY OF STABILIZED SPELTOID X SATIVUM CROSSES

The speltoid form used in the crosses which will be considered now
is commercially known as "Gatineau." The variety originated from a
cross between T. durum and T. sativum. It grades usually from 4 to 7
in the classification used in this study for spike form and so resembles
the commercial spelts in this respect. It does not have the brittle rachis
of spelt, and the grain thrashes from the glumes more easily than the
grain of spelt, being like some of the tight-glumed wheats. It is neither
a typical spelta nor a typical sativum.

The F^ plants of the crosses between Gatineau and T. sativum were
almost like Gatineau. The Fj generation consisted of forms which were
intermediate; typical spelts of classes i to 2 were not found. The classi-
fication of the Fj individuals is reproduced in Table VII.

Table VII. — Classes of F2 generation plants of two speltoid X wlieat crosses. Numbers
of individuals obtained compared with the theoretical expectations

Degree of spelting.

Number of plants.

De-
via-
tion.

Series.

2
0

4
5

9
9

II
9

15
15

15
17

Total.

Obtained.

Calculated.

Prob-
able

Spel-
toid.

Wheat

Spel-
toid.

Wheat

13228a ^

13229a 2

0
0

68
63

Si
46

15
17

51.0
47.2

17.0
IS- 8

2. 0
I. 2

2.4

2-3

> Series 13228a Turkey (C. I. 3375. Sel. 13389) X Gatineau (C. I. 2959. Sel. 13403).
'Series 13339a Seneca Chief (C. I. 3372, Sel. 1338S) XGatineau(C. I. 3959, Sel. 13403).

348 Journal of Aqriculiural Research voi. xxn.No. 7

So far as the ratios of speltoid to wheat forms are concerned, these
crosses segregated in the simple mendeUan fashion. In series 13228a
(Turkey X Gatineau) there were 53 speltoids and 15 wheats, showing
a deviation from the theoretical numbers of 2.0 with a probable error of
±2.4 In series 13229a (Seneca Chief X Gatineau) there were 46 speltoids
and 17 wheats, the deviation here being 1.2 with a probable error of ±2.3.
In both cases the approximations of the figures obtained to those
expected are within the range of their probable erros. Therefore, it can
safely be concluded that the ratio is 3 to i and that there very likely is
but one spelt factor difference.

The question of interest in the inheritance of this speltoid form
(Gatineau) is not so much in its 3 to i ratio as in the way it differs in
details from the spelt X wheat crosses first discussed, which segregated in
the ratio of 3 to i.

In order to compare the Fj curves of these two groups of spelt X wheat
crosses, the Fj frequencies in Table VII (series 13228a and 13229a) and
the first two series in Table I (13255a and 13260a) are represented graphi-
cally (fig. 2).

The comparative characteristics of the curves of these two sets of
crosses are as follows :

The curves of the true spelt X wheat crosses (13255a and 13260a) begin
at class I , where they have their highest spelt frequencies. They gradu-
ally drop until they reach classes 5 to 9, inclusive, where there seems to
be an indefinite fluctuation of frequencies. Then the curves suddenly
rise again at class 10, which contains the spelt-free populations.

As to the curves of the wheat X speltoid crosses, the Fj spelt popula-
tions begin at about class 3, where but a very few individuals are found.
Beginning at class 5, the curves steadily rise until they reach their maxi-
mum height at class 10. The curves produced by the true spelt crosses,
it will be recalled, continuously dropped instead of ascending.

When these two sets of crosses are compared it will be observed that,
although there is but one spelt factor difference in each, the spelt factor
present in crosses 13255a and 13260a is entirely different from the spelt
factor present in crosses 13228a and 13229a. The wheat parent has
had no influence is producing this variation in distribution, the same
wheat parent plant, Turkey, C. I. No. 3375, selection 13389, having been
used in crosses 13260a and 13228a.

GENERAL DISCUSSION OF THE GROUPINGS WITHIN THE SPELT AND

SPELTOID CLASSES

Having discussed the question of ratios, let us turn our attention to
the analyses of the details of the variations within the spelt and speltoid
classes.

The possibility of distinguishing the homozygous from the heterozyg-
ous spelts of the Fj generation is of primary interest. Table VIII has

Genetic Behavior of Spelt Form in Crosses

349

_ .

SER/ES /326ba ^P£^T/9/V^^ TUf^KEY

SERI^ /325Sa fSPEET/^ND p/?L£ ^LORf/^

SERIES i3228a TURKEY /f/yPO^XIf^ ^^f-
SERIES /3229<Z^£N£C/9 CM/£F^NOGffn

-♦••^•^

V£/fu\

', j^

^sJ

/-"

OSOPSEOF SP£LT/Ne

Fig. 2.— Degrees of spelting of F2 population true spelt X sativum crosses (13255a and 13260a) as compared
with the curves of sativum X speltoid crosses (13228a and 13229a) of the same generation.

350

Journal of Agricultural Research voi. xxn. no. ?

been prepared to show these differences. Two sets of frequencies are
represented. One set represents the classes of spelt inheritance of the
Fj plants of series 13255a and 13260a, which produced nothing but
spelt in the F3 generation. The other set shows the plants of the same
generation and series which proved to be heterozygous for the spelt
character and produced spelts as well as wheats in ratios approximating
3 to I. These figures are taken from the data in Tables IX and X.

Table VIII. — Comparison of classes of spelt inheritance of tested homozygous and
heterozygous plants of the F^ generation

Grades of spelting.

Totals.

4 s

Homozygous spelts:

I'ja'iica

I
I

7
8

1. 71

2. 25

13260a

Average of means

1.98

Heterozygous spelts:

i?2cca

2
3

2
2

I
2

14
13

4.64
3-69

13260a

Average of means

4. 16

From Table VIII it is seen that the plants which proved to be homozy-
gous for the spelt character occur from class i to class 4, the average of
their means being 1.98. The Fj plants which proved to be heterozygous,
on the other hand, came from practically all classes, the average of the
mean classes being 4.16.

Table VIII shows that, although there is no sharp difference in the
phenotypic appearances of the homozygous and heterozygous individuals,
yet as a rule the Fj spelts of the speltoid classes (classes 5 to 9) are far
more likely to be heterozygous for the spelt character than those of the
true spelt classes (classes i to 4).

The comparison of the distributions of the F3 populations of the Fj
homozygous spelts of the 3 to i and 15 to i segregating families is of
special interest, as, among other things, it supports the two-spelt-factor
explanations already given. The distributions of the F3 populations of
constant spelt-producing Fj individuals of each of the three series 13126a,
13255a, and 13260a are recorded in Tables V, IX, and X, respectively,
and are represented graphically in figure 3.

Nov. 12, 1921 Genetic Behavior of Spelt Form in Crosses

351

CI/9SSES OFSP£L TEDA/ESS

§<50

•3 <!f- ,S 6 7 8
CL/fSSES OFSPEl TEDNE6S.

—

CO 80
^ 70.

/£

|?y

'S/£ea

?//5

WT^Q(//9/?1

-»

•5V

''iS

(

I—

^ 'f-O

-.,

k ^^

)

r^'

1^0-

-a

■^-

t--

■sj

-15

■18

-w

x*^

•

«/

■/o'

' '\

;

X I

»*j

-V

--.

.^^

r-'

—

■i?

«<

(£*

>»:

—

is t3 ^- ^5" /9 7 A

CLffSSES 0FSP£LTEDN£6a

Fig. 3. — Comparison of Fj generation curves of the progeny of F2 spelts which yielded only spelts
(series 13260a, 13255a, and 13126a).

70494°— 21 2

352

Journal of Agricultural Research voi. xxn. no. 7

Table IX. — Frequencies of spelt and wheat classes in F3 progeny of Fj individuals in
family Ij2^§a, Dale Gloria X spelt

FAMILIES CONSISTING OF SPELTS ONLY

Pedigree

number

Fj.

Class
of F2
parent
plant.

Classes of spelt inheritance.

Number
of spelts

and
speltoids.

Num-
ber of
wheats.

Totals.

I
I
2

4
5
3

10
16

7
16

5
2
6

7
8

I
I

7
I
I

I
I

14
17

43
15
25

17
6

43
25

FAMILIES CONSISTING OF WHEATS ONLY

10
10
10
10
10
10
10
10
10
10
10
10

25
13
17
21

19
38
23
29
20

33
14

II
25
13
17
21

19
38
23
29
20

33
14

FAMILIES CONSISTING OP WHEATS AND SPELTS

15-
16.

17-
21 .

23-
24.

25-
26.
28.
30.

Expectations

Probable error

DcAnation

Ratio between deviation and probable error.

3
3

4
7
5

S
2

5
I

4
2

24
II

4
6

I ^

S
2

rcrnlic

prnrx

2
T? r

>1ftTlt«

15
20
27
44
23

4
28
29

9
33
25
33
32

356

384

±6.61

2. 72

4
4
8

IS
10

24
20

17
II
12
17
3

156
128

Nov. 12, 1921 Genetic Behavior of Spelt Form in Crosses

353

Table X. — Frequencies of spelt and wheat classes in F^ progeny of Fj individuals of
series lj26oa, spelt X Turkey

FAMILIES CONSISTING OP SPELTS ONLY

Pedi^ee

number

F,.

Class
of F2
parent
plant.

Classes of spelt inheritance (F3).

Number
of spelts

and
speltoids.

Num-
ber of
wheats.

Total.

2
2

2
2
3

5
8
6

I
8
6

12
II
20

13
12

3
20

I
I

FAMILIES CONSISTING OF WHEATS ONLY

10
10
10
10
10
10
10
10
10

17
14
15
15
30
20
30
12
24

17
14
15
15
30
20

30
12
24

15
IS
30
20
30

FAMILIES CONSISTING OP SPELTS AND WHEATS

I
I

3
I
2
7
5
2
2

5
I
0

4
8

7
5
7
4
4

5
2
8

13
16
20
10
18
17
13
16
26

13
12
16
22

4
8

7
5
7
4
4
3
4
S
2
8

10
10

5
6

3
5
3

I
I
3

17
23
24
17
20

29
17
17
18

3
10

9
6

I
6

II
6
I

2
6

I
2
3

I
I

I
I
8

I
10

ofhetet
ous F2 ]
Expectal
Probable
Deviatio]
Ratio of

ozyg-
jlants.
ions. .

212

212. 25

±4- 91

■25

•OS

71
70-75

283

error

deviation to crobable erro

The F3 populations of the series segregating in the 3 to i ratio (13255a
and 13260a) have a general tendency to produce the maximum frequen-
cies at class I , sometimes at class 2. In only 4 families out of 15 are there
individuals in classes above the fourth, 10 individuals in 248 receiving the

354 Journal of Agricultural Research voi.xxii.no.

higher classification. The few mdividuals found in these speltoid classes
at present may be regarded as exceptions. Their significance will be
considered later.

The distributions of the families in the series segregating in the ratio
of 15 to I (13 126a) (Table V) are entirely different. They do not take the
general course described above. Some of them have very low frequencies
at classes i and 2. Families 5, 7, and 12 have no individual in class i, the
population of family 5 being composed of class 9 individuals only. Family
25 produced its spelts in classes 6 to 9, inclusive. Of the 12 families
being considered 6 produced class 9 individuals, while among the families
segregating in the 3 to i ratio there is but one instance (13255a, family i)
where class 9 individuals have been produced.

The explanation of the increased variability of the constant spelt-
producing families of series 13126a, as compared with series 13255a and
13260a, will be found in the factorial explanations given for these two
groups of crosses.

Families 13255a and 13260a segregated in the simple monohybrid
3 to I ratio. By hypothesis, all the spelts producing only spelts are sup-
posed to have the genotypic composition SS.

As to the cross 13126a which segregated in the 15 to i ratio, it was
shown that there were five constant spelt forms, namely :

SjvSiSaSg SiSiSjSs S1S1S2S2 SiSjSzSg S1S1S2S2

Although these forms would keep on producing only spelts, they are
not genotypically identical.

The fact that in the families segregating in the 3 to i ratio there was
only one genotypic spelt form and in the family segregating in the 15 to i
ratio five such forms were present may account for the increased varia-
bility among the pure-breeding spelts of the latter cross.

The F3 progenies can not be separated into the five theoretical geno-
typic groups just referred to because, among other things, there is positive
evidence that modifiers also are concerned which have the tendency to
shift the classes one way or the other. This phase of the subject will
next be discussed.

MODIFICATION OF THE DEGREE OF INHERITANCE OF SPELT CHAR-
ACTERS, DUE TO THE PRESENCE OF MODIFYING FACTORS

For the consideration of the subject of modifiers the analyses will be
confined primarily to the spelt classes (i to 9, inclusive) of the progeny
of the heterozygous F.^ individuals of series 13260a, shown in Table X.
This family has been chosen because it represents a simple mode of
segregation. Whatever is said about modifiers for this family will be
found to apply just as well to the other families.

It has been shown that only one spelt factor was concerned in the cross
under consideration. All the Fj heterozygous plants had the formula

Nov. 12, 1921 Genetic Behavior of Spelt Form in Crosses 355

Ss and produced spelts and wheats in the 3 to i ratio. If these F3
heterozygous plants have all the same genotypic constitution with
regard to the spelt factor we would expect to see them produce similar
F3 spelt distributions. These distributions obtained experimentally are
far from being uniform. For instance, plant i326oa-3 (Table X) pro-
duced individuals mainly of classes 8 to 9, and plant i326oa-6 produced
its spelts in classes i and 2 only. The progeny of i326oa-i5, on the
other hand, showed no definite grouping, the curve spreading from one
extreme to the other.

It may be argued that (i) these variations are insignificant; (2) they
may be due to variations in soil and other external conditions; or (3) they
are merely nonheritable fluctuations.

These arguments may be answered easily:

1 . In the first place, let us take the first two frequencies (classes i to
9, inclusive), namely, those of i326oa-3 and i326oa-6 (Table X). The
means are 8.00 ±0.29 and 1.3 1 ±0.08. The difference between the two
means is 6.69 ±0.30. This difference, it is seen, is very significant.
Similar striking differences will be found when the means of the other
distributions are compared.

2. With regard to variations due to soil conditions and external factors,
it is only necessary to mention that these plants were grown on the same
plot of the experimental field. The pedigree numbers following the
family number represent the numbers of the rows in which the progeny
of each of the Fj plants was grown. For instance, the progenies of
plants i326oa-28, 29, 30 (at bottom of Table X) were grown in three
adjacent rows, yet i326oa-28 was composed of individuals contributing
to nearly all classes of spelting, i326oa-29 produced practically all typical
spelts, and the progeny of i326oa-30 were nearly all speltoid forms
approaching the wheat type. So this second objection may also be
dismissed.

3. As to the nonheritability of these variations, the objection may be
settled by comparing the Fj and F3 generations in terms of the coefficient
of heredity. If these variations are nonheritable fluctuations, there
should be no correlation between the Fj and F3. Putting the statement
in the affirmative, if there is a significant correlation in the degree of
spelting of parent and offspring of the F2 and F3, then it is a direct and
indisputable proof that these variations are transmitted to the follow-
ing generations — that is, they are heritable. The accompanying corre-
lation table (Table XI) has been prepared with a view of determining the
validity of this last objection. The x-axis represents the mean classes
of the F3 and the y-axis represents the classes of the F2 individuals which
produced these Fg forms. In this table are included all the progeny of
the heterozygous forms of series 13255a and 13260a in order to have a
sufficient number of individuals. The coefficient of heredity as calculated
is 0.880 ±0.029. As this coefficient is over 0.5 and over 10 times its

356

Journal of Agricultural Research voi. xxu.no. 7

probable error it may be regarded as being very significant. As there
is a significant correlation between these two generations, the variations
under consideration are not fluctuations due to external conditions but
are hereditary variations.

Table XI. — Correlation table showing the classes of spelt inheritance o/Fj heterozygous
plants, with the average degree of spelling of the F^ progeny of each F-^ plant (series Ij2j^a
and 12260a)

Classes of spelt inheritance (Fs)

CoeflBcient of heredity=o.88o±o.o29

If there were no interference due to modifiers, the curve of the spelt
F3 progeny of the heterozygous Fj individuals would follow the spelt
curve of the Fj generation, as both the spelts of the Fj curve and those
of the F3 curves of heterozygous Fg individuals consist of SS and Ss
spelt plants in the proportion of i to 2, respectively.

An examination of the F3 spelts of heterozygous individuals in Table X
shows that the curves of the 13 families vary considerably from the curve
of the F2 generation (Table I, series 13260a), although the curve for the
totals is much the same.

Again, if there were no genetic interference, all the F3 progeny curves
produced by heterozygous F2 plants would be expected to follow more or
less the same course. The experimental results exhibit wide differences,
as the comparison of the classes of individuals i326oa-3, -6, -7, etc.,
will readily show.

Nov. li, 1921 Genetic Behavior of Spelt Form in Crosses 357

As the presence of multiple factors is entirely out of question, it being
proved in this case that the parents of this cross differ in only one factor
for spelting, the following explanation may be given to account for these
variations. One or more sets of modifiers furnished by the spelt, by
the wheat, or by both parents seem to be present where each set of
modifiers was in a homozygous dominant condition in one parent and in
the alternative condition or absent in the other parent. These modifiers
in the presence of the S factor tend to intensify the spelt character.

An example may be given to illustrate the effect which a modifier may
produce in a spelt X wheat cross. The modifier which may cause dilu-
tion of spelting may be represented by the factor D and may be assumed
to be carried by the wheat parent. (If this factor were contributed by
the spelt parent, the latter would have been a dilute spelt, which was not
the case in these crosses.) The wheat parent will then be represented
by ssDD and the spelt parent by SSdd. The genotypic forms of the
successive generations will be as follows :

SSdd
spelt.

X
SsDd

ssDD

wheat

semidilute 1

spelt.

1^2

I SSdd.

2 SSDd.

I SSDD.

I ssdd.

2 Ssdd.

4 SsDd.

2 SsDD.

2 ssDd.
I ssDD.

3 typical

6 semidilute

3 dilute

4 nonspelts.

spelts.

12 spelts. 4 wheats.

This represents a ratio of 3 spelts of different grades to i wheat.

If we assume that the nature of the modifier were to produce intensifi-
cation of spelt inheritance in the presence of factor S, which in this case
may have been carried either by the spelt or by the wheat, we will have,
in the F, generation, 3 intense spelts, 6 semi-intense spelts, 3 normal
spelts, and 4 wheats.

Some of these spelts (intense, normal, or dilute) will breed true to
those conditions ; others will produce some or all of these forms in different
proportions as expected on the factorial hypothesis. If more than one
set of modifiers are present the types and their proportions naturally
become rather complex.

If, in the crosses 13255a and 13260a, a diluting modifier has been intro-
duced, we would occasionally expect among the spelts (homozygous or
heterozygous) some which are grouped in the dilute speltoid classes.
The F3 population of i3i26a-5 and -25 (Table V); i3255a-26 (Table
IX) ; i326oa-3, and -14 (Table X) ; and a number of others represent
such cases. The progeny of i326oa-2o, -6, -10 and others may represent
spelts carrying some intensifying factor.

358 Journal of Agricultural Research Voi.xxn.No. 7

In conclusion, it may be said with certainty tJbat besides tlie S factor
in series 13255a and 13260a and the Si and So factors in 13126a, modifiers
are present which tend to dilute or intensify the spelt character.

In statistical studies of density in wheat, the junior author has found
two characters whose mode of inheritance is almost identical with that
of the spelt character. When a dense wheat (T. compactiim) is crossed
with a lax wheat (T. vulgare) a 3 to i segregation is found in the Fj gen-
eration. The F2 density curve consists of two distinct curves. One of
these is a skew curve in the dense classes which contains 75 per cent of
the individuals. After a gap, the other curve, which is composed of the
lax segregates containing the remaining 25 per cent of the F2 population,
begins. Although the F3 progeny of these heterozygous dense plants of
the F2 generation invariably produce bimodal curves similar to that of
the F2 just described, their modes or the means of the dense and lax
curves shift at times considerably toward the lax classes and sometimes
toward the denser classes, much in the same manner as does spelting. A
similar phenomenon has been observed by Nilsson-Ehle (8) among his
dense X lax wheat crosses.

The other parallel case is density of the type just mentioned, but in
this case the modifier is known to be the spelt factor itself. The curves of
the progeny of the heterozygous dense individuals of dense wheat X spelt
crosses have the general bimodal form, but the populations which, in
addition to density, carry the S factor always have their density curves
shifted toward the lax classes.

With some spelts, the S factor shifts the density curves so much toward
the lax classes that this S has to be regarded also as an inhibiting factor
for density.

Hull-lessness in oats, according to Love and McRostie (7), is inherited
in a similar manner. While this character segregates in the simple
mendelian ratio of i hulled to 2 intermediates to i hull-less, the inter-
mediate forms vary appreciably as regards the percentage of hulled
kernels they produce. By correlating the percentage of hull-lessness of
parent and offspring, they have shown that these variations within the
I to 2 to I ratio are hereditary.

The mode of inheritance of the spelt character as shown in Tables IX
and X closely resembles also Castle's (2) case of hooded rats, which had
for a time aroused considerable controversy for and against the question
of inconstancy of unit characters. In numerous crosses between rats
having the hooded pattern and the wild (totally pigmented) or the Irish
(white belly) types the hooded pattern behaved as a mendelian recessive.
The ratio of nonhooded to hooded F2 offspring was 3 to i , showing that
the hooded condition is dependent upon a single factor difference.
Among the hooded individuals a considerable degree of variation was
observed with respect to the degree of the extension of this pattern. By
making selections for many generations in plus and minus directions

Nov. 12. 1921 Genetic Behavior of Spelt Form in Crosses 359

Castle was able to increase and decrease the pigmented area. His belief
then was that the variations observed in the race of hooded rats were
not mere fluctuations but were hereditary variations in the sense that
the factor for the hooded condition had undergone alterations.

The assumption of unit-factor inconstancy, which Castle applied to
account for variation of pigmentation of his hooded rats of course can not
be applied for the analogous variations in the groupings of the spelt
individuals, for, if this were the case, variations in this same extent
should have been present among the self -fertilized population from which
the parental form was selected. The classes of spelt inheritance in the
parental strain ranged from i to 4, the mode being between classes i
and 2 . No departures nearly as great as those found in the homozygous
extracted spelts of the F3 generation were observed among this parental
population. The study of the F2 generation shows clearly that either
some modifier or modifiers were introduced by the nonspelt parent or
were carried by the spelt parent, but these modifiers were reduced to a
recessive state as a result of crossing.

PRODUCTION OF SYNTHETIC WHEATS BY CROSSING TWO SPELTS, AND
SYNTHETIC SPELTS BY CROSSING TWO WHEATS

The writers frequently have obtained synthetic spelts in interspecific
crosses in wheat. No indication has been observed as to the possibilities
of producing true wheats in crosses between two different nonwheat
species. It is theoretically possible, however, that such forms eventually
will be produced in crosses between certain kinds of spelts. This suppo-
sition may be explained by taking as an example the results of one of the
experiments discussed at length in this paper.

In the case of cross 13126a it Avas shown in detail that two spelt factors
Si and S2 were involved; that the F2 segregates which bred true to the
spelt character were not all genetically identical; and that they were
composed of five genotypic forms, namely:

v3iOiv32^2 wiS202^2 W1D1V52S2 ^1^1^2'^2 Sj^Sj0202»

As long as these forms are allowed to be selfed, as they are in nature,
no wheats ever segregate; but, by hypothesis, in a number of crosses
between these five forms, a certain proportion of wheats are expected
to appear in the following manner :

1. Crosses producing no wheats:

SiSiSaSaXany other genotype; SiSiSoSaXSiSiSgSa; S1S1S2S2XS1S1S2S2.

2. Cross where one out of every four F^ plants will produce 6^ per
cent wheats :

*-'l^l^2^2 X v5iSiv32W2 •

3. Crosses where half of the F^ plants will produce 6^ per cent wheats:

^l*-'l^2^2Xv3iSiv3202; Oiv3i02S2XSiSit52S2-

4. Cross where all F^ plants will produce 6^' per cent wheats:

^1^1^2^2XSiSiv32»32'

360 Journal of Agricultural Research voi. xxii. no. 7

If wheats segregate trom these crosses it will prove further the correct-
ness of the two-factor hypothesis. It will also lead to the expectation
that genotypic forms similar to the above, and other combinations as
well, exist among the so-called pure commercial spelt forms and when the
proper cross is made among these commercial spelts, a certain number
of synthetic wheats may be produced in the Fj generation.

It is easy to understand how the wheat character, being distinctly
hypostatic, may be carried from generation to generation by the spelt
type. But how can the spelt type segregate from a wheat X wheat cross?
How can one conceive the spelt factor, which is so pronouncedly epistatic
to the wheat character, as being carried by a wheat without being mani-
fested phenotypically ? The explanation is simple. It was shown that
modifiers are involved in these crosses. Common wheats occasionally
carry modifiers which tend to dilute the spelt character. Some of these
modifiers were shown to be so effective that they grouped all of the spelts
in class 9. Most of the class 9 individuals, as recorded in the foregoing
tables, resemble wheat so closely that no one would be likely to call
them true spelts.

If a certain diluting modifier can shift the spelts to class 9, a group of
these may readily shift the spelt to classes between 9 and 10. If these
diluting factors are reduced to a homozygous dominant condition, the
dilute spelt which will be classified as 10 will breed true to type and be
considered as wheat, although from a genetic standpoint such a form is
a spelt.

As long as such sorts are allowed to self-fertilize they will produce a
so-called pure line consisting of a constant wheat type. Their spelt
characteristics are exhibited only when crossed with a common wheat
which carries the factor for dilution in a recessive state. In the second
generation of this cross the segregates which carry the S factor with the
factor for dilution in a recessive state; that is, SS dd, will be spelts.

Fortunately, experimental evidence can be cited to support this
statement. One of the writers has observed at the Kansas Agricultural
Experiment Station over 20 spelts among Fj hybrid plants derived
from a number of wheat X wheat crosses where one of the parents was
a rust-resistant variety of winter wheat and the other was Preston,
Marquis, or Haynes Bluestem, well-known varieties of spring wheats.
These parental types and some of the spelt segregates are shown in
Plate 33, B.^

None of the F^ plants in these crosses were spelts, or at least passed for
spelts, although they might have shown some spelt characteristics in a
weak form. In the Fj generation, however, depending upon the cross,
the proportions of wheat to spelts varied roughly from 3 to i to over

' The authors are indebted to Professors John H. Parker and L. E. Melchers for allowing them to photo-
graph these forms and use them in connection with this paper.

Nov. ra, igar Gefietic BehavloY of spelt Form in Crosses 361

100 to I, with some sets of crosses producing no spelt at all. Besides
the true spelts, a number of speltoid forms also segregated.

The absence of the spelt type in the F^ generation shows that the
absence of the spelt character in one of the parents was due to the pres-
ence of an inhibitor in the parent plant which carried the S factor. If
the appearance of the spelt form in the Fj generation was due to com-
plementary factors furnished by both parents, the spelt should have
appeared in the F^ generation. Such was not the case.

The cultures consisted of over a thousand Fg plants. It is not now
absolutely necessary to know how, or exactly in what proportion, these
appeared. The purpose of citing these examples is to substantiate the
views expressed above regarding the possibilities of producing synthetic
spelts from wheat X wheat crosses, which might have been regarded
as a mere speculation in the absence of this experimental evidence.
The fact that there were no spelts grown near the P^ or F^ plants and
that spelts appeared in more than one cross excludes the possibility of
accidental or natural cross-fertilization.

These observations show that common wheats may carry the spelt
factor, but the latter can not express itself because one or more diluting
or inhibiting factors are carried with it. Some of these diluting factors
may be regarded as inhibiting factors which are not totally dominant
but produce intermediacy in a heterozygous state.

From the small percentage of spelts which appeared in the Fj genera-
tion in some cases, it may be inferred that there is one and in some
cases more diluting factors.^

The production of synthetic spelts in wheat X wheat crosses just con-
sidered is similar to the synthetic production of T. dicoccum dicoccoides,
the so-called "Wild Wheat" of Palestine, in the vulgar e X durum cross
(6). The wild character, consisting of a number of interdependent
specific characters, is strongly dominant over both the sativum and the
durum types, as is the spelt type toward sativum. Yet in both instances
the character showing strong dominance toward either of the parental
forms was carried by one of the parents together with a factor inhibiting
in one case the wild and in the other case the spelt characters.

MODIFICATIONS OF MENDELIAN RATIOS

The question of modifiers whose presence in the production of the
spelt character was demonstrated in various ways brings us to the con-
sideration of the modifications of mendelian ratios. As all spelts or all
wheats are not alike with respect to their ability to intensify, repress,
or inhibit the production of the spelt characters, it is natural to expect

' Nilsson-Ehle (.8, q) and Kajanus (j, 4) have also observed the occurrence of spelts in certain sativum X
sativum crosses. They support the correctness of the foregoing observations and tend to preclude the
assumption of the possibility of the occurrence of some accidental or natural crossing of one of the sativum
patents with a spelt.

362 Journal of Agricultural Research voi. xxii. no. 7

certain modifications of the 3 to i ratio. For instance, in certain spelt X
wheat crosses, depending upon the potency of the diluting factors, the
proportion of wheats to spelts may increase in certain amounts. In
some instances, the increase of wheats will be slight, so that the obtained
deviation from the 3 to i ratio, which will be on the side of excess for
the wheat class, will be considered within or near the limits of the probable
error and the inheritance will be regarded as simple mendelian. Perhaps
series 13255a, where an increased proportion of both constant breeding
wheats and F3 wheat segregates of heterozygous Fj plants are obtained,
represents such a case.

It also is possible that the ratio may fall between 3 to i and i to i in
case the diluting factor is very strong. Here, then, will be an excess
in the homozygous wheat class at the expense of the homozygous dom-
inant spelt class. If factors of both dilution and of intensification
are introduced in the same cross, the experimental ratios will defy any
attempt at simple factorial explanations. The writers have obtained a
cross where the Fg generation suggested a possible 3 to i segregation,
but on examining the F3 generation, which was composed of a fairly
large population, neither the individual segregations nor the totals of
.these approached in any way 3 to i or i to 2 to i expectations.

It naturally follows from the foregoing discussion that in spelt-wheat
crosses wide departures from simple ratios occasionally may be expected.

SUMMARY

T. spelta and T. sativum are differentiated by a number of linked
specific characters, which are present in one species and absent in the
other. These characters, so far as observed, are not inherited independ-
ently but are transmitted as a group.

In crosses between a spelt and common wheats the F^ hybrid shows
dominance of the spelt, but this character appears in a somewhat diluted
form. In the second generation all classes of spelt inheritance are ob-
tained. In order to classify these forms, 10 arbitrary classes were
erected, class i representing the true spelt and class 10 the total absence
of this character. The intermediate classes represent diff'erent grades of
spelt inheritance.

In most of the material studied there was but one factor difference for
spelt, but in two cases two spelt factors were present. Both 3 to i and
15 to I ratios were obtained. These ratios were verified after determin-
ing the genotypic constitution of the Fj plants, which gave pure breeding
spelts, inconstant spelts, and pure breeding wheats in the ratios of
I to 2 to I and 7 to 8 to i , respectively. Of the constant spelts produced
in crosses with this latter ratio, approximately half yielded (in the F3
generation) spelts and wheats in the ratio of 15 to i and the other half
in the ratio of 3 to i, as expected on the two factor hypothesis.

Nov. 12, 1921 Genetic Behavior of Spelt Form in Crosses 363

The speltoid form "Gatineau" when crossed with wheats gave also a
3 to I segregation of spelts and wheats, but the curves showing the
classes of spelts produced by this cross were entirely different from the
curves produced by the spelt-wheat crosses which segregated in the same
ratio.

Aside from the factor or factors for spelting, there is positive evidence
showing the presence of intensifying and diluting modifiers which tend
to affect the degree of spelt characters without affecting to any extent
the ratios of spelts to wheat. Some of the diluting modifiers tend to act
as inhibitors.

The progeny of all heterozygous spelts of the Ss type do not produce a
similar spelt curve. There are wide discrepancies in the spelt inheritance
of the progeny of these forms. These variations within the spelt classes
have been found to be hereditary and to be caused by modifiers.

The theoretical possibility of producing synthetic wheats in crosses
between certain pure-breeding spelts is shown.

Experimental evidence also is presented showing that, in spite of the
fact that the spelt character is dominant over the wheat form, the former
may be synthetically produced by crossing certain wheats, provided one
of the wheats carries a spelt factor together with an inhibitor and that
the other wheat carries neither.

It is shown that, if intensifying, inhibiting, and diluting modifiers are
introduced in a cross, wide departures may be expected from the 3 to i
and 15 to I ratios.

LITERATURE CITED
(i) BosHNAKiAN, Sarkis.

1918. THE MECHAmCAL FACTORS DETERMINING THE SHAPE OF THE WHEAT
KERNEL. In Jour. Amer. Soc. Agron., v. lo, no. 5, p. 205-209, fig. 27.

(2) Castle, W. E., and Phillips, John C.

1914. PIEBALD rats and SELECTION ... 56 p., 3 pi. Washington, D. C.
(Carnegie Inst. Wash. Pub. 195). Bibliography, p. 31.

(3) Kajanus, Birger.

1912. 0BER EINEN SPONTAN entstandenen weizenbastard. In Ztschf.
Pflanzenziicht., Bd. i, Heft i, p. 13-24.

(4)

1918. KREuzuNGSSTUDiEN AN winTERWEIzen. In Bot. Notisef, 1918, Haftet
5, p. 235-244.
(5) Love, H. H., and Craig, W. T.

1918. the relation between color and other characters in certain

AVENA crosses. In Amer. Nat., v. 52, no. 620/621, p. 369-383.

(6)

1919. THE SYNTHETIC PRODUCTION oP WILD WHEAT FORMS. In Jonr. Heredity,

V. 10, no. 2, p. 51-64, 10 fig.
(7) and McRosTiE, G. P.

1919. THE INHERITANCE OF HULL-LESSNESS IN OAT HYBRIDS. In Amer. Nat.,

V. S3, no. 624, p. 5-32, 7 fig.

364 Journal of Agricultural Research voi. xxii, no. 7

(8) NitSSON-EHLE, H.

1911. KREuzuNGSUNTERSucHUNGEN AN hafer und weizen. II. In Lunds
Univ. Arsskr., n. f., afd. 2, bd. 7, no. 6, 84 p. Literaturzerzeichnis zu
den Einleitung, p. 20.

(9)

I917. UNTERSUCHUNGEN UBER SPELTOIDMUTATIONEN BEIM WEIZEN. In Bot.

Notiser, 1917, Haftet 6, p. 305-329, i fig.

(10) Saunders, Charles E.

1907. report of the cerEaust. In Canada Exp. Farms Rpts., 1906, p. 235-
256, I pi.

(11) Surface, Frank M.

I916. studies on oat breeding. III. ON THE INHERITANCE OF CERTAIN
GLUME CHARACTERS IN THE CROSS AVENA FATUA X A. SATIVA VAR.

KHERSON. In Genetics, v. i, ixO. 3, p. 252-286, pi. 2-3. Literattire
cited, p. 285-286.

(12) TscHERMAK, Erich von.

1910. WEIZEN (TRITICUM). KORRELATIONEN. BASTARDIERUNG. /n Fruwirth,

C. Die Ziichtung der landwirtschaftlichen Kulturpflanzen, Aufl. 2, Bd.
4, p. 119-139, 164-187, fig. 11-12. Berlin. Bibliographical footnotes.

PLATE 33

A. — Wheat spikes showing different degrees of spelting. i and 2 are intense spelts;
3 to 9 are intermediate forms, showing varying degrees of dilution of the spelt character;
10 is a pure-breeding wheat (sativum) form, showing no trace of spelting. The numbers
represent approximately the types falling in the 10 classes of spelting.

B. — Snythetic spelts produced in Fj generation in wheat X wheat crosses; a, b, c, and
d are the sativum parent plants; b-i spelt form of the progeny of a X b; c-i to c-5
spelt forms of the progeny of a X c; d-i and d-2 spelt forms of the progeny a X d.

Genetic Behavior nf the Spelt Form in Crosses

Plate 33

)

\2 i<9 /^

Journal of Atjricultural Research

Vol. XXII, No. 7

PLUM BLOTCH, A DISEASE OF THE JAPANESE PLUM,
CAUSED BY PHYLLOSTICTA CONGESTA HEALD AND
WOLF^

By John W. Roberts, Pathologist, Fruit Disease Investigations, Bureau of Plant
Industry, United States Departinent of Agriculture

INTRODUCTION

In June, 1905, W. M. Scott of the Bureau of Plant Industry, United
States Department of Agriculture, collected near Fort Valley, Ga., fruits
of the Japanese plum (Prunus triflora Roxbg.) affected with a disease
very closely resembling the apple blotch, due to Phyllosticta solitaria
E. and E. In the diseased areas were spore-bearing pycnidia which were
found also on the leaves in gray papery spots resembling those on apple
leaves caused by Phyllosticta solitaria. On May 27, 1908, the disease was
again observed by Scott on both fruit and foliage of the Burbank plum at
Montezuma, Ga. It was found to be rather common in several orchards
about Montezuma, in some cases causing enough damage to injure
seriously the market value of the fruit. In one orchard a large part of the
fruit was affected, and many specimens bore from 15 to 20 spots each.

On May 29, 191 7, the writer collected near the same locality Japanese
plum fruits and leaves affected with the same disease. In the single
orchard in which the disease was found, most of the fruit was heavily
nfected and rendered nearly worthless. Considerable difficulty was
encountered in finding the disease again, as the Japanese plum industry
in Georgia had about passed out. Lack of demand for the fruit coupled
with the susceptibility of all parts of the tree to various diseases and
insect pests had caused growers either to eradicate their trees or to let
them die. At present there are almost no Japanese plum orchards
remaining in Georgia, and all of the trees in which plum blotch was found
have been eradicated. So far as the writer knows, then, the disease no
longer exists, though it is to be looked for throughout the South as far
west as Texas. Should the growing of Japanese varieties of the plum
be revived in the South, blotch may prove to be one of its most serious
diseases, as it is very destructive, and probably would be exceedingly
difficult to control.

The varieties found to be affected were Abundance, Burbank, and what
was apparently an unnamed seedling.

1 A brief description of this disease was published as an abstract of a paper presented at the Ninth Annual
Meetmg of the American Phytopathological Society. (Roberts, John W. plum blotch. (Abstract.)
In Phytopathology, v. 8, no. 2, p. 74. 1918.)

Journal of Agricultural Research, Vol XXII No

Washington, D. C. Nov. i.. 1921

^^ Key No. G-253

70494°— 21 3 (365)

366 Journal of Agricultural Research voi. xxu. No. 7

DESCRIPTION OF THE DISEASE

The infected parts on the unripe fruit appear as dark-colored raised
areas with fringed margins and are somewhat roughened by the presence
of small blisters and depressions (PI. 34, B). As in the case of apple
blotch, the skin often becomes ruptured as the fruit increases in size.

On the ripe fruit the blotched parts appear as irregular browned areas 3
to 6 mm. in diameter and consist of an aggregation of from 4 to 20 sunken
spots, each separate spot being i mm. or less in diameter. At this stage
the spots have a peculiar light blue cast owing to the "bloom" of the
ripe plum covering the browned epidermis. The diseased area is rather
superficial, extending only slightly below the epidermis. The affected
tissues become hardened and somewhat leathery and show no tendency
to decay.

Small, glistening pycnidia are produced in considerable numbers even
in the younger spots. Quite commonly there are 25 to 30 scattered
promiscuously about in each blotched area. Infection evidently takes
place when the fruits are very young, since the spots found May 29 were
well formed and bore pycnidia with mature spores. Judging from the
writer's inoculation experiments, infection probably took place five to
six weeks earlier, or about the middle of April.

On account of its characteristic appearance on the fruit, the disease has
been given the common name of "plum blotch."

On the upper surface of the leaf blades (PI. 34, A), the spots are angu-
lar, rather small (about 0.5 mm. across), brown when young, but later
becoming gray or silvery in color. They may be numerous, as many
as 200 sometimes appearing on a single leaf. Usually only a single
pycnidium is present in each spot, except where two or more spots have
coalesced to form a single large spot. Affected areas are also found on
the petioles and on the veins of the lower surface, especially on the
midrib. On these the diseased areas are much larger than on the upper
surface of the blade and are black and sunken. Pycnidia, bearing
spores, are present in great abundance.

,, Pycnidia, apparently identical with those found on the fruit and
leaves, were found also in small light-colored, often slightly sunken areas
on the twigs; but, as spores were lacking, positive identification could
not be made. It is possible that these pycnidia had discharged their
spores early in the spring and had brought about the early infections on
the fruit.

CAUSE OF PLUM BLOTCH

By comparison with type specimens, the organism involved in the pro-
duction of plum blotch has been found to be identical with the fungus
described by Heald and Wolf ^ as Phyllosticta congesta. Heald and

1 Heaid, F. D., and Wolf, F. A. new spEaEs op texas fungi. /wMycologiav. 3, no. i, p. 8. 191 1.

Nov. 12, 1921

Plum Blotch

367

Wolf found the fungus on the leaves only of Prunus sp. in Texas Their
description is as follows :

Maculis minutis, .5-.8 mm diam., brunneis numerosis, venis limitatis; pycnidiis
solitariis in quaque area, 50-125 ix diam.; sporulis globulosis vel leniter elongatis,
hyalinis 6-9 m-

On Prunus sp. Boeme (Texas) 1554 (Type).

On the upper surface of the leaf are very numerous brown areolae bounded by the
veins of the leaf. The lower surface may not be discolored. These minute spots fuse,
and each contains at its center a single black pycnidium. The pycnidia contain
globular or slightly oval, clear spores.

Fig. I. — Section through a pycnidium of Phyllosticta congesta, showing spores. Natural
infection on plum fruit, Georgia 1917. X 340.

Heald and Wolf do not mention the fact that the older spots become
gray or silvery, though the type specimens as well as those collected by
Scott and the writer show this to be the case. The spots on these
leaves and those on Georgia specimens collected by the writer show a
marked resemblance, and the fungi found upon them are morphologically
the same. The spots on the leaves collected by the writer have a greater
tendency to fall out. ^iof-:;,

The pycnidia (fig. i) are glistening, lens-shaped, erumpent, on the
leaves 65 to 120 /x in diameter, on the fruit 60 to 120 /x in diameter.
On the average, pycnidia on the fruit are somewhat
larger than those on the leaves. Spores on the leaves
measured 7 to 9 ju in diameter, on the fruit 8 to 9 /i.
Spores from younger spots were invested with gelati-
nous envelops which were sometimes lengthened into
appendages (fig. 2). Spores from older spots do not
show these envelops, and they are not to be found in
the dried herbarium specimens. The young spores
of Phyllosticta solitaria have sucli an envelop. In fact,
P. solitaria and P. congesta resemble one another so
closely that on purely morphological grounds they
might be considered as identical. Since the ascogenous stage of neither
fungus is known, the writer prefers to retain the name P. congesta as a
matter of convenience, unless it is shown by cross inoculations that the
fungus on the apple and that on the plum are identical in every way.

Fig. 2. — Spores of Phyl-
losticta congesta, with
the gelatinous envelops
which are sometimes
present. From pycni-
dia on plum fruit,
Georgia 1917. X 680.

368 Journal of Agricultural Research voi. xxii. no. 7

Of course the final test of identity would lie in whether or not the as-
cogenous stages of the two fungi, assuming them to exist, would prove
to be identical.

Specimens of Phyllostica congesia on fruit and foliage of Prunus
triflora have been deposited in the Pathological Herbarium, Bureau of
Plant Industry, United States Department of Agriculture.

It is not known how the fungus is carried over from one season to
another. If it occurs on the twigs, as the writer is inclined to think, there
would be good reason for believing that production of spores from twig
lesions in the spring would constitute an important infection source. It
is also possible that the fungus survives the winter on leaves and fruit.

On all the ordinary culture media the fungus shows about the same
type of growth. On corn meal agar, beef agar, prune agar, potato agar,
and oatmeal agar growth is very slow, and on all these media it presents the
same appearance. There is a dense black mass of closely woven hyphae
forming a raised and irregular aggregation of shining bead-like bodies
which may be considered as sterile pycnidia, since they are more or
less hollow bodies containing oil drops. The margin of the growth is
often fringed; in fact on the above-named media the growth is almost
as blotch-like as it is on the fruit of the plum. On sterihzed stems of
Melilotus the growth resembles that on the agars, but spores are often
formed though very scantily.

On Japanese plum twigs growth is also very slow. Pycnidia and
spores are formed in about two months. Pycnidia are formed on the
bark and may also be formed at the cut end of the twig, in which case
they are densely aggregated.

Sterilized apple twigs proved to be the best medium for the production
of spores, though two to three months must elapse before spore produc-
tion begins. On this medium the only sign of growth by the fungus is
the formation of a dense mass of hyphae and pycnidia, closely aggregated
at the upper end or at an abraided place on the side of the twig. On all
the media used the type of growth exhibited by Phyllosticta congesia
differs somewhat from that of P. solitaria. On sterile apple twigs, for
instance, the latter produces pycnidia which are scattered over the bark,
whereas the pycnidia of the former are formed only at the cut ends of
the twigs.

In 191 7 the fungus was isolated from both fruit and leaves by the
poured plate method, using spores, and by planting bits of the diseased
tissues in plates.

In the spring of 19 18, no spores had been obtained in cultures, but
inoculations were made by spraying the young fruits and leaves of
Abundance and Burbank plums with bits of hyphae and sterile pycnidia
suspended in sterile distilled water. The results were negative in every
case.

Nov. la, I92I Plum Blotch 369

In 19 19, spores obtained from apple twig cultures and suspended in
sterile distilled water were applied to fruit, foliage, and twig of Abundance
plums on May 15. Where cultures originally obtained from plum fruits
were used, two fruits were found with two typical blotches on each of
them; three leaves were found with scattering spots, each spot typical
of the disease and each bearing a single pycnidium with the character-
istic spores of Phyllostida congesta. Like results were obtained by the
use of cultures obtained from the leaves; one fruit showed three typical
blotches with pycnidia and two others showed one; seven leaves were
successfully infected. From all these artificially inoculated parts, the
fungus was reisolated and proved to be P. congesta.

No lesions were found on the twigs.

Inoculations made upon Japanese plums with spores from pure cul-
tures of Phyllostida solitaria gave negative results in 1918, 1919, and 1920,
though the spores were applied to fruit, foliage, and twigs at frequent
intervals throughout the spring.

Though the inoculation experiments herein reported upon are suffi-
cient to prove Phyllostida congesta the cause of plum blotch on leaves and
fruit and show the fungus on the fruit to be identical with that on the
leaves, they are not as complete as the writer should wish. All the inocu-
lation work was done at Arlington, Va., under conditions probably un-
favorable to the fungus, since it has been found naturally only in regions
much farther south.

It is planned to carry on further inoculation work with both the plum
blotch and apple blotch Phyllostictas. The writer expects eventually to
obtain successful inoculations on plum twigs using Phyllostida congesta as
inoculum.

CONTROL MEASURES

No attempts to control plum blotch have been made. One would
expect that control might be had by spraying with a strong fungicide at
intervals beginning shortly after the petals have been shed as is the case
with apple blotch. Dilute lime-sulphur solution and Bordeaux mixture
injure Japanese varieties of the plum so severely as to preclude their use
during the growing season. It is also doubtful whether or not dilute
lime-sulphur solution would control severe cases of disease, since it will
control only mild cases of apple blotch. Self-boiled lime-sulphur can be
used with safety on the Japanese plum, but it is a fungicide which is
even weaker than dilute lime-sulphur solution. It seems probable,
therefore, that should this disease ever become an important one, its
control will present a problem of considerable difficulty, though it is
realized that the reasoning by analogy in which the writer has just
indulged may easily lead to wrong conclusions.

370 Journal of Agricultural Research voi. xxii. no. 7

SUMMARY

Plum blotch, a hitherto unknown disease of the Japanese plum {Prunus
iriflora), has been found in Georgia. In addition to the fruit, the leaves
and possibly the twigs are affected. The lesions on fruit and leaves
greatly resemble those of the apple caused by Phyllosiicta soliiaria
E. and E.

Varieties Abundance and Burbank were found to be susceptible. An
unnamed seedling, probably also belonging to Prunus iriflora was found
to be severely infected.

From diseased fruits and leaves the fungus, Phyllosiicta congesta Heald
and Wolf, was isolated and grown in pure culture. Spores obtained from
cultures on sterile apple twigs when suspended in distilled water and
sprayed on healthy fruits and leaves produced characteristic lesions of
the disease.

Phyllosiicta congesta Heald and Wolf is to be considered for the present
as different from P. soliiaria E. and E-, though greatly resembling it.
Inoculation experiments on plums using spores from pure cultures of P.
soliiaria were negative.

No attempts have been made to control plum blotch, but the possibili-
ties of control are discussed.

PLATE 34

A. — Plum leaves affected with Phyllosticta congesta, Georgia, 1917.
B. — Plum fruits affected with Phyllosticta congesta, showing the characteristic
' blotches, " Georgia, 1917.

Plate 34

Journal of Agricultural Research

Vol. XXII ,No.7

A COMPARISON OF THE PKCTINASE PRODUCED BY
DIFFERENT SPECIES OF RHIZOPUS

By ly. ly. Harter, Pathologist, and J. L. Weimer, Pathologist, Office of Cotton, Truck,
atid Forage Crop Disease Investigations, Bureau of Plant Industry, United States
Department of Agriculture

INTRODUCTION

Recent investigations by Harter and Weimer ^ showed that Rhizopus
tritici Saito, an organism demonstrated to be parasitic on sweet potatoes,
produces a powerful intracellular and extracellular pectinase which
dissolves the middle lamella so that the cells readily separate without
themselves undergoing any noticeable alteration. A suspension of 0.25
gm. of the enzym powder in 25 cc. of water was found to completely
macerate sweet potato disks i mm. in thickness in two to five hours.
Furthermore, the solution on which the fungus grew was even richer
in pectinase, maceration of the sweet potato disks being completed in
one to three hours.

Since the foregoing results have been published Harter, Weimer, and
Lauritzen ^ have concluded experiments which showed that out of 1 1
species of Rhizopus studied 9 were parasitic on the sweet potato.
Furthermore, these investigators found that the species differed in
degree of parasitism, both as regards the percentage of infection and
the rapidity of decay.

The present investigations, therefore, had for their object to determine
(i) if pectinase is produced by all species of Rhizopus and, if so, to what
extent and (2) if its production is any indication of the parasitism of

the species.

TECHNIC

The methods employed in carrying out macerating experiments
with the different species of Rhizopus were for the most part the same as
those used in previous work to which reference ^ has already been made,
although some slight modifications were necessary to meet certain phases
of the problem. Three sets of experiments were carried out with each
organism. All the species were included in a single experiment and the
macerating action was determined for all at the same time, so that the
results for each species are directly comparable for a single experiment.
The culture medium was so prepared and in sufficient quantity as to make

1 Harter, L. I,., and Weimer, J. L. studies in the physiology of parasitism with speciai, refer-
ence TO THE SECRETION OF PECTINASE BY RHIZOPUS TRITICI SAITO. In Jour. Agr. Research, v. 21, no. 9,
p. 609-625. 1921. Literature cited, p. 624-625.

* Harter, L. I,-, Weimer, J. L-, and Laitritzen, J. I. the susceptibility of the different varie-
ties OF SWEET potatoes TO DECAY BY RHIZOPUS NIGRICANS AND RHIZOPUS TRITICI. In Phytopathology,
V. II. 1921. In press.

Journal of Agricultural Research, Vol. XXII, No. 7

Washington, D. C. Nov. 12, 1921

aam Key No. G-354

(371)

372 Journal of Agricultural Research voi. xxii. no. 7

it uniform in all the flasks for all organisms. Four flasks (2 liters),
containing 300 cc. of the culture medium, were inoculated with each
one of the species in each experiment, and the cultures were incubated
for three days.

The macerating action was determined for the following species of
Rhizopus: chmensis Saito, nodosus Namysl, tritici Saito, maydis Bru-
derl, delemar (Boid) Wehmer and Hanzawa, arrhizus Fischer, oryzae
Went and Pr. Geerligs, nigricans Ehmb., reflexus Bainier, artocarpi
Racib., and microsporus v. Tieg.

It has been shown ^ that the different species of Rhizopus do not all
have the same optimum temperture for growth. Some species thrive
at high temperatures, some at relatively low temperatures, and others
at a temperature intermediate between the two extremes. Therefore,
the 1 1 species studied have been separated into three groups with respect
to their temperature relations. In all the experiments connected with
the present investigations the same grouping of the different species
has been observed, thus subjecting each organism to as nearly the
optimum temperature for its growth as possible.

The cultures of chinensis were incubated at 40° C, those of nodosus,
tritici, maydis, delemar, arrhizus, and oryzae at 30°, and those of nigri-
cans, reflexus, artocarpi, and microsporus at 20°. Although so far as
temperature is concerned the results are not strictly comparable, pre-
liminary experiments showed that more reliable data could be obtained
by growing the different organisms at temperatures suited to their growth
than by subjecting them all to a uniform temperature. Some of the
species, as for example nigricans, which requires a relatively low tem-
perature, make no growth or only a feeble growth at 30° and none at 35°.
On the other hand, chinensis, a high temperature form, makes a reduced
growth at 30° and a feeble growth at 20°. |

At the close of the incubation period (three days) the mycelial growth
was lifted from the culture flask and the substrate was filtered through a
fine grade of muslin. The mycelium was treated subsequently by
acetone and ether according to the method previously described.^ The
solutions from the different flasks in which the same species had grown
were made into a compound sample thoroughly shaken, and 25-cc.
portions were used for maceration experiments. Likewise all the fun-
gous felts of the same organism grown in the different flasks were brought
together and treated as one sample, a weighed portion of the dried
mycelium being used for maceration of the raw disks. Two types of
controls were run with each set of experiments, as follows: (i) Sweet-
potato decoction on which the fungus had grown for three days, which
after the removal of the mycelium was steamed for 15 minutes to inac-
tivate the enzym; (2) decoction which was identical with that used for

1 Harter, X,. h; Weimer, J. L., and Lauritzen, J. I. op. cit.

2 Harter, I^. 1,., and Weimer, J. 1,. op cix

Nov. 12, 192 1

Pectinase Produced by Different Species of Rhizopus 373

inoculation purposes but which had not supported a fungous growth.
Maceration by the enzym in the solution and in the mycelium was carried
out at a temperature of 40° C. for all the species. Before the addition
of the raw sweet-potato disks the solutions and suspensions of the my-
celium were held for one hour at 40° in order to bring them to the tem-
perature at which maceration was to take place.

A/o^o,sa<s
r/?/T/c/

Af/C/?OSPORUS
A//(9/?/0/9A/iS

O / 2 <^ ^ <5 e 7

Fig. I. — Graph showing the time required by the different Rhizopus species to completely macerate
sweet-potato disks by the enzym in the solution on which the fungi had grown for three days; also the
comparative rate of maceration in the three experiments.

Maceration with the mycelium was carried out by the use of 0.5 gm.
ground in sand and suspended in 25 cc. of water. All the sweet potato
disks (i mm. thick and 1.5 cm. in diameter) required for maceration in
the solution on which the fungus grew and in a water suspension of the
mycelium were cut from a single potato for an entire experiment of all
the species. pJxism

374

Journal of Agricultural Research voi. xxn. Na»

EXPERIMENTAL DATA

The results obtained in the different experiments both as regards the
maceration in the solutions and in a water suspension of the mycelium
are represented graphically in figures i and 2. Each of the vertical lines
of a single group represents the results obtained for a particular organism
in a single experiment. The length of the vertical lines indicates the

OE^£:M/P/r'

C/y/ZV/ETA/iS/tS
T/?/T/C/

m^mmm ^^mmm wammmm — — Mi— 1— ■■ 1— oi— — aa»

^mmm^ ■hbbkb watm^

wtm^ma — apif jawii wa^mmm i^^km mm^imm mw— hmmm

O / 2 3 ^ s e

7 e s /o

Fig. 2.— Graph showing the time required by the differeut Rhizopus species to completely macerate
sweet-potato disks by the enzym contained in Yi gr. of the mycelium; also the comparative rate of macera-
tion iu the three experiments,

length of time in hours required to complete maceration of the disks,
I cm. being equivalent to one hour. From these figures a direct com-
parison can be made of the results obtained from the different species as
well as the variation in the results of the same species in different experi-
ments.

Nov. 12, 1921 Pectinase Produced by Different Species of Rhizopus 375

DISCUSSION OF RESULTS
VARIATION

A comparison of the results as shown by figures i and 2 indicates that
under the conditions of these experiments maceration was completed by
the enzym exuded into the solution in a shorter length of time than by
that contained in the mycelium. This difference, however, does not
mean that the enzym is more powerful or more abundant in the solution
than in the mycelium since no attempt was made to employ an amount of
mycelium that would be equivalent in macerating power to the enzym
of the solution. In these experiments maceration was regarded as
complete when the disks pulled from opposite sides separated without
any perceptible resistance. In completely macerated tissue coherence
of the cells is entirely lost and the tissue can be readily pulped between
the thumb and finger.

The data show that a considerable amount of variation exists in the
results obtained in the different experiments with a single species both in
respect to the solution and the mycelium. There are probably several
factors responsible for these variations. In the first place a dififerent
supply of the sweet-potato decoction was prepared for each set of experi-
ments. In spite of the fact that the dififerent solutions were prepared
to be alike as nearly as it is possible to make them, it can not be said, in
view of the fact that different potatoes were used each time, that the
various preparations were identical. After the cultures were inoculated
incubation was carried out at a temperature which varied very little
but possibly enough to influence slightly the rapidity and volume of
growth. At the close of the incubation period the substrate and mycel-
ium were handled as nearly alike as possible in all the experiments, but
in spite of such precautions some variations might result. It would seem
that the greatest source of error might be attributed to a variation in the
composition of the potatoes from which the raw disks were cut. In this
connection preliminary experiments showed that different potatoes are
macerated in a dififerent length of time the variations, however, being
within relatively narrow limits. Furthermore, it is probable that the
composition of the sweet potato is gradually changing with the increase
in the length of time after digging. So far as their susceptibility to
maceration is concerned it is interesting to note that a comparison
between newly dug potatoes and those stored for several months showed
that the latter are macerated in a shorter time than the former. Although
the present experiments were carried out with a single variety the various
experiments were conducted in sequence so that the later experiments
were made on what might be termed older potatoes.

376 Journal of Agricultural Research voi. xxii. no. 7

COMPARISON OF SPECIES
MACERATION OP DISKS IN THE SOLUTIONS

It appears from figure i that two species, nigricans and artocarpi,
macerate raw sweet potato disks more slowly than any of the others,
followed by chinensis and reflcxus in the order mentioned. The other
species complete maceration in a relatively short time, the most rapid
being arrhizus, tritici, and maydis.

MACERATION OF DISKS IN A SUSPENSION OF MYCELIUM

With respect to maceration by the mycelial enzym, four species,
nigricans, micros porus, chinensis, and artocarpi, stand out as being conspic-
uously slow. So far as the mycelium is concerned delemar, a species inter-
mediate between the slow and rapid forms, is less active than reflexus but
more active than the latter when the solutions are used. Likewise the
enzym contained in the mycelium of micros porus macerates slowly, while
that in the solution, on the other hand, disintegrates the tissue rapidly.
Chinensis shows a similar relationship existing between the enzym of the
mycelium and that of the solution, although this species does not stand
out as conspicuously as microsporus.

From the few illustrations cited it is evident that there is no complete
correlation between the activity of the mycelial enzym and the activity
of that exuded into the substrate. An examination of figures i and 2
shows that the different species do not secrete an equivalent amount of
pectinase, since the completion of maceration by the enzym of both the
mycelium and solution may vary greatly. It is conceivable and the
results of these investigations seem to indicate that some species give up
their enzym to the solution more readily than others. For example, the
solution on which tnicrosporus grew is relatively rich in pectinase while
the enzym contained in the mycelium acts slowly. Delemar is another
outstanding example of the same phenomenon.

PECTINASE PRODUCTION IN RELATION TO PARASITISM

If a relationship between the production of pectinase by the different
species of Rhizopus and their parasitism could be shown to exist, con-
siderable light might be thrown on the physiology of parasitism, espe-
cially among fungi which are characterized by their ability to dissolve
the middle lamella in advance of their growth. In a previous publica-
tion ^ it was pointed out that all the species of Rhizopus studied were
parasitic on sweet potatoes with the exception of microsporus and chinen-
sis. These two species were studied in connection with the others.
They were given equal opportunity to cause decay, but in no case was
there any evidence to indicate parasitism. However, both of these

iHarter, L. L., Weimbr, J. L., and Lauritzen, J. I. op. cix.

Nov. 12, 1921 Pectinase Produced by Different Species of Rhizopus 377

species produced pectinase. The amount of pectinase in the myceHum at
the end of the growth period was relatively small, but microsporus and
to a lesser degree, chinensis, exuded enough into the culture solution to
cause maceration in a much shorter time than either nigricans or arto-
carpi, both of which are parasites. Maceration of sweet-potato disks
by means of the mycelial enzym of the two parasitic and nonparasitic
species just mentioned was completed in about the same length of time.
Nigricans is the most commonly isolated species and is probably respon-
sible for most of the loss to sweet potatoes caused by this group of fungi.
At a suitable temperature it decays sweet potatoes and other vegetables
rapidly. The middle lamellae of sweet potatoes decayed by this species
are dissolved some distance in advance of the growth of the mycelium,
so that coherence is lost. In the early stages, at least, the cells them-
selves are not invaded by the fungus. The same may be said of artocarpi.
However, in cultures nigricans and artocarpi, in contrast to the other
species, exude a very small amount of enzym into the substrate and
retain very little in the mycelium. Delemar, a species which readily
decays sweet potatoes, seems to give up most of its pectinase to the
substrate, so that maceration by means of the mycelium is relatively
slow, at least within the limits of these experiments. All the other
species are vigorous parasites, decaying the sweet potato within a few
days under favorable conditions. They also produced large quantities
of pectinase, relatively speaking, some of which is exuded into the solu-
tion and some retained by the mycelium, as shown by the fact that
maceration, by both the mycelium and solution, is comparatively rapid.

SUMMARY

(i) The secretion of pectinase by 11 species of Rhizopus has been
studied. All the species were found to produce pectinase and to exude
some of it into the culture solution.

(2) The amount of pectinase produced varies with the species, grown
under identical conditions. The mycelium of four species — nigricans,
microsporus, chinensis, and artocarpi — and the solution on which two —
nigricans, and artiocarpi — are grown is comparatively weak in pectinase.
Chinensis and microsporus, whose mycelial enzym is weak, secrete it
abundantly into the substrate.

(3) Two species, nigricans and artocarpi, both of which are parasitic
on the sweet potato, secrete a relatively small amount of pectinase.
On the other hand, chinensis and microsporus, two nonparasitic species,
while retaining a small amount of enzym in the mycelium, secrete a
comparatively large quantity of enzym into the culture solution.

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Vol.. XXII NOVEMBER 19, 1921 No. «

JOUKNAI> OF

AGRICULTURAL

RESEARCH

CONTENTS

Pago

Hemotoxins from Parasitic Worms - - - - - -379

BENJAMIN SCHWARTZ

( Contribution from Bureau of Animal Industry )

Ash Content of t^ie Awn, Rachis, Palea, and Kernel of Barley
during Growth and Maturation - - - - - - 433

HARRY V. HARLAN and MERRITT N. POPE

<Contilbution rom Bureau of Plant Industry)

PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE

WITH THE COOPERATION OF THE ASSOCIATION OF

LAND-GRANT COLLEGES

WASHINGTON, D. C.
GOVERNMENT PRINTING OFFICE

1921

EDITORUL COMMITTEE OF THE

UNITED STATES DEPARTMENT OF AGRICULTURE AND

THE ASSOCIATION OF LAND-GRANT COLLEGES

FOR THE DEPARTMENT

KARIy F. KELLERMAN, Chairman

Physiologist and Associate Chief, Bureau
of Plant Industry

EDWIN W. ALI.EN

Chief, Office of Experiment Stations

CHARLES h. MARLATT

Entomologist and Assistant Chief, Bureau
of Entontology

FOR THE ASSOCIATION

J. G. LIPMAN

Dean, State College of Agriculture; and
Director , New Jersey Agricultural Experi-
ment Station, Rutgers College

W. A. RILEY

Entomologist and Chief, Division of Ento-
mology and Economic Zoology, Agricul-
tural Experiment Station of the University

of Minnesota

R. L. WATTS

Dean, School of Agriculture, and Director;
Agricultural Experiment Station, The
Pennsylvania Stale College

All correspondence regarding articles from the Department of Agriculture should be
addressed to Karl F. Kellerman, Journal of Agricultural Research, Washington, D. C.

All correspondence regarding articles from State Experiment Stations should be
addressed to J. G. Lipman, New Jersey Agricultural Experiment Station, New
Brunswick, N. J.

JOINAL OF AGRldlllAlRESEARCH

Vol. XXII Washington, D. C, Nove;mber 19, 1921 ^\^^'y^^O- 8
HEMOTOXINS FROM PARASITIC WORMS

By Benjamin Schwartz '

Zoological Division, Bureau of Animal Industry, United States Department of

Agriculture

I. INTRODUCTION

Aside from the purely mechanical injuries which parasitic worms may
inflict upon their host as a consequence of their migrations, displacement
of a certain amount of the host's tissue, bites and laceration of the mucosa,
obstruction of ducts, and various other mechanical disturbances, it has
been generally assumed that they may also produce harmful effects as a
result of their toxic secretions. Despite the fact that the data on which
the view that parasites secrete toxic substances is based, so far as they
have been recorded heretofore in the literature, are somewhat contra-
dictory, they have been accepted by a large number of investigators
as affording a more plausible explanation of the cliemical pathology of
helminthiasis than the data with reference to any other theory that has
thus far been advanced. With reference to the subject of toxic products
of parasitic worms. Wells {igiSy states:

The subject has received much less consideration than its importance deserves, as
we are quite in the dark as to how much of the effects produced by animal parasites
are not merely mechanical, but are due to soluble poisons that they secrete or excrete.
Some of the parasites probably cause harm mechanically and in no other way, but
with most of them there is more or less evidence of the formation of poisonous
substances.

While it must be admitted that the evidence in favor of the view that
parasites secrete products toxic to the host is as yet rather incomplete, the
fact of the existence of such toxic products can not be denied. So
far as they have been investigated, the serological reactions of hosts har-
boring parasites afford proof that parasitic worms liberate products
against which the host develops defense or "immunity" reactions. It
has been known for a relatively long time that in cases of infestation
with species of Trichinella, Schistosoma, Necator, and Anyclostoma a

' Resigned December is, 1920.

2 Dates in parenthesis refer to " Literature cited," p. 428-432.

Journal of Agricultural Research, Vol. XXII , No. 8

Washington, D. C. Nov. 19,1921

aan Key No. A-62

(379)

380 Journal of Agricultural Research voi. xxii, no. s

high eosinophilia is commonly present. An increase in the number of
eosinophile leucocytes has also been observed, although not as regularly,
in cases of infestation with species of Ascaris, Oxyuris, Strongyloides,
and other nematodes. Similar conditions have also been encountered in
cases of infestation with Taenia solium, T. saginata, Fasciola hepatica,
Clonorchis sinensis, and other cestodes and trematodes. As a matter of
fact, eosinophilia is so commonly associated with parasitic infestation
tliat the finding of a high eosinophile content in the peripheral blood is
generally considered as presumptive evidence of parasitic infection. In
a recent extensive review of the literature on the subject of eosinophilia,
Schwarz {191 4) states that an increase in the number of eosinophile
leucocytes in the peripheral circulation in cases of parasitic infestation is,
from an etiological viewpoint, the most clear-cut illustration of general
eosinophilia.^

Aside from the cellular inmiunity reactions, as shown by the increase
in the number of eosinophile leucocytes in the blood in cases of infesta-
tions with parasitic worms, there appears to be evidence of a humoral
immunity as well. In the case of hydatid (Echinococcus) disease of man
and animals, it has been shown by a number of investigators that specific
antibodies are present in the blood of the host, demonstrable by the
technic of complement fixation and precipitate formation. That such
immunity reactions are not limited to hydatid disease is the opinion of
certain investigators, who support their views by experimental evidence
which shows that specific antibodies are also present in cases of infesta-
tions with species of Ascaris, Fasciola, Schistosoma, and other parasitic
worms. ^

The facts cited in the preceding paragraphs appear to indicate that
hosts harboring parasitic worms develop typical defense or "immunity"
reactions to the absorption of foreign and presumably toxic substances
of parasitic origin. A logical corollary to the study of the serological
reaction of animals to secretions of parasitic worms is the study of the
secretions themselves with reference to their chemical and physiological
properties. This subject has recently received considerable attention
in studies on the causes of pernicious or infectious anemia of horses, a
disease of unknown etiology, which Seyderhelm and Seyderhelm (1914)
attribute to a secretory product of an internal parasite (the larvae of
Gastrophilus) . Although the assumption of the Seyderhelms has not been
confirmed, numerous experiments by diff'erent investigators have shown
that injection into animals of extracts of various parasitic Avorms may
lead to serious consequences, frequently terminating in death. Despite
the fact that these experiments are in a general way confirmatory of the
work of earlier investigators on the physiological effects of extracts of

• " Die Vermehrung der a-Zellen in peripheren Blut bei Anwesenheit von Parasiten aus dem Stamme
der Wiirmer ist vielleicht die atiologisch am meisten klargestellte Form der allgemeinen Eosinophi'ie "
' References to and a summary of this phase of the subject may be found in an article by G. Ghedini.

Nov. 19,1921 Hemotoxins from Parasitic Worms 381

parasitic worms, the experimental evidence on the subject is somewhat
contradictory, due no doubt to the fact that different investigators
experimented under different conditions. The study of the effects of
extracts of parasites on hving animals presents numerous difficulties and
complications and may lead to contradictory results unless suitable
precautions are taken to control various extraneous factors. More accu-
late studies on the effects of toxic products may be carried out in vitro,
provided the toxic substance in question has affinity for tissues and cells
available for such experiments. As is well known, red blood cells serve
as excellent indicators of test-tube reactions in v/hich hemotoxic sub-
stances are involved, and in the case of toxic products of parasitic origin,
experiments with red blood cells are of great importance in view of the
fact that in many parasitic infestations anemia is a characteristic symp-
tom. The effects of extracts of parasitic worms on red blood cells,
especially of extracts of those parasites that are known to cause anemia,
are thus of interest with reference to the possibility that the parasites in
question secrete specific toxins for the blood (hemotoxins).

II. REVIEW OF LITERATURE ON HEMOTOXINS IN PARASITIC WORMS

The same year in which Ehrlich (iSg8) announced the discovery of a blood toxin
produced by Bacillus tetanus, Schaumann and Tallqvist (i8g8) reported the discovery
of a blood toxin in the broad tapeworm of man {Diphyllohothrium latum). Ehrlich 's
discovery in the field of bacteriology served as a stimulus to the study of bacterial
hemolysins by various investigators and was followed by a series of investigations into
the nature and action of these hitherto unknown products of bacterial growth. Al-
though the discovery of Schaumann and Tallqvist did not arouse the same degree of
activity in parasitology as Ehrlich 's discovery aroused in bacteriology, the results of
their investigations were not without influence on subsequent researches in parasit-
ology, the influence being especially marked in connection with studies on the causes
of the anemia that occurs in cases of infestation with hookworms.

The facts published by Schaumann and Tallqvist {1898) may be briefly summarized
as follows:

Macerated material of Diphyllobothrium latum contains a hemolytic substance active
in vitro as well as in vivo. Peptic digestion liberates the hemolysin from the tissues
of the parasites. The introduction of D. lattim material into dogs parenterally or peros
leads to a marked reduction in the number of erytlirocytes.

In a later paper Tallqvist {1907) gives a more detailed account of the nature of the
hemotoxic secretions of Diphyllobothrium latum. The hemolytic principle is closely
bound to the cells of the parasite and is but slightly soluble in water and physiological
salt solution. By means of peptic digestion and alcohol or ether extraction it becomes
dissociated from the tissues and goes into solution. The hemolysin is thermostabile
and does not cause the development of antibodies when injected into animals. In
these respects it resembles normal tissue hemolysins. Tallqvist fotmd, moreover,
that D. latum contains not only a hemolysin but also a hemagglutinin. The latter is
soluble in water in contrast to the water-insoluble lipoidal hemolysin. The hemagglu-
tinin as well as the hemolysin is nonspecific. The potency of these agents varies,
however, for different species of red blood corpuscles.

Faust and Tallqvist (1907) studied the Diphyllobothrium hemolysin as to its chemi-
cal nature. These investigators found that extraction of the entire worm material in
ether removed all the hemolysin from the tissues of the parasite, since the removal of

382 Journal of Agricultural Research voi. xxii.no. s

the ether-soluble fraction left a fraction entirely devoid of hemolytic activity. The
ether-soluble fraction was then freed from its lecithin and cholestrin content without
injuring its hemolytic activity. In the remaining ether fraction (freed from lecithin
an'd cholestcrin) I^aust and Tallqvist identified tlu-ee fatty acids, namely, paln:itic,
stearic, and oleic acids. The first two substances did not exhibit any hemolytic
properties, whereas oleic acid was found to be markedly hemolytic. These investi-
gators therefore concluded that oleic acid is the active principle of Diphyllobothrium
hemolysin.

In a later paper Faust {1908) records the results of experiments on the effects of oleic
acid on dogs when introduced per os. In brief, this investigator concluded that pro-
Icftiged feeding of oleic acid to dogs produced anemia in the latter, as evidenced by a
reduction in the number of red blood corpuscles. Beumer {igig), however, has
found, on the contraiy, that animals may be fed daily with considerable quantities of
oleic acid for long periods without permanent ill effects, and has failed to substantiate
the harmfulness of oleic acid.

In this connection it is of interest to recall the experiments of Dascotte (cited by
Weinberg, 1912), who states that extracts of Taenia solium and T. saginata, cestodes
parasitic in man, dissolve human red blood corpuscles. Dascotte found, moreover,
that the hemolysin from these parasites is soluble in alcohol and resistant to heat at
temperatures of 100° to 120° C. Calamida {igoi) found that extracts of two species of
cestodes from carnivores {Dipylidiuvi canimim and Multiccps muUiceps) are hemolytic
to the red blood corpuscles of rabbits and guinea pigs and that the hemolysin goes
through the pores of a Berkefeld filter. According to Weinberg {igoy), physiological
salt-solution extracts of two species of tapeworms parasitic in horses (Anoplocephala
plicata and A. perfoliata) have no deleterious effects on the blood corpuscles of the
horse. Tallqvist {igoy), on the other hand, denies the presence of hemolysins in ces-
todes other than Diphyllobothrium latum. He states that he worked with a number of
species, including T. saginata. He admits that he sometimes observed slight hemolytic
effects of extracts of these parasites but expresses the opinion that they are to be
ascribed to secondary degeneration products associated with acid formation.

While Diphyllobothrium latum is capable of causing severe anemia in man, clinically
indistinguishable from pernicious anemia and, according to many investigators, differ-
ing from the former in one respect only, namely, by the disappearance of the symptoms
and recovery of the patient after expelling the parasite, there are numerous cases on
record in which the presence of D. latum in man was not accompanied by anemia.
In fact, grave blood disturbances in cases of D. latum infection are, according to the
observations on record , not as common as the incidence of infection with this tapeworm,
a fact which has given rise to considerable speculation as to the causes of the occasional
appearance of anemia in the course of infection witli this parasite. These speculations
will be referred to elsewhere in this paper.

In contrast to the occasional appearimce of anemia in cases of infection with Diphyl-
lobothrium /atowinfections with hookworms (Necator and Ancylostoma) are generally
accompanied by severe anemia. That the causes of anemia in hookworm disease are
due to a toxin is a view which was adopted by a number of investigators on a purely
a priori basis, because the direct abstraction of blood by these parasites, even when
present in large numbers, fails to account for the severity of the clinical picture usually
present in such cases. This fact was recognized early in the study of the disease, and
led to the postulation of the "toxin theory."

Luscana (iSgo) found that as a result of injecting rabbits with urine taken from
patients suffering from hookworm disetise, the animals developed symptoms of anemia.
It was not until 1905 , however, that the toxin theory received more direct experimental
support from Calmeftte and Breton. These investigators found that salt-solution ex-
tracts of the Old World hookworm of man (Ancylostoma duodenak) are hemolytic to the
red blood cells of man. Alessandrini (zpo^) had already foxmd by direct microscopic

Nov. 19, 1921 Hemotoxins from Parasitic Worms 383

observation that human red blood corpuscles are destroyed when placed in contact
with the cervical glands isolated from hookworms (species not given but presumably
A. duodenale), but subsequent investigation showed that the hemolysin is not limited
to the cervical glands.

Loeb and Smith (1904) in the course of experiments wath salt-solution extracts of
the dog hookworm (Ancylosioma caninum), found that these extracts showed no hemo-
lytic properties and left the blood still intact and uncoagulated after being in contact
with it for 17 hours. Reference to the work of these investigators on the anticoalgula-
ting property of hookworm extract {A . caninum) will be made elsewhere in this paper.

Liefmann (7905) found that in two out of three experiments salt solution extracts of
Ancylosioma caninum produced slight hemolysis of dog blood. This writer observed
intact erythrocytes in the intestines of the worms and therefore came to the conclusion
that the parasites do not secrete a hemolysin. Liefmann fails to state whether or not
he washed the blood corpuscles before testing them against hookworm extract.

Preti {1908) foimd that the Old World hookworm of man {Ancylosioma duodenale)
contains a hemolysin insoluble in salt solution but soluble in ether and alcohol. He
states that tryptic digestion liberates the hemolysin and renders it soluble in water.
He found the hemolysin to be resistant to boiling for three hours and nonspecific, since
it was equally potent against the blood corpuscles of several other species of animals
as well as man.

In the course of his investigations concerning ancylostomiasis and beriberi, Noc
{1908) found that physiological salt-solution extracts of the hookworm of man {Necator
dmericanus) are hemolytic to the washed red blood corpuscles of man. He states that
the hemolysin withstands a temperatiu-e of 80° C. for one hour without injury to its
potency. Noc found that whereas the blood serum of patents suffering from severe
ancylostomiasis and beriberi contained no antihemolysins, that of normal persons and
of those recovering from these diseases was antihemolytic.

De Blasi {1908) examined the blood serum of 12 human subjects infested with hook-
worms {Ancylosioma duodenale) and found that after the serums were heated for 30
minutes at 56° to 62° C. they acquired hemolytic properties. Before heating, the
serums in question were not hemolytic. Heating the serum evidently destroyed some
antibodies which neutralized the potency of the hemolysin. The heated serum of
normal persons, according to this writer, did not contain any hemolysins.

Whipple {1909) records tests of salt-solution eyLtractsoi A ncylostotna caninum, A.
duodenale, and Necator americanus on unwashed citrated blood of man, dog, and rat.
He states that he found a weak hemolysin in the three species of hookworms exhibiting
similar properties, namely, nonspecificity, susceptibility to boiling which destroys it,
and distribution in all parts of the body of the worms. According to Whipple, the
hemolysin is only demonstrable in concentrated extracts, and probably bears no
relation to the secondary anemia of ancylostomiasis.

lyoeb and Fleisher {1910) state that a salt-solution extract of Ancylosioma caninum,
containing as much as 5 mgm. of the powdered worm material in i cc. of salt solution
did not produce any hemolytic effect on the washed erythrocytes of the dog. These
writers also state that lecithin used in doses in which it alone produced no hemolytic
effect failed to activate A. canimim extract. Loeb and Fleisher admit the possibility
that the temperature at which the specimens were dried (42° to 50° C.) may have had
an injurious effect on the hemolysin, but they do not consider this very probable.

Recently Usami and Mano {1919) have studied the effects of hookworm extracts on
red blood cells. These writers state that hookworm hemolysin is thermostabile,
insoluble in water, and soluble in alcohol, ether, and acetone.

It will be seen from the foregoing summary with reference to hookworm extracts
that Loeb and Smith {1904) and Loeb and Fleisher {1910) are the only investigators
who failed to observe hemolysis in the presence of these extracts. As will be shown
elsewhere in this paper, the negative results of Loeb and Smith may have been due

384 Journal of Agricultural Research voi. xxn. no. s

to the antilytic action of normal blood serum. The negative results recorded by
Loeb and Fleisher {1910) may have been due to insufficient or faulty extraction of
the worm material, insufficient quantity of powder used in the experiments, or possibly
to the destruction of the hemolysin by drying at temperatures between 42° and 50° C.
The results recorded by Preti (iQoS) as regards the insolubility of the hemolysin in
salt solution and its resistance to boiling are at variance with those of other investi-
gators, and, as will be shown in the following pages, are not in harmony with the
results obtained by the present writer. Moreover, Preti 's results can not be accepted
as conclusive, owing to his failure properly to control his experiments. Alessandrini 's
attempt {1904) to associate the secretisn of hemolysin with the cervical glands of the
parasites is not sustained by Whipple {igog), who found the hemolysin in all parts
of the worm.

It is interesting to observe that the different species of hookworms referred to in
the foregoing summary have the common biological property of secreting a substance
destructive to red blood cells. Inasmuch as hookworm disease is characterized by
severe anemia, the presence of a blood-destroying substance in the parasites is highly
significant.

In addition to the hemolytic substance which is present in hookworms, Loeb and
his collaborators have shown that the hookworm parasitic in dogs {Ancylostoma
caninum) also secretes a substance which inhibits coagulation of blood in vitro (IvOeb
and Smith, 1904; Loeb and Smith, 1906; Loeb and Fleisher, 1910). The results of
experiments by these investigators with reference to the anticoagulins of hookworms
may be summarized as follows: In A. caninum a substance is present which retards
coagulation of blood in vitro. This substance which is present in the anterior part of
the worm and practically absent in the posterior part is not destroyed but is markedly
weakened by boiling for 15 minutes. The substance does not resemble hirudin, a
toxic constituent of the leech, but appears to resemble cobra venom so far as its physi-
ological properties are concerned. It is of interest to note in this connection that
Liefmann {1905), who rejects the view that the hookworm secretes a hemolysin, like-
wise rejects the view that this parasite secretes an anticoagulin, since he obtained
positive results in but one out of three experiments which he performed. Liefmann
ascribes his positive results to substances from the intestine which may have adhered
to the worms, namely, pancreatin and peptone. Loeb and Smith {1906) point out,
however, that in view of the fact that they washed the wonns carefully and that
neither peptone nor pancreatin is known to inhibit coagulation of dog's blood in
vitro, and further, in view of the fact that the posterior parts of the hookworms showed
but a slight anticoagulating effect on dog blood and that extracts of ascarids and tape-
worms from dogs did not retard the coagulation of dog blood, Liefmann 's contention
can not be sustained.

The carefully controlled experiments of Loeb and his collaborators leave no room
for doubt as to the presence of a hemotoxin in A ncylosioma canimtm which inhibits the
coagulation of dog blood. Loeb and Smith ascribe etiological significance to this
toxin and believe that it has the power of causing small hemorrhages in regions of the
intestine that have been lacerated by the worms.

The pathological role of the whipworm {Trichuris trichiura) parasitic in man is empha-
sized by Askanazy (1896), who states that this parasite feeds on blood, basing his
assertion on the presence of iron pigment in the intestine of the worm demonstrable
by the Berlin blue reaction. Askanazy assumed, of course, tliat the iron found in the
worm is obtained from the hemoglobin of the host's blood. Schultze (1905) rejects
Askanazy 's interpretation and considers tliat the pigment in question is obtained from
the host's intestine rather than from the blood.

Guiart {1908) presents conclusive evidence as regards the bloodsucking habit of
Trichuris trichiura, since he found blood-engorged specimens in a human patient.
Guiart's observation has been confirmed by a number of investigators, including Garin,

Nov. 19. 19" Hematoxins from Parasitic Worms 385

Seidelin, and Leon (Guiart, IQ14). Guiart and Garin (iQog) found that the presence
of Trichuris eggs in the feces of human subjects is correlated with the presence of blood
in the feces as shown by a positive Weber test.

As to the presence of hemotoxic secretions in whipworms, Whipple (1909), who ex-
perimented with extracts of these parasites, found that they contained a hemolytic
substance destructive to the red blood cells of the dog and of man. Whipple states
that the hemolysin left some samples of human red blood cells intact but was destruc-
tive to others. Garin (1913) performed similar experiments with Trichuris extracts
and confirmed the presence of a hemolysin in these parasites. According to Garin,
the whipworm hemolysin is tliermostabile, being destroyed by 30 minutes' heating at
56° C. The inactivated hemylosin can not be reactivated by normal guinea-pig serum
(complement) , according to this investigator. Garin states , furthermore , that whereas
he obtained positive results with human red blood corpuscles the results of experiments
with the erythrocytes of rabbits and guinea pigs were doubtful.

A survey of tlie literature relating to tlie pathogenic role of Ascaris lumbricoides
reveals the fact that this parasite may be responsible for anemia, which is sometimes
mistaken for hookworm anemia or for pernicious anemia. The clinical reports of
Demme (iSgi) have become a classic illustration of this fact. In brief, Demme found
a child suffering from severe intestinal catarrh, with a high-grade pernicious anemia
showing a red blood coiuit of 2 ,450,000 and a hemoglobin content of 40 per cent. Two
weeks after numerous worms (A. lumbricoides) had been expelled from the child's
intestine the red blood corpuscle count rose to 4,200,000 and the hemoglobin content
reached 70 per cent. In a second case of apparent pernicious anemia, which resulted
in death and in which the erythrocytes had diminished to 1,650,000 per cubic milli-
meter, numeroiis ascarids were found on post-mortem examination which were appar-
ently responsible for the death of the child . Kuttner {1865) found that in a girl aged 12
blood destruction occurred and that this was cured by expelling a number of ascarids.
According to Filatoff {189'/), Karaven cured a case of pernicious anemia in a child by
expelling a number of ascarids from its intestine. Francois {1906) , in the course of his
investigations on anemia of miners, found many cases of severe anemia in which hook-
worms were not present but which showed numerous Ascaris eggs in the feces. A
number of observations by different investigators on hogs and horses infested with
ascarids and on man infested with A . lumbricoides bear out the fact that symptoms of
anemia are frequently associated with such infestation.

As to the manner in which species of Ascaris cause anemia two views have been
advanced, which are not mutually exclusive. Guiart {1899), who accepts the view
that worms of this genus secrete a hemolysin, inclines strongly to the view that they
also lacerate the mucosa, thus causing hemorrhages. In support of this view Guiart
describes and figures Ascaris conocephala attached to the stomach of a dolphin, the
head of the parasite being deeply embedded in the mucosa. Guiart refers to the ob-
servations of Leroiix, who found lesions in the intestine of a human being infested with
ascarids resembling lesions produced by ascarids on the mucosa of the dolphin . Fried-
berger and Frohner {1895) also support this view and state that dogs that harbor
numerous ascarids show on post-mortem examination of the intestine numerous
roimd, dark spots, surrounded by an inflamed zone, due, in their opinion, to bites of
the worms. According to Garin {191 3), several observers, including Weinberg, have
found ascarids attached to the mucosa. Garin admits, however, that despite the
fact that he made numerous post-mortem examinations of human subjects infested with
A. lumbricoides and of dogs and cats infested with ascarids, in the latter cases shortly
after death, attached parasites were never observed by him. He confirms, however,
the presence of reddish points sturounded by an ecchymotic area in the mucosa of the
intestine of infested subjects, both human and animal. Thaler {1918) has recently
reported a case of persistent intestinal hemorrhages in a human subject which did not

386 Journal of Agricultural Research voi. xxii. nos

yield to symptomatic treatment and which was cured only after removing several
ascarids.

The view that Ascaris secretions contain hemotoxins was first advanced by Schim-
melpfennig {1902), who found that in the presence of the coelomic fluid of Ascaris
equorum red blood corpuscles of the horse became crenated and were ultimately
destroyed. Schimmelpfennig furthermore discovered oxyhemoglobin in the coelomic
fluid of the parasite , a fact which led him to regard this worm as a bloodsucker. Wein-
berg {1907), Whipple (1Q09), and Alcssandrini (191 3) failed to observe any toxic
efi'ect of salt-solution extracts of species of Ascaris on red blood cells. Flury (1912),
on the other hand, records the presence of strong hemolysins in the coelomic fluid of
species of Ascaris. Flury ascribes the hemolytic action of Ascaris secretions to free
fatty acids, of which oleic acid is the most active principle. In the course of his
studies on the pharmacology of salt-solution extracts of worms of the genus Ascaris,
Brinda (1914) found that injection of the extracts into guinea pigs brings about a
reduction in the number of erythrocytes and a diminution in the hemoglobin content
of the blood. Recently Shimamura and Fujii {1917), in the course of their investiga-
tions on "askaron," a toxic constituent of worms of the genus Ascaris, state that
ether-soluble and alcohol-soluble fractions of Ascaris material contain a hemolytic
agent. The present writer (Schwartz, 1919), in a preliminary paper on the hemolytic
effects of Ascaris extracts, has briefly described the properties of the hemolysin.

A number of investigators have found, moreover, that the coelomic fluid of worms
belonging to the genus Ascaris contains a substance that inhibits the coagulation of
blood. Weil and Boye {1910) found that as a result of injecting the fluid of Ascaris
equorum into rabbits the blood of the latter when drawn remains uncoagulated for 20
minutes longer than blood from a normal rabbit. Experiments with rabbit blood and
Ascaris fluid in vitro yielded negative results, according to these investigators. Leroy
{1910) likewise observed that the blood of dogs which had been injected with the body
fluid of A. equorum coagulated more slowly than blood from normal dogs. Flury
{1912) observed that Ascaris fluid delayed the coagulation of dog blood and of human
blood in vitro. That Loeb and Smith {1904) failed to observe anticoagulins in extracts
of dog ascarids that are active in vitro has already been mentioned.

Worms belonging to the genus Strongylus (frequently referred to as Sclerostomum)
are parasitic in the large intestine of horses. These nematodes attack the mucosa, to
which they may be found adhering by means of their buccal capsule. In view of the
fact that these parasites somewhat resemble hookworms in their attacks on the intes-
tinal mucosa and in the effects which they produce on the host, Weinberg (1907)
investigated their hemotoxic .secretions primarily with a view of throwing light on the
causes of anemia due to hookworms. This investigator found that physiological salt-
solution extracts of freshly collected vStrongylus material dissolves erythrocytes of
horses, cattle, sheep, rabbits, and guinea pigs. The parasites secrete, therefore, a
nonspecific hemolysin. Weinberg determined that the hemolysin is thermostabile,
resisting heat at a temperature of 115° to 120° C. for 15 to 20 minutes. In addition to
the hemolysin, Weinberg found that these parasites secrete a substance which inhibits
the coagulation of horse blood in vitro. He also found that salt-solution extracts of
worms of the genus Strongylus contain a substance which when brought in contact
with the blood serum of the horse causes the formation of a precipitate. The precipi-
tin, too, is nonspecific in its action, since it was found by Weinberg that it produces a
precipitate when added to rabbit-blood serum.

Bondouy {1908, 1910) studied the chemical composition of worms belonging to the
genus Strongylus, with special reference to their hemolytic constituents, and con-
firmed in the main the results obtained by Weinberg as regards the presence of a soluble
hemolysin in these parasites. The new facts discovered by Bondouy may be briefly
summarized as follows: The parasite contains soaps and free fatty acids which exert a
destructive effect on red blood cells in vitro. Bondouy states, however, that the

Nov. 19. 1921 Hemotoxins from Parasitic Worms 387

presence of these substances in the parasite is due to its blood sucking habit, basing
his assertion on the fact that blood serum contains neutral fats, fatty acids, and soaps.
This writer found a lipolytic enzym in worms of the genus Strongylus which apparently
converts the storage fat into fatty acid. It is of interest to note also that Bondouy
found neither lecithin nor cholesterin in the parasite. Lecithin, as is known, has the
property of activating certain hemolytic agents, namely, snake venoms, whereas
cholesterin inhibits hemolysis of blood by active hemolysins. Contrary to Weinberg's
experience (Weinberg, igoy), Bondouy found that Strongylus hemolysin is soluble in
alcohol. From the alcohol-soluble fraction of the parasite this writer isolated an
extremely active hemolysin which he identified as an alkaloid. He also found a
ptomain in the parasites which exhibited hemolytic properties.

Brumpt and Joyeux (quoted by Brumpt, ipio) found that a watery extract of the
stomach worm of sheep (Haemonchus contortus) produced a slight hemolytic effect ^
after 2}^ hours and a total hemolysis after 12 hours. Cuill6, Marotel, and Panisset
(iQii) state that extracts of sheep strongyles (species, of which apparently several
were involved, not given) did not exert any effect on sheep red blood corpuscles from
either healthy or sick animals. These writers also state that extracts of these parasites
contained hemoglobin.

According to Garin {1913) Graphidium sirigosum and Trichostrongylus retortaeformis,
nematodes parasitic in the stomachs of hares and rabbits, secrete hemolysins. With
reference to the hemolysin of G. strigosuvi, Garin found that it is secreted by the living
worm in vitro. He found, furthermore, that the hemolysin is apparently a complex
substance and acts on the blood not directly but in combination with complement.
Heating at 55° C. for 30 minutes does not destroy but merely inactivates the hemolysin,
which may be reactivated by normal serum, according to this investigator. In view
of the limited number of experiments which Garin performed, his conclusions can be
accepted anly with reservation. The work requires confirmation. As for the hemoly-
sin from T. retortaeformis, Garin found it to be far less potent than that of G. sirigo-
sum. He also states that the hemolysins from the two species have far greater affinity
for the blood cells of rabbits than for those of other species of animals and are therefore
relatively specific.

Yagi {1910) found that salt-solution extracts of the blood fluke. Schistosoma japoni-
cum, are hemolytic to erythrocytes of cattle, sheep, and rabbits. He found, fiuther-
more, that this hemolysin is soluble in ether and concluded that it is probably a fatty
acid. Yoshimura (191 3) experimented with salt-solution extracts of the same species
and found them to be destructive to rabbit erythrocytes. Human blood cells, accord-
ing to this writer, are refractory to these extracts. Yoshimura also experimented
with ether extracts, which he found destructive to rabbit red blood corpuscles and
to a lesser extent destructive to human red blood corpuscles.

According to Guerrini (1908), Fasciola hepaiica secretes a hemolysin which is ab-
sorbed by the host and is demonstrable in the blood serum of the latter.

Alessandrini (1913) records the results of experiments with extracts of Macracantho-
rhynchus hirudinaceus, the thorn-headed worm of the hog. He tested the body fluid
and extracts of various parts of the worm and found them to be destructive to the red
blood cells of swine, cattle, and sheep. Alessandrini states that the hemolysin from
M. hirudinaceus is a colloidal substance insoluble in alcohol, soluble in water, and highly
sensitive to heat, since a temperature of 40° C. diminished its potency and a tempera-
ture of 515° destroyed it entirely.

Although the larvae of species of Gastrophilus which occur in the stomach of the
horse are in a zoological sense not parasitic worms, the results of a study of their toxic
secretions may be included in this review because these larval parasites are biologically
more closely related to parasitic worms than they are to free-living insect larvae. At
any rate their secretions may be absorbed by the host and give rise to disturbances

• No details are given as to kind and quantity of blood corouscles used.

388 Journal of Agricultural Research voi. xxn. No. 8

similar to those produced by the secretions of helminths. Weinberg (1908) investi-
gated the hemotoxic properties of the fluid of these parasites and obtained the following
results: Extracts of the intestine and of the red cells of the fatty bodies of the larvae
contain a soluble hemolysin, nonspecific in its action and susceptible to heating for
}4 hour at 56° C. , which does not destroy it but merely weakens its potency. Weinberg
found, moreover, that these extracts have an inhibiting action on the coagulation of
the blood of several species of animals.

SUMMARY

Summarizing the results of hitherto recorded investigations on hemo-
toxins from parasitic worms, it may be stated that while there is more
or less contradictory evidence in the literature the following facts have
apparently been established:

1. Certain parasitic worms secrete substances that affect the blood
of their host deleteriously. These substances, which may be designated
as hemotoxins, are in general nonspecific in the sense that they are also
active toward blood of animals other than their normal host.

2. Diphylloboihriu7n latum, a tapeworm which is known to cause severe
anemia, contains a hemolytic agent. It appears questionable that this
agent is oleic acid, as claimed by Faust.

3. Concerning hemolysins in cestodes other than DiphyUobothrium
latum no definite conclusions can be drawn from the literature on the
subject, but that hemolysins are present in several species appears
probable.

4. Schistosoma japonictmi secretes an ether-soluble hemolysin.

5. Hookworms (Ancylostoma and Necator) secrete a hemolysin and
an anticoagulin.

6. Whipworms {Trichuris trichiura) apparently secrete a hemolysin.

7. Worms belonging to the genus Ascaris contain a hemolysin which is
closely bound to the tissues of the worms and is therefore but slightly
soluble in water, which fact accounts for the negative results obtained
by certain investigators. These parasites also appear to secrete a feeble
anticoagulin.

8. Worms of the genus Strongylus secrete a hemolysin and an anti-
coagulin. The hemolytic principle of these parasites is apparently an
alkaloid, although other substances found in them show hemolytic power.

9. Haemonchus contortus apparently secretes a weak hemolysin.

10. Extracts of Macracanthorhynchus hirvdinaceus are apparently de-
structive to erythrocytes.

1 1 . Hemolytic and anticoagulating properties are found in extracts of
the larvae of species of Gastrophilus.

12. Hemolytic substances from parasites are soluble in alcohol* and
ether, thus resembling lipoids.

13. With respect to their resistance to heat, hemolysins from animal
parasites vary, but in general they are thermostabile.

' According to Alessandrini the hemolysin lxoraMacracanthorhynchushirudinaceusKms.o\nh\& in alcohol.

Nov. 19, 1921 Hemotoxins from Parasitic Worms 389

Owing to the fact that the direct abstraction of. blood by parasites
appears to be inadequate as an explanation of the causes of anemia in
parasitic diseases, and in view of the fact that in tapeworm infections
which are accompanied by anemia due entirely to the presence of the
parasites the direct abstraction theory is inapplicable, the view that
hemolysins from parasites are of etiological significance in parasitic
diseases appeared to be entirely justified.

III. TECHNIC

Unless otherwise indicated, the experiments described in the following
pages were performed with washed red blood cells. In most cases the
blood was defibrinated, filtered through gauze, centrifuged to remove the
serum, and washed in physiological salt solution at least three times to
free it from traces of serum. In a few cases a somewhat different pro-
cedure was followed. The blood was collected in a 2 per cent solution of
sodium citrate or in physiological salt solution containing i per cent
sodium citrate. The removal of the serum and subsequent washing in
physiological salt solution were carried out as in the case of defibrinated
blood. Unless otherwise stated, a 5 per cent suspension of corpuscles,
made by suspending i part of washed red blood corpuscles in 19 parts of
physiological salt solution, was used.

Blood serum used in these experiments was obtained as follows: In
the case of rabbits blood was obtained by severing the marginal ear
vein, and in the case of the larger domestic animals it was obtained at an
abattoir from animals that were being bled and was allowed to drop
into a sterile centrifuge tube. The tube containing the blood was
allowed to remain at room temperature for a few hours. By means of
a sterile platinum wire the clot was loosened from the sides of the tube
to which it adhered and the tube was then centrifuged. The clear
serum was pipetted off, and if the serum was to be kept for more than
three days sufficient phenol was added to give a phenol content of 0.25
to 0.5 per cent; otherwise no preservative was added.

Extracts of parasites were made from fresh material and from dried
material. In both cases the living specimens were obtained shortly after
they had been removed from the host. Certain writers who deny the
presence of toxic substances in parasitic worms base their objection to
the evidence in favor of the view that parasitic worms secrete toxic
substances on the grounds that extracts are frequently made from para-
sites that are obtained as a result of anthelmintic medication and that
the toxicity may be due to traces of anthelmintic which adhere to the
surface of the parasite or to secondary degeneration products of dead
worms. The present writer has been careful to use fresh specimens in
order to avoid complications of the sort just mentioned. It should also
be stated that the parasites obtained from the intestines and other organs
were washed in physiological salt solution and were transferred tliree or

390 Journal of Agricultural Research voi. xxnNo, 8.

four times in succession to fresh salt solution. In this manner the sur-
face of the worms was freed from adhering intestinal material. In the
case of salt-solution extracts that were allowed to remain at room tem-
perature or in an incubator for several hours or for a few days, a pre-
servative, usually a few drops of chloroform, was added to the extract
to inhibit bacterial growth.

Specimens were dried as follows: After having been washed a number
of times in physiological salt solution, the surface of the worms was
dried with filter paper. The specimens were then placed in a single
layer in a glass dish and allowed to dry either at room temperature in
an incubator or in vacuum over sulphuric acid. Small worms dry in a
few hours, even at room temperature, and become sufficiently crisp to
be pulverized. Larger specimens dry more slowly and are usually crisp
in about 48 hours. The dried material was triturated in a mortar and
stored in bottles, usually in a dark place.

Special points in technic are covered in connection with the different
series of experiments and are not taken up in this connection.

As used in this paper, the terms physiological salt solution and salt
solution refer to an 0.85 per cent solution of sodium chlorid in distilled
water.

Controls on all samples of blood corpuscles used in the experimental
work described in the following pages were maintained in connection
with each experiment or series of experiments.

IV. EXPERIMENTS WITH HEMOLYTIC EXTRACTS OF ASCARIS I.UM-

BRICOIDES

I. METHOD OP OBTAINING FI,UID FROM WORMS

Body fluids and extracts were obtained from specimens of Ascaris
lumbricoides from swine. A supply of these parasites is available in
abattoirs during all seasons of the year.

The fluid which is present in the body of the worms was usually
obtained by cutting off the posterior end of medium-sized to large-sized
specimens and allowing the pinkish liquid to drop into a test tube.
Fluid obtained in this manner does not keep well and is available only
for immediate use. Allowed to stand, even at a low temperature, the
body fluid thus collected undergoes bacterial decomposition in about 24
to 36 hours. Weinberg and Julien (1911) describe a method of col-
lecting Ascaris body fluid under aseptic precautions. Briefly, the
method consists in drying the worms with filter paper, holding the ends
of each specimen and passing the middle region of the worm through the
flame of a Bunsen burner until the cuticle bursts. The first two or three
drops of fluid which ooze out are discarded and the remaining fluid is
allowed to drop into a sterile tube. This procedure was tested by the
present writer with inconstant results so far as the keeping qualities of

Nov. 19. I92I Hemotoxins from Parasitic Worms 391

the fluid were concerned. In some cases sterile fluid was obtained in
this manner, but more often the fluid became contaminated. The con-
tamination was extraneous and not inherent in the body fluid of the
worms, since a number of experiments performed by the writer showed
quite conclusively that the intact body fluid of Ascaris is sterile.

In the course of the experimental work described in this paper speci-
mens were kept alive in vitro for a few days. This necessitated infor-
mation as to the conditions that are favorable to the survival of the
parasites outside of the host. The customary procedure of keeping
parasitic worms at a low temperature is not applicable to Ascaris lumbri-
coides when considerable periods, generally in excess of 24 hours, are
involved. Incubator temperatures (37.5° C.) are more favorable than
refrigerator temperatures, but so far as longevity of the worms outside
the host is concerned, a temperature ranging from above 25° to 32° was
found to be the most favorable. The worms were kept in shallow dishes
and in beakers, and sufficient salt solution was added to cover the worms.
Fluid from worms that had thus been subjected to starvation was
obtained in the same manner as fluid from fresh worms.

2. EXPEIRIM^NTS WITH TH:^ BODY Fl^UID OF ASCARIS I^UMBRICOIDEIS

In nematodes the space between the body wall and the gut wall is filled
with a fluid which in the case of such large-sized worms as those of the
genus Ascaris is available in quantities sufficient for investigation.
According to Flury the body fluid of Ascaris equorum consists largely of
water (95 per cent). Other substances present in this fluid, according to
the same investigator, are albumin, globulin, and other proteins, soaps, free
fatty acids, various katabolic products of proteins, purin bases, and their
derivatives, sodium, chlorid and other inorganic substances, as well as
digestive and oxidizing enzyms. Flury found that the body fluid of
A. lumbricoides is physically and chemically indistinguishable from that
of A. equorum.

The fact that the body fluid of Ascaris lumbricoides, which in fresh
specimens has a bright pinkish color, contains oxyhemoglobin is of great
significance. The presence of oxyhemoglobin in the worms may be
readily demonstrated by means of the spectroscope. Schimmelpfennig
{1902) appears to have been the first investigator to note this fact, on the
basis of which he ascribed to worms of the genus Ascaris the r61e of blood-
suckers. This investigator also states that worms belonging to this genus
liberate their oxyhemoglobin content into the physiological salt solution
in which they are kept alive in vitro. The presence of iron granules in the
gut wall of ascarids was affirmed by Askanazy (i8g6) , who bases his view
on positive Berlin blue tests, the inference being that the pigment in
question is obtained from the blood of the host. Flury (19 12) refers to
the presence of hemoglobin in ascarids and states that he observed it in
worms which had been kept for two weeks in an incubator. Flury

392 Journal of Agricultural Research voi. xxii, no. s

inclines to the view that oxyhemoglobin is a normal constituent of these
worms. Dobemecker {191 2) records the presence of oxyhemoglobin in
ascarids, which he determined by means of the spectroscope. Faure-
Fremiet {1913) expresses the view that the oxyhemoglobin present in
the intestine of worms belonging to the genus Ascaris is obtained from the
blood of the host and that the iron pigment in the intestinal cells is
derived from disintegration products of hemoglobin. Galli-Valerio
{191 5) affirms the presence of blood in ascarids and states that the body
fluid of a female ascarid gave a positive benzidin test for blood. The
present writer (Schwartz, 191 9) found that Ascaris lumbricoides loses its
oxyhemoglobin when kept in vitro for a number of days and that coinci-
dent with the loss of this substance the worms become sluggish and die.
Magath (1919) has made a similar observation in the case of another
nematode (CamaUanus americanus) which contains a "reddish fluid."
Magath also notes the presence of pigment granules in the gut wall of
this worm.

It has already been stated in another section of this paper that Schim-
melpfennig {1902) and Flury (191 2) found that the body fluid of worms
belonging to the genus Ascaris is destructive to red blood cells. Fol-
lowing are the observations and experiments of the present writer on this
question.

Fluid collected from fresh specimens of Ascaris lumbricoides within 24
hours after removing the parasites from the host is not hemolytic.
Such fluid was tested on the washed erythrocytes of cattle, sheep, hog,
rabbit, and guinea pig without producing any appreciable dissolving
action. In one case it was found tliat fluid which had been kept in a
refrigerator for three days was destructive to sheep erythrocytes, but a
repetition of this experiment with fluid from another lot of worms yielded
negative results. Fluid collected under aseptic precautions and kept
in a refrigerator for two or three days failed to hemolyze red blood
corpuscles.

On the other hand, fluid from worms which had been kept alive in
vitro for a number of days was found to be hemolytic. In one case
worms were kept alive in a physiological salt solution for eight days at a
temperature of 32° to 33° C. At the end of this period fluid was obtained
from the worms and tested on the washed red blood cells of the hog, with
positive results. A repetition of this experiment on a different sample of
washed erythrocytes from the hog likewise yielded positive results. In
another case worms which were kept alive for six days yielded a fluid
which was destructive to washed sheep corpuscles. Fluid from another
lot of worms which had been kept in the laboratory for four days was but
slightly although quite unmistakably hemolytic to sheep blood cor-
puscles. A portion of this fluid was boiled and the clear liquid after
being separated from the coagulum was still hemol)rtic. Fluid from

Nov. 19, 1921 Hemotoxins from Parasitic Worms 393

worms which has been kept ahve for eight days was strongly hemolytic
to washed sheep blood corpuscles.

In the course of these experiments it was observed that whereas fresh
specimens of Ascaris lumbricoides from swine are pink in appearance
they become white as they are kept in the laboratory. Spectroscopic
examination of the fluid showed that the pink appearance is correlated
with the presence of oxyhemoglobin and the white appearance is corre-
lated with the absence of that substance. In other words, worms kept
in vitro lose their oxyhemoglobin, a fact which appears to indicate that
this substance is not a constant constituent of the worm but that it is
obtained from the host, the supply evidently being renewed from time
to time. Inasmuch as Schimmelpfennig {1902) states that the oxyhemo-
globin is eliminated in vitro, the present writer made spectroscopic
examinations of physiological salt solution in which ascarids had been
kept alive for 24 hours or longer, and found that such solutions did not
show the oxyhemoglobin spectrum. Tests for iron in such salt solutions
showed but slight traces of this substance. That these traces were
excretion products of the parasite was shown by the fact that a quantity
of salt solution from the same supply which was added to the beakers in
which the worms were kept gave negative results. It may be concluded,
therefore, that when removed from the host and kept in a physiological
salt solution living ascarids lose their oxyhemoglobin content not by
excreting it as such but probably by breaking it down into simpler sub-
stances and storing the iron in their tissues. The fact that ascarids are
rich in iron and that this substance enters in considerable quantities into
the composition of the eggs (Schimmelpfennig, igos) is decidedly signifi-
cant in this connection.

On the basis of certain experiments Flury (191 2) states that salt
solutions in which living ascarids have been kept for 24 hours have
absorbed the hemolysin which the parasites excrete. The observations
of the present writer on this point do not bear out Flury's view, as the
following experiments will show.

A number of swine ascarids were kept in a beaker for 24 hours in a
quantity of physiological salt solution sufficient to cover the worms.
Ten cc. of this salt solution produced no dissolving effect on i cc. of a 5
per cent suspension of guinea-pig red blood corpuscles. A similar experi-
ment was performed with a different lot of worms, the salt solution in
this case being tested on washed hog erythrocytes, with negative results.
Negative results on sheep erythrocytes were also obtained with salt
solution in which another lot of worms had been kept for 24 hours. In
a similar way negative results were obtained on several other occasions
with salt solution in which living ascarids had remained from 18 to 36
hours.

In the experiments mentioned above the parasites were examined and
found to be still alive before the salt solution was tested as to its hemolytic

394 Journal of Agricultural Research voi. xxii. No. s

property. In another series of experiments in which some of the worms
were found to be dead it was observed that the salt solution in which
they had been kept was destructive to red blood corpuscles. That the
hemolytic effects of salt solution in which dead ascarids had been kept was
independent of bacteria was shown by the fact that the salt solution
was free from putrefactive odors associated with decay, due to the pre-
cautions which were taken to free the parasites from bacteria by immers-
ing them in 2 per cent formalin and washing them first in water and then
in salt solution before subjecting them to these experiments. In one
experiment which was conducted under strictly aseptic precautions the
worms were thoroughly washed in running water, in formalin, and in
sterile salt solution in the order indicated and then placed in sterile flasks
containing an 0.85 per cent solution of sodium chlorid. These flasks
were placed in an incubator at 37° C. for several days. The worms died,
but the fluid showed no cloudiness. Transfers of portions of this fluid
to culture media (nutrient broth and agar) failed to produce bacterial
growth despite the fact that the tubes containing the media were kept
in the incubator for a week. The sterile salt solution in which the
ascarids died was hemolytic to washed sheep corpuscles.

These facts appear to indicate that Ascaris hemolysin is closely bound
to the cells of the parasites and becomes dissociated from them rather
easily after death of the worms, a view which is in harmony with the
observation of Tallqvist (1907) with reference to the hemolysin from
Diphyllohothrium latum,. The fact that the body fluid of worms which
have been kept in vitro for a number of days becomes hemolytic is entirely
in harmony with that view, since, under conditions of starvation, autolysis
of the tissues of the parasites undoubtedly takes place, especially after
the storage products, largely glycogen,^ are consumed.

3. expe;rime;nts on the; possibi^e; presence of complement in the

BODY FEUIDS of ASCARIS EUMBRICOIDES

Experiments with body fluid from fresh specimens of Ascaris lumhri-
coides were performed with a view to determining whether it contains a
substance capable of activating a hemolytic system. As is well known,
washed red blood corpuscles to which a specific inactivated antiserum is
added will not hemolyze unless a certain quantity of normal fresh blood
serum is added. The substance in the normal blood serum which in
itself has no hemolytic power but which activates inactivated antiserum
is known as alexin or complement. Comparatively little is known of
this body except that it is a normal constituent of blood serum, that it
deteriorates rapidly in vitro, and that it is destroyed by heating at 56° C.
for 30 minutes. According to Noguchi (1907), soluble soaps to which

' Schulte and Krummacher(79/5) have showrl that starving ascarids do not consume their fat content
and have confirmed Weinland's views with reference to the role of glycogen in the metabolism of the worms
in vitro.

Nov. 19. 1921 Hemotoxins from Parasitic Worms 395

inactivated serum is added act as complement; in other words, a mixture
of inactivated serum and soap can activate a hemolytic system (washed
red blood corpuscles plus specific antiserum) .

The present writer endeavored to answer the following questions:
Is the fresh body fluid of Ascaris lumbricoides , which, as has already been
shown, has no hemolytic power, capable of activating a hemolytic sys-
tem? In dther words, does it contain complement? Second, can a
combination of inactivated serum and an alcoholic extract of body sub-
stance of A. lumbricoides from which the ether-soluble fraction has been
removed, and which contains whatever soluble soaps the parasite has,^
activate a hemolytic system? The answers to these questions will be
found in the results of the following experiments.

One cc. of washed sheep red blood corpuscles was mixed with a unit
of specific inactivated antiserum (amboceptor) determined by previous
titration. To one tube containing this mixture a certain quantity of
fresh guinea-pig serum (complement) was added, sufficient to activate
the amboceptor — that is, to cause it to combine with the blood corpuscles
and to produce hemolysis. The quantity of complement necessary to
activate the hemolytic system was determined by previous titration.
Hemolysis was produced in 30 minutes at 37° C. To a series of 10 tubes
containing the mixture of amboceptor and sheep red blood corpuscles
various quantities of body fluid collected from living swine ascarids under
aseptic precautions shortly after the worms had been removed from their
hosts were added. The quantities of fluid added to these tubes ranged
from 0.1 cc. to 10 cc. These tubes were shaken and incubated at
37° C. for one hour. No hemolysis was observed in any tube. The
tubes were then put in a refrigerator for 20 hours longer, but the blood
corpuscles remained intact. It should be stated in this connection that
the body fluid in question was free from bacteria, since a portion of it was
thoroughly mixed with melted agar which was plated and incubated.
The plates remained sterile. Ascaris fluid lacks, therefore, a substance
(complement) which is capable of activating a hemolytic system.

As to the combination of inactivated normal serum with an alcoholic
extract of Ascaris lumbricoides, the following experiment was performed:
Dried ascarids were powdered, extracted in warm alcohol, and the alco-
holic extract after evaporating the alcohol was washed with ether. The
ether, as is known, removes neutral fats, fatty acids, lecithin, cholesterin,
and other lipoids. The ether-insoluble substance was then dissolved in
salt solution and combined with normal guinea-pig serum that had been
heated to 51° C. to determine whether this combination can act as com-
plement, that is, whether it can activate a hemolytic system.^ To one

^ The presence of soaps in ascarids is affirmed by Flury (.1Q12).

' According to Noguchi, similar chemical fractions of mammalian tissues combined with inactivated
normal serum act as complement.

70495°— 21 2

396 Journal of Agricultural Research voi. xxii, no. s

tube containing i cc. of a mixture of washed sheep red blood corpuscles
and specific antiserum in the proper proportion as determined by previous
titration, one unit of normal guinea-pig sei'um (complement) was added.
(The unit of complement was determined by titration.) Hemolysis
resulted. To a second tube containing a mixture of washed sheep red
blood cells and spscific antiserum one unit of inactivated complement
(heated to 51°) was added. No hemolysis resulted. To a series of
tubes containing washed sheep red blood corpuscles and specific anti-
serum various combinations of inactivated guinea-pig complement and
alcoholic extract of A. lumhricoidcs were added. No hemolysis was
produced in any of these tubes. It is evident, therefore, that A. lum-
hricoides not only lacks complement but that an alcoholic extract of
the worm freed from all ether-soluble substances combined with inacti-
vated normal serum can not act as complement.

In this connection it is of interest to note that Holland (19 19) found
that the blood of insects lacks complement and that this substance is
also absent from the blood of mollusks. Cantacuzene {1919) examined
the fluids of a number of invertebrates as well as of tunicates but failed
to find complement. He succeeded, however, in producing complement
in a crab (a species of Eupagurus) by artificial immunization with sheep
red blood corpuscles.

Summarizing, Ascaris lumbricoides in common with other inverte-
brates lacks complement, a substance that is known to play an important
role in the immunity processes of higher vertebrates. That A. lumbri-
coides and other internal parasites which live in parts of the body where
bacteria are more or less abundant protect themselves against bacterial
invasion is probable. The intestine of A. lumbricoides contains bacteria,
as has been recorded by several investigators. The present writer found
bacteria in the intestine, but the body fluid of fresh ascarids when col-
lected under aseptic precautions was found to be sterile. That the body
fluid and tissue extracts of ascarids and of other internal parasites con-
tain bactericidal substances has been afiirmed by a number of writers
(Alessandrini, 191 3).

4. EXPERIMENTS WITH EXTRACTS OE ENTIRE WORMS

It has already been stated that Weinberg {1907), Whipple (1909), and
Alessandrini (1913) failed to find hemolysins in salt-solution extracts of
ascarids. Garin {1913) records the results of 10 experiments with extract
of worms of the genus Belascaris, of which 8 yielded negative results and 2
yielded positive results on dog-blood corpuscles. These investigators
experimented with extracts of fresh specimens made by macerating the
worm material in physiological salt solutions. The present writer found
that as a result of extracting Ascaris lumbricoides material by macerating
fresh worm substance in salt solutions the hemolysin is seldom liberated

Nov. 19. 1921 Hemotoxins from Parasitic Worms 397

from the tissues of worms. Better results were obtained by grinding up
fresh worm material with sand and shaking the mixture of worm frag-
ments and sand for a number of hours, followed by extraction in an
incubator for a number of days. This procedure necessitated the addi-
tion of a preservative to the extract in order to prevent bacterial contam-
ination. In experiments in which this procedure was followed, sufficient
carbolic acid was added to make a 0.25 per cent solution; and in hemo-
lytic tests controls involving the use of salt solution containing a similar
quantity of carbolic acid were included. F'ollowing the procedure
described above an extract of fresh worm material was made as follows :
A few pieces (10 gm. by weight) of worm material from a number of dif-
ferent specimens were ground up with sand and suspended in 100 cc. of
physiological salt solution containing 0.25 per cent of phenol. The
mixture was shaken for a few hours in a shaking machine and then in-
cubated, usually for three days, at 37° C. The extract was then filtered
and a clear filtrate tested on various samples of red blood corpuscles as
follows.

The filtrate was tested on washed erythrocytes of a number of cattle,
sheep, hogs, rabbits, guinea pigs, and rats, with positive results. In
most experiments it was found that 0.4 cc. of the extract hemolyzed
I cc. of a 5 per cent suspension of red blood corpuscles. In a number
of tests 0.2 cc. of the extract hemolyzed i cc. of the suspension of cor-
puscles. As a control on the phenol which was added as a preservative,
0.5 cc. and i cc. of a salt solution containing }4 per cent of phenol was
tested on each sample of blood corpuscles used in the hemolytic tests,
with negative results. Tests to determine whether normal serum con-
tains antibodies were nearly always positive. From 0.2 to 0.5 cc. of
serum was sufficient to inhibit hemolysis of i cc. of corpuscle suspension
by from 0.2 to 0.4 cc. of the extract. Sometimes o.i cc. of serum brought
about the same results.

That the activity of the hemolysis is independent of the acidity of the
solution was shown by the fact that as a result of neutralizing the extract
its activity was not destroyed. Furthermore, the hemolytic potency
of the extract was not due to secondary degeneration products asso-
ciated with acid production, because the hemolytic power of the extracts
remained intact for a long period (several months), during which it was
tested from time to time against different species of corpuscles. More-
over, filtrates of extracts of worms that were prepared by thoroughly
triturating the specimens and adding a few drops of chloroform to inhibit
bacterial growth during the few hours that the extracts were kept in
a refrigerator were found to be hemolytic. An example of the results
of experiments with salt-solution extracts of A scar is lumhricoides on
red blood ceils is given in Table I, in which a few experiments are sum-
marized.

398

Journal of Agricultural Research voi. xxii. No. a

Table 1.— Effect of salt-solution extract of Ascaris lumbricoides on red blood corpuscles "

Kind of erythrocytes.*'

Quantity of extract. <=

Results after
two hours at

37° C.

Guinea pig.

Do...

Rat e

Do...

Do...
Hog/

Do....

O. I cc

. 2 CC.<*

Salt solution

. I cc. Hjoiled).

. 2 cc. (boiled).

. I cc

. 2 cc

. .^ cc

Salt solution <^

. 4 cc

Salt solution d.

Cattle / .
Do.
Do.

. 4 cc. . . .
Salt solution <'.

+ + +

+ +

+ + +

+
+ + +

+ + +

a — indicates total absence of hemolysis. + indicates slight hemolysis. + + indicates marked but incom-
plete hemolysis. + + + indicates complete hemolysis.

f> One cc. of a 5 per cent suspension of defibrinated blood washed three times in physiological salt solu-
tion was used in experiments.

c The extract used in these experiments was made by suspending lo gm. of fresh worm material in loo
cc. of 0.85 per cent NaCl.

d Two controls — 0.5 cc. and i cc. of salt solution containing 0.5 per cent phenol were tested on i cc. of the
suspension of corpuscles.

« Pooled blood from six rats.

/ Four samples of corpuscles were tested.

5. EXPERIMENTS WITH ASCARIS LUMBRICOIDES POWDER

The hemolytic principle of Ascaris lumbricoides may be preserved by
drying the parasites. Specimens collected at a local abattoir were
washed in salt solution to remove adhering interstinal debris, dried
superficially with filter paper, and then placed in vacuum over sul-
phuric acid. When the specimens were sufficiently crisp they were
powdered in a mortar and stored for future use. Ascaris lumbricoides
powder when added to a suspension of washed blood cells of cattle,
sheep, swine, etc., produces rapid hemolysis. As ip the case of extracts
of the parasite, the hemolytic action is inhibited by normal serum. The
hemolytic substance may be more easily obtained from dried than from
fresh ascarids by extracting the worm material in physiological salt
solution. This is no doubt due to the fact that the dried material can
be readily crushed and the hemolytic substance which, as has already
been indicated, is rather closely bound to the parasite, may be more
readily liberated. The following experiments performed by the writer
illustrate this point: Several swine ascarids were broken tip into small
fragments but were not powdered in a mortar. A portion of this ma-
terial was extracted in salt solution for a few hours and filtered. The
filtrate was tested on washed sheep corpuscles with negative results.
The remaining portion of dried worm material was thoroughly ground

Nov. 19, 192 1

Hemotoxins from Parasitic Worms

399

in a mortar, extracted in physiological salt solution, filtered, and the
filtrate tested on sheep corpuscles. The results in this case were positive.

A number of experiments were made with salt-solution extracts of
powdered Ascaris lumbricoides. Rabbit and sheep corpuscles were
used in nearly all experiments with these extracts. The results of these
experiments were positive when the extracts were made from thoroughly
powdered material; otherwise the extracts were only slightly hemolytic.

Extracts of powdered material of Ascaris hmihricoides were usually
prepared as follows: A definite quantity of powder was added to a
definite volume of physiological salt solution in a flask, the latter was
shaken thoroughly, and the material was extracted for a few hours to
two days in a refrigerator without the addition of any preservative, or
extracted in an incubator, in which case a few drops of chloroform were
added. The mixtures were then filtered, and in cases in which chloro-
form had been added the filtrate was left in an open receptacle in order
to get rid of the chloroform by evaporation. The salt-solution filtrates
were then tested as to their hemolytic power.

An example of results of these experiments is given in Table II, in
which a number of tests are summarized.

Table II. — Effects of salt-solution extracts of powdered Ascaris lumbricoides on red blood

corpuscles «

Experi-
ment
No.

Kind of
erythrocytes.''

Rabbit

...do

•3.

...do

Sheep

...do

... do

...do

Quantity of extract.

5 drops

8 drops

8 drops (boiled)

10 drops (boiled)

10 drops (heated at 60° C. 30 minutes)

xo drops salt solution

8 drops

10 drops

10 drops salt solution

8 drops

10 drops

10 drops salt solution

Results

after 2 hours

at 37° C.

+ -H +

+ +
+ + +

+ + +

+
+ +

Results

after 20
hours.d

+ + +
+ + +

+ + +

1— indicates negative results. + indicates slight hemolysis. ++ indicates marked but incomplete
hemolysis. + + + indicates complete hemolysis.

^ Five drops of a s per cent suspension of washed rabbit erythrocytes and a 3 per cent suspension of
washed sheep erythrocytes were used in these experiments.

c In experiments i to 8, inclusive, the following extract was used; 0.85 gm. of powder were suspended in
85 cc. of salt solution and extracted in an incubator for 24 hours. In experiments 10 to 12 the extraction
was made as follows: i gm. of powder was extracted in 10 cc. of salt solution in a refrigerator.

<* After remaining in an incubator for 2 hours the tubes containing the corpuscles and extracts were in
some instances transferred to a refrigerator (8° C.) where they were kept for 18 hours longer before the
final reading was taken.

400 Journal of Agricultural Research voi. xxn. no. 8

6. EXPERIMENTS WITH EXTRACTS OF DIFFERENT ORGANS OF ASCARIS

I^UMBRICOIDES

It has already been stated that the body fluid of fresh specimens of
Ascaris lumbricoides is not hemolytic and that this fluid acquires
hemolytic properties as the parasites are kept in vitro. Extracts of
entire worms, on the other hand, were found to contain a hemolytic
substance which is apparently firmly bound to the tissues of the parasite.
These facts appear to indicate that the hemolytic substance is liberated
in rather small quantities and that it ultimately finds its way into the
body fluid. That the liberation of hemolysin from the tissues and cells
of the parasite is associated with metabolic processes of the worms is
advanced as a plausible explanation of the facts. In the host animal
the body fluid of the worm contains blood and blood products by which
the hemolysin is apparently neutralized. In vitro, on the other hand,
the blood elements disappear, as judged by the disappearance of
oxyhemoglobin ; and meanwhile fresh hemolysin which has found its way
into the fluid remains unbound.

The question as to which morphological elements of Ascaris lumbri-
coides secrete the hemolytic substance or substances is interesting. A
number of specimens of the parasite were therefore dissected and the
intestine, reproductive organs, and body wall were separated into
different lots. Physiological salt-solution extracts from each lot were
tested on hog blood, and in a few cases on sheep blood.

In one series of experiments it was found that the extracts of the
intestine were strongly hemolytic, whereas extracts of the body wall
showed no hemolytic eftects. Extracts of the reproductive organs were
moderately hemolytic. In a second series of experiments extracts of the
intestine were found to be very markedly hemolytic, whereas extracts
of the body wall and reproductive organs showed weak hemolytic power.

In another series of experiments a number of worms were dissected,
and the body wall, reproductive organs, and chyle intestine were sepa-
rated into different lots. Each lot was washed in physiological salt
solution and dried with filter paper. The material in each lot was then
put in an incubator at 40° C. and allowed to remain there for 24 hours.
Pulverized material from each lot was then suspended in physiological
salt solution and tested on washed sheep corpuscles. Extract of the
intestine produced rapid hemolysis at 37° (in about i hour), whereas
extract of body wall of approximately the same strength as that of the
intestine produced no hemolysis even after 3 hours at 37° followed
by 18 hours in a refrigerator. Extract of the reproductive organs pro-
duced no hemolysis after 3 hours at 37° but after an additional
period of 18 hours at 8° a slight indication of hemolysis was observed.

Nov. 19, 1921 Hemotoxins from Parasitic Worms 401

It may be concluded, therefore, that the hemolytic agent of Ascaris
lumbricoides is primarily a secretory product of the intestine and that
part of this substance finds its way into the body fluid where it is ap-
parently neutralized by blood elements that are obtained from the
host.

7. EXPERIMENTS WITH DIFFERENT CHEMICAL. FRACTIONS OF ASCARIS

I^UMBRICOIDES

In contrast to the comparatively slight solubility of the hemolytic
substance of Ascaris lumbricoides in physiological salt solution is its
ready solubility in lipoid solvents, especially in alcohol. Equal quanti-
ties of powder were suspended in 5 cc. each of physiological salt solu-
tion, 95 per cent alcohol, ether, and acetone for 48 hours. The filtrates
were evaporated and redissolved in 5 cc. of physiological salt solution.
These extracts were then tested on a 5 per cent suspension of washed
rabbit red blood cells. The alcoholic extract was the most potent
from the point of view of hemolysis. Acetone and ether extracts were
about as potent as the physiological salt-solution extract. In a second
series of experiments in which A. lumbricoides powder was extracted
in the substances referred to above, the extracts were tested on sheep
red blood cells. In those experiments the alcoholic extract was the
most potent, while the physiological salt-solution extract and the ether
extract were the least potent.

Further experiments with different fractions of Ascaris lumbricoides
were performed. Dried worm material was ground up in a mortar and
extracted in four volumes of ether in a flask for 48 hours at 37° C. The
ether was then removed from the worm material and saved and fresh
ether was added to the flask. This was allowed to extract for 24 hours,
the ether being removed at the end of that period and added to the
first ether extract. To the worm material fresh ether was again added,
and after 24 hours of extraction the mixture was filtered. The last
ether filtrate was practically free from any extract, A portion of the
ether extract was then evaporated and a brownish yellow fatty sub-
stance left behind. This substance had the characteristic odor of A.
lumbricoides. A small quantity of this substance was emulsified in
physiological salt solution and tested on washed rabbit blood corpuscles,
which it hemolyzed. A second portion of ether extract in solution was
shaken with an equal quantity of distilled water and allowed to remain
at room temperature for two hours. Two layers — ^namely, an ether
layer (fraction i) and a water layer (fraction 2) — were separated. The
ether layer (fraction i) was evaporated, and a fatty substance was left
behind which was hemolytic to washed sheep corpuscles. A portion
of this substance was redissolved in ether, and to this solution an equal
quantity of a solution of sodium bicarbonate was added and the mix-
ture was thoroughly shaken. The ether layer (fraction la) was removed

462 journal of Agricultural Research voi.xxn.TJo.ft

and evaporated. A fatty substance free from the characteristic odor
of A. lumbricoides was left after evaporating the ether. This substance
had no hemolytic power. Inasmuch as sodium bicarbonate saponified
the free fatty acids in the ether, it is evident that the hemolytic effect
of the ether extract free from the water-soluble fraction is due to fatty
acids. Flury {191 2) , in fact, came to the conclusion that the hemolytic
power of ascarids is to be ascribed to free fatty acids of which the un-
saturated fatty acids are of prime importance. Flury stated further-
more that oleic acid is probably the most active principle of Ascaris
hemolysin because of the known hemolytic powers of this substance.
The watery layer (fraction 2) was opalescent and contained a thick
suspension of a grayish substance which was found to be slightly hemo-
lytic to sheep cells.

The ether extract contains therefore two fractions, (i) a water-insoluble
fraction which consists of neutral fats and fatty acids, and (2) a water-
soluble fraction, both of which are hemolytic. The composition of the
water-soluble substance was not definitely determined. This substance
was tested and found to be soluble in 95 per cent alcohol and in hot and
cold water. By acidifying a watery solution of the substance and shaking
it with an equal volume of ether it was made to go into solution and was
recovered in the ether layer. Another portion of the water-soluble sub-
stance was salted out from water by adding a few drops of a strong solu-
tion of sodium chlorid. It rose to the surface, where it formed a thick
layer which was insoluble in salt solution. Bondouy {1908, 1910), who
experimented with a similar chemical fraction of a species of Strongylus,
identified it as consisting of soluble soaps, substances that are known to
have hemolytic power.

To recapitulate, an ether extract of Ascaris lumbricoides was divided
into the following fractions : ( i ) An ether-soluble and water-insoluble frac-
tion, and (2) a water-soluble fraction. Both fractions were hemolytic,
the latter, however, only to a moderate degree. The fatty acid in the
first fraction (fraction i) was saponified. The fatty acid-free fraction
which was extracted in ether was not hemolytic. This fraction consists
largely of neutral fats. The hemolytic potency of the ether extract of
A. lumbricoides is therefore due largely to free fatty acid. That the
water-soluble part of the ether fraction (fraction 2) is a mixture of soaps
is probable.

A portion of the remaining Ascaris lumbricoides powder (free from
ether-soluble fraction) was extracted in distilled water for 48 hours in an
incubator. The mixture was then filtered. The filtrate had a brownish
color and a sweetish odor. Tests for proteins were positive. The residue
was evaporated at 40° C. A portion of the residue was taken up in salt
solution, to which it gave a yellowish coloration. Tested for its hemo-
lytic power on sheep blood corpuscles, it produced rapid hemolysis. The
remaining portion of the residue was extracted in 95 per cent alcohol for

Nov. 19, 1921 Hemotoxins from Parasitic Worms 403

24 hours. It was only partly soluble. After filtering off the alcohol, fresh
alcohol was added and the extraction continued for 24 hours longer.
The alcoholic extracts were evaporated and the residue was taken up with
a small quantity of physiological salt solution. Tested for its hemolytic
power, the results were strongly positive on sheep erythrocytes. The
alcohol-insoluble fraction was not hemolytic even when large quantities
were employed.

These experiments are rather significant in view of the fact that they
show quite conclusively that the hemolytic potency of Ascaris lum-
hricoides extracts are due not to fatty acids alone but that another sub-
stance or substances, soluble in alcohol and water, must be involved.

The experiments described above were repeated several months later
with similar results.

Extracts of powdered ascarids in 95 per cent alcohol were made by
adding about 6 volumes of alcohol to i volume of powder and removing the
alcohol by filtration at intervals of two to three days and adding fresh
alcohol. After evaporating the filtrates, which were all mixed together,
a brownish residue was left behind which was only partly soluble in ether.
The ether-soluble portion as well as the ether-insoluble portion was
hemolytic. A portion of the powder, free from the alcohol-soluble frac-
tion, was extracted in etlier, but when the latter was removed and evap-
orated no residue was left behind. The remaining portion of the powder
free from the alcohol-soluble portion was extracted in physiological salt
solution, and this extract when tested on red blood cells was found to be
nonhemolytic. These experiments show, therefore, that the hemolytic
substances of Ascaris lumhricoides are all soluble in alcohol, and confirm
the results of the earlier series of experiments with reference to the fact
that the ether-soluble fraction of A . lumhricoides contains but a portion
of the hemolytic substance.

Part of the alcoholic extract was divided into two fractions — namely,
an absolute alcohol-soluble fraction and an absolute alcohol-insoluble
fraction. The latter was hemolytic, whereas the former showed no hemo-
lytic power.

An ether extract of Ascaris lumhricoides powder was redissolved in
ether and divided into two fractions by adding acetone in excess, which
resulted in the formation of a whitish precipitate. The precipitate was
separated from the solution and found to be nonhemolytic. The acetone-
ether solution was evaporated and taken up in salt solution. It was also
found to be nonhemolytic, whereas prior to precipitation with acetone
the ether extract was hemolytic. The precipitate was obtained in quan-
tities insufficient to determine its nature. That it was probably largely
lecithin ^ can hardly be doubted. As is known, lecithin in quantities in
which it alone produces no hemolytic effect can activate other substances
and cause them to produce hemolysis. That this actually occurs in the

' The presence of lecithin in ascarids was demonstrated by Flury Oiii^)-

404 Journal of Agricultural Research voi. xxii, no. 8

case of the ether-soluble hemolytic substance of A . lumhricoides appears
probable from the experiments described above.

It should also be stated that a 95 per cent alcohol extract of Ascaris
lumhricoides developed a precipitate when kept in solution in 95 per
cent alcohol at 8° C. This precipitate went into solution when the
alcohol containing the extract was transferred to room temperature.
The removal of this precipitate by filtering in a refrigerator yielded a
whitish substance which had no hemolytic power, nor did the removal
of this substance interfere with the hemolytic potency of the extract.

8. PROPERTIES OF* ASCARIS lyUMBRICOlDES HEMOI^YSIN

At low temperatures ranging from 6° to 10° C. hemolytic extracts of
Ascaris lumhricoides lose their potency. Mixtures of extracts and sus-
ceptible corpuscles that showed complete hemolysis after 2 hours' incu-
bation at 37° showed no trace of hemolysis after 24 hours at 8°.
After being removed from the low temperatures and transferred to an
incubator hemolysis occurred rapidly in such mixtures.

In order to determine whether the hemolytic substance of Ascaris
lumhricoides is absorbed by the red blood cells at low temperatures the
following experiments were performed.

Mixtures of washed red blood cells (rabbit and sheep) and hemolytic
extracts were put in a refrigerator at 8° C. After 24 hours the super-
natant fluid was removed from the corpuscles and the latter were washed
three times in succession to free them from traces of extracts; to the
washed corpuscles from which the supernatant fluid had been removed
an equal quantity of salt solution was added, and the tubes were thor-
oughly shaken and placed in the incubator. Hemolysis set in slowly.
The supernatant fluid which was removed from the corpuscles was also
tested as to its hemolytic potency, with inconstant results. In some
cases it was found that it had lost its hemolytic potency completely, but
in a number of cases it still retained its blood-destroying power. That
the potency of the fluid that had been in contact with susceptible cor-
puscles had been considerably reduced was evident, since it had but
slight hemolytic power as compared with that of intact extract. Whether
the hemolytic substance in contact with susceptible corpuscles at a low
temperature becomes fixed to the cells or whether it is precipitated at a
low temperature and escapes removal despite repeated washing has not
been determined.

Hemolytic extracts of Ascaris lumhricoides are highly resistant to heat.
Heating at temperatures ranging from 56° to 60° C. for 30 minutes did
not weaken the potency of the extracts. An exposure to 70° for two
hours failed to destroy the hemolytic substance. Salt-solution extract
as well as alcoholic extracts were heated to boiling, and after cooling
they were tested on susceptible red blood cells. It was found that as a

Nov. 19, 1921 Hemotoxins from Parasitic Worms 405

result of boiling the potency of the extracts was weakened but not
destroyed.

The hemolysin goes through the pores of Berkefeld, Chamberland,
and diatomaceous filters. The filtrates are less potent, however, than
nonfiltered solutions.

V. EXPERIMENTS WITH AGGLUTINATING SUBSTANCES FROM
ASCARIS LUMBRICOIDES

In the course of experiments on hemolysis of red blood cells by ex-
tracts of Ascaris luvibricoides it was observed that the cells frequently
became agglutinated before hemolysis set in. The agglutinating effect
of the extracts was especially marked on rabbit red blood cells and was
observed only occasionally on sheep erythrocytes. Several experiments
on hog erythrocytes showed them to be refractory to the agglutinating
substance of the parasite.

The agglutinating property of Ascaris lumhricoides with respect to
rabbit-blood corpuscles was present almost invariably in physiological salt-
solution extracts. Alcohol and ether extracts of entire worms were not
entirely free from agglutinating properties, however. Unlike the hemo-
lytic substances which are entirely removed from the worm material by
alcohol and ether extraction, the agglutinating substance resists ex-
traction in these solvents and may be recovered in the fraction of the
worm material from which the alcohol-soluble and ether-soluble frac-
tions have been removed. The salt-solution-soluble hemagglutinin does
not appear as firmly bound to the cells of the parasites as the lipoidal
hemolysin. The latter, as has already been stated elsewhere in this
paper, is but slightly soluble in physiological salt solution unless the
material is thoroughly triturated. Salt-solution extracts of coarsely
powdered worm material that yield but a small quantity of hemolysin
were found to contain a considerable quantity of agglutinating substance.
In physiological salt-solution extracts of Ascaris lumhricoides that con-
tain the hemolysin and the hemagglutinin the potency of the former may
be suppressed by low temperatures {6° to 10° C), whereas that of the
latter remains unaffected by those temperatures.

The hemagglutinin from Ascaris lumhricoides is relatively thermo-
stabile and differs in this respect from the hemagglutinin which Tallqvist
(1907) isolated from Diphyllohothrium latum. The latter is injured by 30
minutes' heating at 55° C, whereas that of A. lumhricoides with-
stands heating at temperatures ranging from 56° to 60° for 30 minutes.
Hemagglutinating extract of A . lumhricoides was passed through a Cham-
berland filter without injuring its potency.

Summarizing, it may be stated that in contrast to the lipoidal hemoly-
sin, which is inactive at 6° to 10° C. and which is but slightly soluble in
physiological salt solution, the agglutinin of Ascaris lumhricoides is
readily soluble in salt solution, slightly soluble in ether and alcohol, and

4o6 Journal of Agricultural Research voi.xxn.No. s

active at low temperatures. It also differs from the hemolysin in its rela-
tive specificity for certain species of erythrocytes.

VI. THE EFFECT OF ASCARIS LUMBRICOIDES FLUID ON COAGULA-

TION OF BLOOD

As has already been stated, Weil and Boye {19 lo) found that as a re-
sult of injecting the fluid of Ascaris equorum into rabbits the coagulation
of the blood was retarded by 20 minutes. These investigators state,
however, that they obtained negative results with rabbit blood in vitro.
Leroy (19 10) likewise observed that the blood of dogs which had received
injections of the body fluid of A. equorum exhibited a delayed coagula-
tion time. Flury (1912) made observations on the coagulation of dog
blood in contact with the fluid of ascarids in vitro and records a decided
delay. His experiments with human blood were likewise positive.

In view of the contention of Weil and Boye with reference to rabbit
blood in contact with Ascaris equorum fluid in vitro, the writer tested
freshly drawn rabbit blood to which various quantities of A . lumbricoides
fluid were added, in order to determine if the coagulation power would
be affected. The addition of 3 to 5 drops of the fluid to lo drops of blood
delayed the coagulation time about 15 minutes as compared with that
of normal blood. The addition of 8 drops of fluid to 10 drops of blood
produced a 35-minute delay, whereas the addition of 10 drops of fluid to
an equal quantity of blood resulted in a delay of 42 minutes.

The body fluid of Ascaris lumbricoides retards the coagulation of blood
in vitro as well as in vivo, but its power in this respect is rather limited.

VII. EXPERIMENTS WITH HOOKWORM HEMOLYSIN (ANCYLOSTOMA

CANINUM)

The anemia which occurs in cases of infestation with hookworms has
been ascribed to several different factors. The direct abstraction of
blood by the parasites, the possible absorption of toxic substances from
the digestive tract as a result of the ulceration of the mucosa, hemorrhages
following the laceration of the mucosa by the worms (Loeb and his collab-
orators), and the absorption by the host of hemolysins secreted by the
parasites have been advanced as explanations. The last view was ac-
cepted as a plausible explanation before any experimental evidence in
favor of it had been advanced. That the data with reference to the pro-
duction of hemolysins by hookworms appear to show that such absorp-
tion probably occurs has already been pointed out elsewhere in this
paper.

Nov. 19, 19" Hematoxins from Parasitic Worms 407

I. EFFECTS OF SAI^T-SOI,UTlON EXTRACT OF FRESH WORMS ON RED BI,OOD

CORPUSCI^ES

In the following experiments the hemolysin was obtained from about
100 specimens of Ancylostoma caninutn collected from three dogs. The
parasites were put into a bottle containing a physiological salt solution
and kept in an ice box for about 24 hours after removal from the hosts,
without any apparent loss of vitality.

The extract designated as extract of fresh worms was prepared as
follows: The parasites were ground up in a mortar containing a small
quantity of a physiological salt solution, and the macerated material
was then suspended in about 20 cc. of salt solution, shaken vigorously
for a few minutes, and placed in a refrigerator overnight. The super-
natant fluid was found to be hemolytic, as the following experiments
will show.

Experiment i. — ^To each of three tubes containing 0.5 cc. of a 2 per
cent suspension of washed dog erythrocytes there were added, respec-
tively, 5, 8, and 10 drops of the extract of fresh worms. As a control,
to a fourth tube containing the same quantity of suspension of cor-
puscles there were added 10 drops of a salt solution. The tubes were
shaken thoroughly and placed in the incubator at a temperature of
37° C. At the end of 30 minutes the tube containing 10 drops of the
extract showed complete hemolysis. The tube containing 8 drops of
extract showed complete hemolysis 15 minutes later, while the tube
containing 5 drops of extract showed partial hemolysis at the end of
an hour. The control tube showed no hemolysis. The tubes were kept
in a refrigerator overnight and no further change was noted.

Experiment 2. — ^To three tubes each containing 10 drops of a 5 per
cent suspension of washed sheep corpuscles there were added, respec-
tively, 5, 8, and 10 drops of the extract of fresh worms. It was neces-
sary to incubate the tubes at 37° C. for two hours before hemolysis was
produced in any tube. The tube containing 10 drops of extract showed
complete hemolysis; the tube containing 8 drops of extract showed
partial hemolysis, while the tube containing 5 drops of extract showed
no hemolysis. A fourth tube containing 10 drops of corpuscle suspen-
sion and 10 drops of salt solution showed no hemolysis. These tubes
were kept in a refrigerator overnight with practically no change in
results except that hemolysis was complete in the tube containing 8
drops of extract and was faintly indicated in the tube containing 5
drops of extract.

Experiment 3. — ^The extract of fresh worms was tested against
washed rabbit corpuscles as in experiments i and 2. Ten drops of a
3 per cent suspension of washed corpuscles were completely hemolyzed
by 5 drops of extract in 20 minutes at a temperature of 37° C. This
experiment was controlled as usual.

4o8 Journal of Agricultural Research voi. xxn. no. s

ExPBRiMEjNT 4. — ^Twelve drops of extract of fresh worms were heated
for 30 minutes at a temperature ranging from 56° to 58° C. The addi-
tion of 0.5 CO. of washed dog corpuscles from the same lot as that used
in experiment i resulted in partial hemolysis after one hour of incuba-
tion at 37°. The tube was kept in a refrigerator overnight and showed
almost complete hemolysis the next day.

Experiment 5. — ^A quantity of extract of fresh worms was heated at
60° to 65° C. for 50 minutes. To two tubes each containing 10 drops
of extract that had been thus heated there were added 5 drops of rabbit
and sheep corpuscles, respectively, of the same concentration as noted
in experiments 2 and 3. No hemolysis was produced after two hours'
incubation at 37°. The tubes were kept in a refrigerator overnight
and showed slight hemolysis the following day.

Experiment 6. — ^Twelve drops of extract of fresh worms were heated
to boiling, and after cooling they were added to 0.5 cc. of suspension
of dog corpuscles of the same concentration as in experiment i and
were incubated for one hour, but no hemolysis was produced. After
remaining in an ice box overnight the tube showed but a trace of hemo-
lysis. Similar results were obtained when rabbit and sheep corpuscles
were used. Control tubes showed no hemolysis.

A second series of experiments with extracts of fresh worms was per-
formed several weeks later. The details of these experiments follow.

The extract referred to as extract II of fresh worms was prepared by
macerating 29 live specimens of Ancylostoma caninum ^ obtained from
five dogs shortly after the animals had been killed. The macerated
material was suspended in 3 cc. of physiological salt solution, shaken
vigorously, and allowed to extract at room temperature for about an
hour before it was tested for its hemolytic power. Part of the extract
was kept overnight in a refrigerator and was used the following day.
The suspension of corpuscles and extract was incubated at 37° C. for
periods shown in the table, the results were noted, and the tubes were
then placed in a refrigerator for an additional period of 18 hours, when
the final results were read.

The data presented in Table III show that rabbit and dog corpuscles
are more susceptible to hookworm hemolysin than the corpuscles of
swine and cattle. Despite the fact that the latter were not hemolyzed
by the extract used in these tests, they are not absolutely resistant to
extracts of dog hookworms, as will be shown in another section of this
paper.

' These specimens were washed several times in physiological salt solution.

Nov. 19, 1921

Hemotoxins from Parasitic Worms

409

Table III gives a record of the experiments performed with this extract.

Table III. — Effect of extract II of fresh worms (Ancylostoma caninum) on washed red

blood corpuscles «

Kind of corpuscles. &

Rabbit
Do
Do

Dog...
Do
Do

Hog...,
Do
Do

Cattle. .
Do
Do

Quantity of extract.

5 drops. .
8 drops. .
Control c .
5 drops. .
8 drops. .
Control c.
5 drops. . .
8 drops. . ,
Control c.
5 drops. .,
8 drops. . ,
Control*:.

Period of incubation.

1 hour. . . .
....do...
....do...

2 X hours.
....do...
....do...
....do...
....do...
....do...
....do...
....do...
....do...

Results at
end of in-
cubation
period.

+ + +
+ + +

+ +

Results
after 18
hours addi-
tional in re-
frigerator
(S^C).

+ +
+ + +

+ + +
+ + +

o ++ indicates marked though incomplete hetaolysis. -H-+ indicates complete hemolysis. — indi-
cates absence of hemolysis.

''0.2 cc. of a 5 per cent suspension of washed blood corpuscles were used in all experiments summarized
in this table.

" Eight drops of physiological salt solution were added to the washed blood corpuscles in order to control
the experiment.

The sediment in the tube containing the extract of hookworms when
shaken with 3 cc. of physiological salt solution yielded additional hemoly-
sis, as the following experiments will show.

Experiment 7.— After the supernatant fluid from the extract (extract
II of fresh worms) had been removed the sediment was shaken up with
about 3 cc. of physiological salt solution, v/hich was tested against a 5
per cent suspension of washed dog corpuscles from the same lot as that
referred to in Table I. Three drops of corpuscles were completely
hemolyzed by three drops of the extract after one hour's incubation at
37° C. This experiment was controlled as usual.

Experiment 8. — Five drops of the same extract were boiled for about
one minute. After cooling, three drops of dog erythrocytes from the
same lot as that used in experiment 7 were added and the mixture
incubated for 1% hours at 37° C. No hemolysis was produced. The
tube was kept 18 hours in the refrigerator without any change.

EXPERIMENTS WITH EXTRACTS OE DRIED WORMS

The experiments recorded below were performed with the following
extract :

Fifty-eight mgm. of coarsely powdered worm material {Ancylostoma
caninum) dried at 37° C. and kept in a small vial for about two years were
extracted in 10 cc. of physiological salt solution for several hours. Unlike

4IO Journal of Agricultural Research voi. xxii. no. 8

the extract of fresh worms, which is opalescent, the extract of powdered
material remained quite clear.

Experiment 9. — To four tubes labeled from i to 4, each containing 5
drops of a 5 per cent suspension of washed rabbit corpuscles, there were
added, respectively, 3, 5, 8, and 10 drops of the extract. To a fifth tube
containing an equal quantity of corpuscles there were added 10 drops of
physiological salt solution in order to control the results of the experi-
ment. The tubes were incubated for i hour at 37° C, and kept for 18
hours longer in a refrigerator, after which the final results were read.
Tube I showed no hemolysis, while tubes 2, 3, and 4 showed complete
hemolysis. The control tube showed no hemolysis.

Additional experiments with the same extract and the same corpuscles
showed that the hemolytic action was very slow, since 10 drops of the
extract in contact with 5 drops of the suspension of corpuscles failed to
produce hemolysis after 2 hours' incubation at 37° C, but after an
additional period of 18 hours in a refrigerator the tube showed complete
hemolysis, whereas the control tube showed no trace of hemolysis.

Experiment 10. — The extract of dried worms was tested on a 5 per
cent suspension of washed corpuscles of cattle and swine as follows: To
four tubes each containing 0.2 cc. of corpuscles there were added, respec-
tively, I, 2, 3, and 5 drops of the extract, and the tubes were incubated
for I hour. None of the tubes showed hemolysis. After remaining in a
refrigerator overnight the following results were noted.

Cattle corpuscles : The tubes containing i and 2 drops of the extract
showed partial hemolysis, whereas the tubes containing 3 and 5 drops of
extract showed complete hemolysis.

Hog corpuscles: No hemolysis was observed in any tube.

The foregoing experiments were controlled as usual.

In the experiments described above the extract was not filtered but
was added to the suspension of corpuscles together with some particles of
worm material.

In a second series of experiments performed several weeks later it was
found that washed rabbit blood corpuscles were unaffected when placed
in contact with an extract of dried hookworms, incubated for 3 hours,
and then kept in a refrigerator for an additional period of 18 hours. While
no record was made as regards the introduction of particles of worm
material into the tubes containing the suspension of corpuscles, it is
probable that the clear supernatant fluid alone was added.

A repetition of the experiment on a later date yielded the following
results.

Experiment m — ^A small quantity of coarsely powdered worm mate-
rial was extracted in physiological salt solution, filtered, and the filtrate
tested on washed rabbit blood corpuscles. No hemolysis was produced.
To the material which had thus been extracted a small quantity of physio-
logical salt solution was added, the contents were thoroughly agitated,

Nov. 19, 1921 Hemotoxins from Parasitic Worms 411

and a few drops containing worm particles were added to 0.5 cc. of a 5
per cent suspension of washed rabbit erythrocytes. After one hour's
incubation hemolysis was complete. A tube containing corpuscle sus-
pension alone showed no hemolysis. A repetition of this experiment
yielded similar results.

From the foregoing experiments it appears that the hookworm hemoly-
sin is firmly bound to the cells of the parasite. In fresh worms a con-
siderable quantity of free hemolysin is probably present in tlie tissues
and fluids of the body, which is absorbed by the salt solution in the
course of extraction. Since the sediment of extracts of fresh worms has
been found to yield additional hemolysin after the first extraction, it is
evident that salt solution does not absorb all the hemolysin present in the
worms. The observation of Preti (1908) that tryptic digestion liberates
the hemolysin is further evidence of a close union between the hemolysin
and the cells of the worm.

3. EXPERIMENTS WITH EXTRACTS OF ALCOHOLIC SPECIMENS

The experiments described below were performed with extracts
obtained from specimens of Ancylostoma caninum which had been pre-
served in alcohol for about three years. Unless otherwise stated the
extracts were prepared as follows: The specimens were washed several
times in distilled water, dried at room temperature, and powdered in a
mortar; o. i gm. of the powder was suspended in 10 cc. of an 0.85 per cent
solution of sodium chlorid and extracted in a refrigerator for about 24
hours. The supernatant fluid was then tested on the washed erythro-
cytes of rabbit and sheep as follows.

Experiment 12. — Five drops of a 5 per cent suspension of rabbit
corpuscles plus 3 drops of extract showed complete hemolysis at a tem-
perature of 37° C. in 2 hours. Equal parts of extract and corpuscle
suspension showed complete hemolysis in 1^2 hours. This experiment
was controlled as usual.

Experiment 13. — Five drops of a 5 per cent suspension of washed
sheep corpuscles were mixed with 10 drops of extract and incubated for 2
hours without producing any hemolysis. A similar experiment was
performed a few months later with negative results, despite the fact that
after incubating the mixtures of corpuscles and extract for 2 hours
they were kept in a refrigerator for 18 hours longer.

Experiment 14. — Five drops of a 5 per cent suspension of rabbit
corpuscles were not hemolyzed by 5 drops of extract.

Experiment 15. — A 5 per cent suspension of washed guinea-pig
corpuscles resisted hemolysis after remaining in contact for 3 hours at
a temperature of 37° C. with an extract of alcoholic specimens made by
extracting 200 dried specimens in 6 cc. of physiological gait solution and
mixing 3 drops of extract with 2 drops of the suspension of corpuscles.
Fifteen drops of the extract in contact with 3 drops of the blood suspen-
70495°— 21 3

j^i2 Journal of Agricultural Research voi. xxii. no. s

sion for 2 hours at 37° C. followed by 48 hours in a refrigerator resulted
in partial hemolysis. Several controls in which the suspension of corpus-
cles alone and equal quantities of the suspension of corpuscles and extract
were employed showed complete absence of hemolysis.

These experiments indicate that alcoholic specimens are much less
potent in their hemolytic action than fresh specimens. This is doubt-
less due to the loss of hemolytic substance to the alcohol. In confir-
mation of this view the writer found that dried hookworms from the
dog freed from their ether-soluble and alcohol-soluble fractions were
not hemolytic to washed erythrocytes of rabbits. The ether-soluble
fraction left rabbit corpuscles intact. The alcoholic extract was un-
fortunately lost before it was tested for its hemolytic potency.

4. EFFECT OF NORMAL SERUM ON HOOKWORM HEMOLYSIN

Experiment 16. — To each of four tubes containing 0.5 cc. of blood
corpuscles from the same lot as that used in experiment i there were
added 5 drops of fresh hookworm hemolysin described elsewhere in this
paper, and i, 2, 3, and 5 drops of dog serum, respectively. The tubes
were incubated for i hour at 37° C. No hemolysis was observed in any
of the tubes. After the tubes had remained in an ice box overnight it
was found that with the exception of the tube to which but i drop of
serum was added and which showed a faint trace of hemolysis, inhibition
of hemolysis was complete.

Experiment 17. — Five drops of a 5 per cent suspension of washed
rabbit corpuscles from a lot which was susceptible to extract of alcoholic
specimens were only partially hemolyzed when 3 drops of normal rabbit
serum were added. It was also found that as a result of heating the
serum for 30 minutes at a temperature of 56° C. the antihemolytic prop-
erty was neither destroyed nor impaired.

Experiment 18. — ^Washed rabbit corpuscles, which were completely
hemolyzed when equal parts of a 5 per cent suspension of cells and equal
parts of fresh salt-solution extract were mixed and incubated for 20
minutes at37° C, were found to resist a double quantity of the hemolysm
in the presence of various inactivated sera, as follows : In each of three
tubes there were placed 5 drops of the suspension of corpuscles, 10 drops
of the extract, and 2 drops of heated rabbit, horse, or dog serum (60°
to 65° for 30 minutes). The mixtures were incubated for 2 hours
without any resultant injury to the blood corpuscles. After having
been kept in a refrigerator for 18 hours after incubation, the tubes con-
taining dog and rabbit serum showed faint traces of hemolysis, while
the tube containing horse serum showed no hemolysis.

Experiment 19. — To each of two tubes containing 3 drops of unwashed
rabbit blood there were added 7 drops of physiolo^cal salt solution.
These mixtures were incubated for 2 hours with 5 and 10 drops of fresh
^tract, respectively, at 37" C. No hemolysis was produced. The

Nov. 19, 193 1

Hemotoxins from Parasitic Worms

413

tubes were kept 18 hours longer in a refrigerator, with a resultant faint
indication of hemolysis. Washed erythrocytes from the same rabbit
were highly susceptible to the extract, since 10 drops of a 3 per cent sus-
pension of corpuscles were completely hemolyzed by 5 drops of extract
in about 20 minutes.

Experiment 20. — ^To a series of tubes each containing 3 drops of a
5 per cent suspension of washed dog erythrocytes used in an earlier ex-
periment and included in Table I there were added 5 drops of extract
II of fresh worms and various blood sera diluted with an equal quan-
tity of physiological salt solution and heated at 59° C. for 30 minutes.
The data and results of these experiments, including the controls, are
given in Table IV.

Table IV.— Effects of various sera on hookworm hemolysinO'

Tube

N0.6

Kind and quantity of diluted sera.

3 drops (horse serum)
3 drops (dog serum) . .
3 drops (rabbit serum
No serum

Results after
3 hours' in-
cubation at
37° C.

+ + +

Results after

18 hours
longer in re-
frigerator.

+
+ + +

(I + -I- -f indicates complete hemolysis, -f indicates slight hemolysis. — indicates absence of hemolysis.
b Three drops of a 2 per cent suspension of washed dog corpuscles and 5 drops of extract II of fresh worms
were used in this series of experiments.

5. EFFECT OF COLD ON HOOKWORM HEMOLYSIN

Experiment 21. — Dog corpuscles which were found to be highly
susceptible to an extract of fresh worms at 37° C. remained intact after
being kept for 5 hours on ice in contact with a quantity of extract suffi-
cient to destroy the corpuscles at 37° in 30 minutes. The removal of
the supernatant fluid following rapid centrifugation showed that it had
completely lost its hemolytic potency, since it failed to hemolyze sus-
ceptible dog corpuscles after remaining in contact with them for 2 hours
at a temperature of 37° followed by 1 8 hours at a temperature of about 10°

Experiment 22. — The foregoing experiment was repeated, substitut-
ing susceptible rabbit corpuscles for dog corpuscles, with similar results.

The loss of the hemolytic property of the extract in contact with sus-
ceptible corpuscles at a low temperature can not be attributed to a
possible injurious effect of cold, since it was found that the hemolytic
potency of the extract was not injured after standing directly on the ice
for 18 hours. Washed sheep corpuscles were readily hemolyzed by the
refrigerated extract, whereas a control tube containing corpuscles alone
showed no hemolysis.

Experiment 23. — Six drops of dog blood corpuscles from the same lot
as that described in experiment 7 were mixed with 10 drops of extract II

414 Journal of Agricultural Research voi. xxn. no. s

of fresh worms and placed on ice for 3^^ hours. The mixture was
centrifuged and the supernatant fluid was removed and to it there were
added 2 drops of washed dog corpuscles from the same lot as used in the
first part of the experiment. After 1 hour's incubation followed by 18
hours in a refrigerator the corpuscles remained intact. The corpuscles
from which the supernatant fluid was originally removed were washed
three times in salt solution and then incubated with a small quantity of
salt solution for i hour. Complete hemolysis was produced. A control
tube containing a similar quantity of corpuscles without any hemolytic
extract showed no hemolysis when placed in an incubator. While this
experiment appears to indicate that the hemolysin was fixed to corpuscles
and was not removed by repeated washing, this conclusion must be
accepted with caution, because the possibility remains that some frag-
ments of worms which were introduced into the tube together with the
hemolysin may have been responsible for the hemolysis of the corpuscles
after the removal of the supernatant fluid. The fact that the latter had
lost its hemolytic power affords, however, strong presumptive evidence
of an absorption of the hemolysin by the blood corpuscles.

6. DISCUSSION

The results of experiments with reference to the presence of a soluble
hemolysin in hookworms (Necator and Ancylostoma) show quite con-
clusively that when living specimens are macerated in physiological
salt solution they yield a considerable quantity of hemolysin. The latter
is characterized by relative thermolability, nonspecificity, and suscepti-
bility to normal serum, in the presence of which it loses its potency.
So far as its physiological properties are concerned, hookworm hemolysin
resembles strep tocol5^sin, staphylolysin, tetanolysin, and other hemoly-
sins of bacterial origin. It differs from the hemolytic substances of
Diphyllohothrium latum in that it is destroyed by boiling. The con-
clusion of Preti {igo8) that hookworm hemolysin is resistant to boiling
is not sustained by Whipple {1909) and is also contradicted by the
results of the present writer's experiments. Unfortunately, Preti has
not published a full account of his experiments. His general conclusions
are unsupported by details, and judging from the statements that he
makes it does not appear that he controlled his experiments.

The present writer's experiments indicate that the hookworm hemolysin
is rather firmly bound to the tissues of the parasites, which probably
accounts for the difficulty of obtaining strong hemolytic filtrates from
salt solution extracts of powdered specimens. That the living worm
secretes the hemolysin is evident, however, from experiments with ex-
tracts of fresh worms. The unbound hemolysin from fresh specimens
evidently disappears in the course of drying. This comparative insolu-
bility of the hemolytic substance from dried specimens in physiological
salt solution is perhaps the basis of the contention of Preti {1908) and of

Nov. 19. 19" Hemotoxins from Parasitic Worms 415

Usami and Mano {191 8) concerning the insolubility in water of the hook-
worm hemolysin. That Loeb and his collaborators used dried material
has already been stated.

The fact that normal blood serum has antilytic properties and inhibits
the action of the hookworm hemolysin accounts for the negative results
obtained by Loeb and Smith {1904) and for the weakly positive results
obtained by Whipple {1909). In this connection it is important to
recall the observations of Noc {1908) with reference to the presence of
antihemolysins in the blood serum of normal persons and of those recover-
ing from hookworm disease and from beriberi, and the absence of anti-
hemolysins in patients suffering from these diseases. Noc's observa-
tions are decidedly significant and do not bear out Whipple's view that
the hookworm hemolysin probably bears no relation to the secondary
anemia of ancylostomiasis. De Blasi's observations with reference to
the presence of hemolysins in the blood serum of patients infected with
hookworms and Noc's discovery that under certain conditions the anti-
lytic action of the blood serum may become impaired appear to indicate
that the hookworm hemolysin has potentialities of causing anemia and
that in severe infections it probably plays an important role in the disease.

Since cold (6° to 8° C.) inhibits the action of the hookworm hemolysin
in vitro, and the supernatant fluid from tubes in which susceptible blood
corpuscles and potent hookworm extract have been in contact for a num-
ber of hours at a low temperature no longer has hemolytic properties, the
view that the hemolysin is a complex organic substance, not unlike a
toxin, in that it apparently consists of haptophore and toxophore groups,
appears to be justified. By means of the haptophore group union
between the hemolysins and blood corpuscles takes place, but the dis-
solving or lytic action is produced by the toxophore group. Inasmuch
as low temperatures do not appear to interfere with the absorption of
the hemolysin by the corpuscles despite the fact that the latter remain
undissolved, it is permissible to believe that the toxophore and hapto-
phore groups of the hookworm hemolysin act independently of each
other. This view is purely speculative, however, and further experi-
mentation is required before it may be accepted without reservation.

VIII. EXPERIMENTS WITH EXTRACTS OF CA.TTLE HOOKWORMS
(BUSTOMUM PHLEBOTOMUM)

Hookworms belonging to the genus Bustomum occur as parasites in
the small intestine of ruminants. Bustomum phlebotomum is the species
that infests cattle. According to observations of several investigators,
cattle infested with hookworms show symptoms not unlike those of
human beings that harbor species of Ancylostoma or Necator.

Experiments with extracts of Bustomum phlebotomum similar to those
performed with extracts of Ancylostoma caninum showed that the former,
like the latter, contain a powerful hemolytic agent. The extracts

41 6 Journal of Agricultural Research voi. xxn. no. s

referred to below were prepared as follows : Living worms were removed
from the intestine of a calf, washed a number of times in physiological
salt solution, and kept in a refrigerator at a temperature of 8° C. over-
night. The following day the worms which were still alive were trans-
ferred to fresh salt solution and crushed in a mortar. The crushed
material was then suspended in about two volumes of physiological salt
solution, shaken thoroughly, and centrifuged. The supernatant fluid
which was opalescent was removed and tested as to its hemolytic power.
Tested on a 3 per cent suspension of washed sheep blood corpuscles, it
was found that 5 drops of the extract hemolyzed 5 drops of the suspen-
sion of blood corpuscles in i hour at a temperature of 37°. Even i
drop of extract hemolyzed 5 drops of the suspension of corpuscles after
a few hours. Controls, that is, 5 drops of suspension of corpuscles plus
5 drops of salt solution, remained intact. It was observed that before
hemolysis set in the contents of the tubes assumed a dark red hue.

An extract from another lot of Bustomum phlehoiomum prepared as has
already been described was tested on a 5 per cent suspension of washed
rabbit cells. Five drops of extract produced hemolysis rather slowly
upon 5 drops of suspension of corpuscles.

In a third experiment an extract prepared from worms that had been
kept in a refrigerator overnight was tested on four different tubes of cat-
tle erythrocytes and on four different tubes of hog erythrocytes. The
extract in question was prepared from living specimens as follows:
Forty-five specimens were ground up in a mortar and suspended in 2 cc.
of physiological salt solution. The suspension was centrifuged, and the
opalescent fluid was removed and tested on a 5 per cent suspension of
washed blood corpuscles at 37° C. Three drops of extract were added
to 5 drops of corpuscle suspension. The experiments with each sample
of corpuscle suspension were controlled by adding 3 drops of physiological
salt solution to 5 drops of suspension of blood cells. The results of these
experiments follow.

Cattle blood corpuscles. — ^After i hour one tube of blood was par-
tially hemolyzed and three tubes were intact. After 2% hours two tubes
were completely hemolyzed and two were intact. After 3 hours three
tubes were hemolyzed ; one was intact. The tubes containing the mixtures
were placed in a refrigerator at 8° C. until the next day. When examined
hemolysis was complete in all tubes. The controls showed no hemolysis.

Hog blood corpuscles. — ^After i hour all tubes were intact. After
1^4 hours two tubes were partially hemolyzed and two intact. After 2}^
hours two tubes were partially hemolyzed and two completely hemolyzed.
After 3 hours all tubes showed complete hemolysis. Controls were intact.

Inasmuch as it was found that the hemolysin could be preserved by
drying the worms, powdering the dried material, and storing it in a dark
place, further experiments with Bustomum phlebotomum hemolysin were
performed with dried material. The details of these experiments follow.

Nov. 19. igai Hemoioxins from Parasitic Worms 417

To each of four tubes of defibrinated blood (3 drops of physiological
salt solution plus 2 drops of blood) a small quantity of the powder was
added, and the tubes were shaken thoroughly and placed in an incubator
at 37° C. After 2 hours hemolysis was produced in all tubes. Two lots
of cattle blood from different animals were collected in a 2 per cent
solution of sodium citrate (about 2 volumes of blood to i volume of a 2
per cent sodium citrate). Tested against dry Bustomum phlebotomum
powder the unwashed citrated blood became hemolyzed in about 2 hours
at 37°.

Small quantities of powder were also tested on each of fotur lots of
washed cattle blood corpuscles with positive results. Hemolysis set in
rapidly and was complete after i hour at 37° C.

A few drops of a 3 per cent suspension of washed sheep corpuscles
were hemolyzed by a small quantity of Bustomum phlebotomum powder.
Similar results were obtained with washed rabbit erythrocytes.

Bustomum phlebotomum powder extracted in physiological salt solution
yields but a small quantity of hemolysin, as the following experiments
will show.

Eighty-five mgm. of powder were suspended in 5 cc. of physiological
salt solution. A few drops of chloroform were added as a preservative.
The mixture was kept at a temperature of 35° to 37° C. for 2 days and
then filtered. The clear filtrate was tested on a 5 per cent suspension of
washed rabbit cells. Equal parts of filtrate and suspension of cells
yielded negative results. It was necessary to add 10 drops of filtrate to
3 drops of corpuscle suspension to produce hemolysis. Evidently the
hemolysin is firmly bound to the parasite material and is but slightly
soluble in salt solution. In fact, the powder which had been extracted
was dried and retested on rabbit blood cells, which it hemolyzed rapidly.

An alcoholic extract of fresh specimens of Bustomum phlebotomum was
found to be decidedly hemolytic. The extract was prepared as follows :
About 100 specimens were washed a number of times in physiological
salt solution after they had been removed from the host. The specimens
were then triturated in a mortar and extracted in about 2 volumes of
95 per cent alcohol for about a week at 37° C. The alcohol was sepa-
rated from the worm material by filtration. The filtrate was evaporated
and the residue was shaken with a small quantity of physiological salt
solution, in which it dissolved, producing an opalescent solution. Tested
on sheep red blood corpuscles this solution produced hemolysis. A
quantity of the solution which hemolyzed 5 drops of a 5 per cent suspen-
sion of washed sheep corpuscles in about 2 hours at 37° failed to produce
hemolysis on an equal quantity of blood corpuscles in 20 hours in a
refrigerator (8°), thus showing that low temperatures paralyze the
action of the hemolysin. Likewise, normal horse serum (2 drops) inhib-
ited hemolysis of 5 drops of washed sheep corpuscles to which sufficient
hemolytic solution had been added to cause hemolysis in the absence of

41 8 Journal of Agricultural Research voi. xxn, no. 8

normal serum. The worm material from which the alcohol-soluble sub-
stance had been removed was dried and pulverized. A portion of this
powder was added to washed sheep corpuscles but failed to produce any
hemolytic effect, showing that the hemolytic substances of Bustomum
phlehotomum are completely soluble in alcohol.

In a few experiments the effect of normal serum was tested with a
view of determining when it contained bodies capable of inhibiting the
action of Bustomum phlehotomum hemolysin. Washed rabbit corpuscles,
belonging to a lot that were rapidly hemolyzed by a small quantity of
the powder, resisted hemolysis in the presence of a few drops of normal
rabbit serum.

The effect of heat on the hemolysin was found to be the same as the
effect of heat on Ancylostoma canimim hemolysin. A salt-solution
extract of fresh worms was completely inactivated by heating it for 40
minutes at 60° C.

IX. EXPERIMENTS ON THE POSSIBLE PRESENCE OF ANTICOAGUUNS

IN HOOKWORMS

A series of experiments was performed with a view of determining
whether the two species of hookworms discussed in the foregoing pages
(Ancylostoma caninum and Bustomum phlehotomum) secrete a substance
that has the power of inhibiting the coagulation of rabbit blood. Salt-
solution extracts of fresh and dried material from the two species were
tested as follows.

Into a series of tubes containing varying doses of extract, rabbit
blood drawn directly from the marginal ear vein was allowed to drop.
Bach experiment was controlled by allowing an equal quantity of blood
to drop into tubes containing physiological salt solution. So far as the
rapidity of coagulation of the blood was concerned, appreciable but not
very marked differences were detected between the test and control
tubes. These experiments were performed on the blood of several
rabbits with uniformly negative results.

Inasmuch as Loeb and his collaborators tested the anticoagulin from
Ancylostoma caninum on dog blood and obtained positive results, it would
appear that the writer's negative results may indicate that the anticoag-
ulins in hookworms are either strictly specific for the blood of their host
or that they are perhaps only relatively specific. Further data bearing
on hookworm anticoagulin as well as anticoagulins from other nematodes
are given in a separate paper (Schwartz, 1921).

X. EXPERIMENTS WITH EXTRACTS OF HAEMONCHUS CONTORTUS

Haemonchosis or stomach- worm disease is a disease of cattle and sheep
due to the presence in the fourth stomach of a nematode parasite known
as Haemonchus contortu^. Young animals are especially susceptible to

Nov. 19, i92t Hemotoxins from Parasitic Worms 419

stomach-worm disease, and among other symptoms they show those of
a rather severe anemia. As in hookworm disease, the direct abstraction
of blood by the parasites undoubtedly plays a part in bringing about the
train of morbid symptoms associated with loss of blood, but that other
factors are involved — ^namely, a chronic intoxication of the host by toxic
substances liberated by the parasites — appears probable. Furthermore,
it is by no means unlikely that as the suceptible animals grow older they
become more or less immune to the effects of the parasites, although they
are by no means immune to infestation with the worms. Whether the
immunity is developed as a result of a previous infestation or whether
it is a natural immunity associated with maturity is not known. In fact
the clinical phase of haemonchosis is still an almost unexplored field in
parasitology.

The following experiments were performed by the present writer with
reference to the presence of a soluble hemotoxin in this parasite.

A number of specimens of Haemonchus contortus (about 100) that had
been removed from a calf shortly after death were washed a number of
times in physiological salt solution and kept in a refrigerator overnight.
The following day the specimens were still alive. They were ground up
in a mortar with a small quantity of physiological salt solution. The
crushed material was transferred to a test tube and allowed to remain at
room temperature for about two hours. The supernatant fluid was then
tested on a 5 per cent suspension of washed sheep corpuscles. After
2 hours at 37° C. a number of tubes containing graded quantities of
extracts and 5 drops of washed red blood cells showed no trace of
hemolysis. The tubes were then transferred to a refrigerator, where they
remained 18 hours longer. A faint trace of hemolysis was present in
the tube containing the largest quantity of extract. The control tube
was intact.

A second experiment of a similar nature was performed with another
lot of fresh worms. In this case the extract was tested on washed
sheep blood corpuscles, with negative results. Alcoholic specimens of
Haemonchus contortus from sheep were washed in salt solution to re-
move traces of the alcohol and then dried at 37° C. The dried mate-
rial was pulverized, and part of it was extracted in salt solution and
tested on washed sheep corpuscles, with negative results. The remain-
ing portion of the dried material was extracted in 95 per cent alcohol
and the extract suspended in salt solution. Tested on sheep corpuscles,
this extract likewise yielded negative results.

A number of fresh specimens of Haemonchus contortus were dried at
37° C. and pulverized in a mortar. Graded quantities of the powder
were added to washed sheep blood corpuscles. After 2 hours at 37°
followed by 18 hours in a refrigerator slight hemolysis was produced.

420 Journal of Agricultural Research voi. xxii. No. s

To one tube a small quantity of carbolized^ horse blood serum was
added. The serum inhibited hemolysis.

Haemonchus contortus powder was also tested on four samples of
washed cattle blood cells. The results were slightly positive after 2
hours at 37° C. followed by 18 hours in a refrigerator.

Inasmuch as in the experiment described above washed red blood
cells were used, a series of tests were performed in which unwashed
defibrinated blood was used. In this series six samples of cattle blood
were involved. The addition of various quantities of Haemonchus
contortus powder yielded negative results after 3 hours at 37° C.

Summarizing, salt-solution extracts of Haemonchus contortus are very
slightly hemolytic to sheep and cattle erythrocytes. The faint hemo-
lytic property is preserved by drying. The weakly positive results
obtained by experiments in vitro do not favor very strongly the view
which has been commonly accepted as regards the secretion of a hemo-
toxin by H. contortus. It is quite possible, however, that the appar-
ently weak hemolysin requires some activator which is supplied by the
host blood. The fact that experiments in vitro were only slightly
positive by no means precludes the possibility that an absorption by
the host of the secretions of H. contortus is followed by a marked hemo-
lysis. Another possibility, which has already been mentioned, is that
only the blood of young animals is susceptible to the secretions of H.
contortus. The subject requires further investigation.

XI. EXPERIMENTS WITH TRICHURIS DEPRESSIUSCULA EXTRACT

A small series of experiments with an extract of Trichuris depressi-
uscula was performed as follows: About 60 specimens collected from
several dogs were thoroughly washed in physiological salt solution and
dried in an incubator. The dried specimens were then triturated and
extracted in 3 cc. of salt solution overnight at 8° C. The clear filtrate
was tested on rabbit and sheep erythrocytes. Five drops of a 5 per
cent suspension of rabbit blood cells were hemolyzed by 3 drops of
extract in about 2 hours at 37°. Equal mixtures of sheep erythroc}'tes
and extract showed no hemolysis. The extract was boiled for about a
minute, and after it had cooled it was tested on rabbit erythrocytes.
It produced a faint indication of hemolysis, showing that boiling prac-
tically destroyed the hemolysin.

XII. EXPERIMENTS WITH CESTODE HEMOLYSINS

It has already been stated that while an active hemolytic agent has
been shown to occur in Diphyllohothrium latum, evidence that other
species of tapeworms secrete hemolytic substances is rather incomplete.
The presence of a hemolytic agent in D. latum is significant in view of the
fact that this parasite is capable of producing a severe anemia under

' 0.25 per cent solution of carbolic acid in senun.

Nov. 19, 1921 Hemotoxins from Parasitic Worms 421

certain conditions that are not yet understood. Inasmuch as ces-
todes are not capable of causing anemia by direct abstraction of blood
or by lacerating the mucosa, the etiological role of a hemotoxin, if such
a substance can be demonstrated in forms that cause anemia, can hardly be
denied. The discovery of Schaumann and Ta\lqvist{i8g8) and the subse-
quent studies of Tallqvist (1907) and Faust and Tallqvist {1907) with
reference to the D. latum hemolysin are of great significance and mark
the beginning of the study of the pathogenicity of parasitic worms from
the point of view of intoxication. Despite the fact that D. latum ap-
pears to stand alone among cestodes capable of setting up a severe type
of anemia, there is some evidence that other cestodes are also capable of
bringing about anemia, perhaps not so intense as that produced by D.
latum. Railliet {1895), Neveu-Lemaire {1912), Hutyra and Marek
{19 1 3), and other writers on veterinary parasitology state that cattle
and sheep that are parasitized by tapeworms show clinical symptoms of
anemia. Adult cestodes parasitic in these ruminants belong to the
genera Moniezia and Thysanosoma. Only one species of the latter
genus is known in the United States, namely, Thysanosoma actinioides ,
whereas several species of Moniezia occur in this country. Investiga-
tions by the present writer with reference to hemolysins in worms be-
longing to the genera Moniezia and Thysanosoma have yielded the
following results.

A salt-solution extract of Thysanosoma actinioides powder made by
adding the powder to salt solution and allowing the extract to remain
at 8° C. for about 24 hours was found to be hemolytic to washed sheep
blood cells and rabbit blood cells. In one experiment 150 mgm. of
powder were extracted in 5 cc. of physiological salt solution overnight
at a temperature of 8°. The supernatant fluid was filtered and the
filtrate tested on washed rabbit blood cells. Equal parts of extract of
suspension of corpuscles showed complete hemolysis after 2 hours at
37°. Further experiments with salt-solution extracts of dried material
on washed sheep and rabbit blood corpuscles confirmed the presence of a
soluble hemolysin in this parasite. Thus, an extract prepared by adding
0.2 gm. of powder to 2 cc. of salt solution was tested on rabbit and
sheep blood corpuscles and yielded positive results. The action of the
hemolysin was comparatively slow. To tubes each containing 5 drops
of washed blood cells 5 and 10 drops, respectively, of the extract were
added and incubated at 37° for 2 hours; hemolysis was not evident in
the tubes. After an additional period of 18 hours during which the tubes
were kept in a refrigerator hemolysis was complete in the tube to which
10 drops of extract had been added and marked but incomplete in the
tubes to which only 5 drops of extract had been added. It should be
stated in this connection that in several instances salt-solution extracts
of dried T. actinioides were not destructive to red blood cells of sheep.
Whether the red blood cells of certain animals are more resistant than

42 2 Journal of Agricultural Research voi. xxn.No. s

others, or whether the different extracts used in these experiments varied
in their hemolytic content, has not been determined. At any rate,
salt-solution extracts of T. actinioides , so far as the experiments referred
to above are concerned, are not strongly hemolytic.

A quantity of Thysanosoma actinioides powder was extracted in four
volumes of ether. The ether extract after it had been freed from all
traces of ether was emulsified in physiological salt solution and tested on
sheep blood corpuscles with positive results. After a second extraction
of the powder in ether a quantity of powder free from the ether-soluble
fraction was extracted in physiological salt solution, and this extract
was found to be nonhemolytic. The remaining powder was extracted
in 95 per cent alcohol. After filtration the alcohol was evaporated, and
the residue, which had a waxy appearance and consistency, was dis-
solved in physiological salt solution and tested on sheep red blood cells
with positive results. Boiling did not destroy the hemolytic potency
of this extract ; neither did cold inhibit its activity. Normal horse serum
inhibited its action completely.

After alcohol extraction the pow^der was extracted in physiological salt
solution and tested on sheep red blood cells. It was only faintly
hemolytic.

Another lot of powdered Thysanosoma actinioides was extracted in 95
per cent alcohol three times in succession, each extraction lasting
48 hours. After the last extraction only a slight residue was left when
the alcohol had completely evaporated. The residues were dissolved in
physiological salt solution and tested on sheep and rabbit blood cells
with positive results. Boiling did not destroy them and low tempera-
tures had no inhibiting effect on them. The powder freed from the
alcohol-soluble fraction was extracted in salt solution, and this extract
was nonhemolytic.

It may be concluded, therefore, that a hemolysin is present in
Thysanosoma actinioides, soluble to some extent in physiological salt
solution and completely soluble in alcohol. Ether extracts of T. actini-
oides are hemolytic, but worm material freed from ether-soluble fractions
still retain the hemolytic agent. That substances other than fatty acids
are involved in the hemolytic effects of T. actinioides extracts is evident,
since the ether extracts remove whatever fatty acids are present in the
worms. The alcohol-soluble and ether-insoluble fraction of T. actini-
oides resembles rather closely tissue lysins so far as the chemical and
physiological properties of tissue lysins are known. Noguchi {1907)
found that tissue tysins are soluble in 95 per cent alcohol, are not removed
by ether extraction, and that they have the chemical properties of solu-
ble soaps. In common with the latter they are destructive to red blood
cells even at 0° C, are neutrahzed by normal serum, and are resistant to
boiling. While the chemical nature of the ether-insoluble and alcohol-
soluble fraction of T. actinioides has not been determined, its resem-

Nov. 19. 1921 Hemotoxins from Parasitic Worms 423

blance to tissue lysins appears to be very close. The Ascaris lumhricoides
hemolysin as well as the Bustomum phleboiomum and Ancylostoma
caninum hemolysins are not active at low temperatures, as shown else-
where in this paper.

Experiments with a species of Moniezia similar to those performed
with Thysanosoma actinioides have yielded negative results. The addi-
tion of various quantities of powdered Moniezia material to suspension of
washed red blood cells of rabbit and sheep produced no destructive
action on the cells. A salt-solution extract of Moniezia powder was
likewise nonhemolytic when tested on washed sheep blood cells. An
ether extract was only slightly hemolytic, but after removing from the
ether extract the acetone-insoluble fraction, presumably lecithin, its
hemolytic potency was no longer manifest. The acetone-insoluble frac-
tion had no destructive effect on sheep blood corpuscles. A quantity of
Moniezia powder freed from the ether-soluble fraction by repeated ex-
traction with ether was extracted for 72 hours in 95 per cent alcohol at
38° C. The alcohol was separated from the alcohol-insoluble powder
by filtration and evaporated. The residue was taken up in physiolog-
ical salt solution, in which it was only partly soluble, the insoluble por-
tion forming a coarse suspension. This solution had a decidedly acid
reaction. Tested on washed sheep red blood corpuscles, it produced
no hemolytic effect.

XIII. RESULTS OF EXPERIMENTS

The data presented in the foregoing pages have already been sum-
marized in connection with each separate topic. The discussion which
follows is for the purpose of correlating, comparing, and differentiating
the results obtained with various species of parasitic worms that have
been referred to in this paper, and to consider the general bearings that
the results have on the nature of parasitic infection.

Hemotoxins present in parasitic worms contain one or more active
principles. Of the latter, hemolysins stand out as of prime importance.
Hemagglutinins and anticoagulins may be associated with hemolysins.

Hemagglutinins have thus far been observed in Diphyllohothrium
latum by Tallqvist (1907) and in Ascaris lumhricoides by the present
writer. Tallqvist describes the hemagglutinin from D. latum as a water-
soluble, alcohol- and ether-insoluble substance, decidedly thermostabile.
The hemagglutinin observed by the present writer in extracts of Ascaris
lumhricoides is resistant to heat and soluble in lipoid solvents, such as
ether and alcohol, as well as in physiological salt solution. It is, there-
fore, quite a different substance from the agglutinin of D. latum. Anti-
coagulins have been found in species of Strongylus (Weinberg, 1908), in
the larvae of Gastrophilus (Weinberg, 1908) , in species of Ascaris (Weil
and Boye, Leroy, and the present writer), in Ancylostoma caninum (Loeb

424 Journal of Agricultural Research voi. xxn.No. s

and his collaborators) , and in several other species by the present writer
{tq2i.) The anticoagulin in Ancylosioma caninum is the most active of
the anticoagulins observed in parasitic worms and is doubtless a factor
in the anemia that is present in hookworm disease. The anticoagulin of
Ascaris lumbricoides has but a feeble action, so far as available experi-
mental data show.

Hemolysins from parasitic worms, so far as they have been described
in the literature, have certain properties in common with hemolysins of
bacterial origin as well as with hemolysins that have been obtained from
normal tissues by Korschum and Morgenroth, Noguchi, and others.
These properties may be characterized as nonspecificity in action and
relative simplicity of structure as compared with hemolysins that may
be artificially produced in animals by immunization with red blood
corpuscles. The experiments recorded in this paper do not in any case
contradict these facts. Different species of blood corpuscles may show
differences in resistance to hemolytic extracts of parasitic worms, but
absolute resistance of any species of corpuscles has not been established.
Similarly, extracts from different parasitic worms differ in their resist-
ance to heat, but once their potency has been destroyed it can not be
reactivated by normal serum. The only apparent contradiction to this
statement is the result of a small series of experiments of Garin with
Graphidium sirigosum, which, as has already been indicated, can not
be accepted as conclusive in view of the small number of experiments.
Hemolysins produced in an animal as a result of immunization with
red blood corpuscles, are, as is well known, specific in their action,
affecting only corpuscles against which the animal has been immunized,
and complex in structure, since they act in combination with comple-
ment and may be reactivated by normal serum after the complement
has been destroyed.

So far as their resistance to heat is concerned, hemolysins from para-
sitic worms differ considerably. Heat-resisting hemolysins have been
recorded by Tallqvist from Diphyllobothrium latum, by Weinberg from
species of Strongylus, and by the present writer from Ascaris lumbricoides
and Thysatiosoma actinioides. Hookworm hemolysins from worms of the
genera Ancylostoma, Necator, Bustomum, and the hemolysin from Tri-
churis depressiuscula are not as resistant to heat. The relatively thermo-
labile hemolysins from these parasites resemble in this respect bacterial
hemolysins, whereas the thermostabile hemolysins resemble in this respect
tissue extracts.

The solubility of hemolysins from parasitic worms in lipoid solvents,
especially in alcohol, is another property that they have in common with
tissue lysins. A property of the latter is also the nonimpairment of their
activity at low temperatures, even at 0° C. So far as the results of
experiments recorded in this paper are concerned, hemolysins from

Nov. 19, 1921 Hemotoxins from Parasitic Worms 425

worms belonging to the genera Ascaris, Ancylostoma, and Bustomum are
inhibited at 8°. The hemolytic effect of Thysanosoma actinioides extract
is not inhibited at this temperature, however. This fact is important and
clearly differentiates hemolysins of nematodes from the hemolysin of
T. actinioides. In this respect, too, nematode hemolysins resemble bac-
terial hemolysins.

Finally, the action of hemolysins from parasitic worms is inhibited by
normal serum. The antilytic property of the serum is thermostabile
(Weinberg, igo8, and the experimental results obtained by the present
writer). Tissue lysins, too, are inhibited by normal serum. Certain
bacterial hemolysins are similarly susceptible to normal serum.

On the basis of this discussion nematode hemolysins may be character-
ized as relatively simple substances, thermolabile or thermostabile,
depending on the species from which they are obtained, inactive at low
temperatures (6° to 8° C), inactive in the presence of normal serum,
nonspecific, soluble in alcohol and in physiological salt solution.

Cestode hemolysins, so far as they have been investigated, are rela-
tively simple bodies, thermostabile, active at low temperatures, inactive
in the presence of normal serum, nonspecific, soluble in alcohol.

The question naturally arises whether toxic products from parasitic
worms are liberated from the bodies of the latter and get into the circula-
tion of the host. Blanchard (1903), while accepting the evidence in
favor of the view that parasitic worms elaborate toxic products, appears
to doubt the etiological significance of these toxic substances because
of the possibility that they are either not liberated by the worms or if
liberated may be thrown out of the body before they can injuriously
affect the host. The available evidence on this question appears to indi-
cate that hosts harboring parasitic worms actually absorb the toxic
products of the latter. The serological evidence in favor of this view has
already been referred to. It may be added that the fact reported by
Guerrini {1908) with reference to the presence of hemolysins in the blood
serum of hosts harboring Fasciola hepatica and the findings of De Blasi
that hemolysins occur in the blood serum of hosts harboring Ancylostoma
duodenale tend to confirm the belief that parasites liberate their toxic secre-
tions and that these secretions get into the circulation of the host. Wein-
berg {1908) has made some interesting observations on the tissues of hosts
harboring parasitic worms which argue directly in favor of the absorption
by the host of toxic products liberated by the worms. Weinberg exam-
ined histologically the organs of 32 horses infested with strongyles and
obtained the following results: In the blood vessels he found a large
number of mononuclear leucocytes containing iron granules. He also
found similar granules in the spleen, liver, in the conjunctival tissue, in
the Malpighian tubules and in the convoluted tubules of the kidneys,
and in the canals of the right kidney. Histological examinations by the
same investigator of organs from 30 monkeys infested with a species of

426 Journal of Agricultural Research voUxxn.No. s

CEsophagostomum yielded similar results. In another paper Weinberg
{1909) records that the injection of extracts of worms of the genus
Strongylus into guinea pigs leads to a pigmentation of the spleen but seldom
of the liver. From this, it appears that erythrocyte destruction takes
place in animals that harbor hemotoxin-producing parasitic worms and
that the disintegration products of the erythrocytes are ingested by
leucocytes, arrested in certain organs, and eliminated through the
excretory system.

Whether the hemotoxic substances from parasitic worms are liberated
during the normal life of the worms, or whether they are liberated only
when worms sicken or degenerate, as appears to be the case in Diphyllo-
bothrium latum, can not as yet be stated with certainty. In the case of
D. latum the view that only certain specimens secrete the hemolysin has
been advanced by a number of investigators, especially by Leichten-
stern (1896). Tallqvist's experiments show that hemolysins are present
in specimens of D. latum expelled from patients that show no symptoms
of anemia as well as in specimens obtained from cases of severe anemia.
Tallqvist's hypothesis that the hemolysin is eliminated when the worms
disintegrate finds confirmation in numerous cases in which eggs of
D. latum are present in the feces of patients, and anthelmintic medication
fails to expel any worms and merely yields a mass of eggs. Another
factor which may be of importance, and which, so far as the present
writer is aware, has been entirely overlooked, is the fact that certain
individuals may lack antilytic constituents in their blood and are thus
susceptible to the toxin which other individuals are capable of neutralizing.
That the antilytic properties of the blood may under certain conditions
be absent is probable from the observations of Noc {1908) with reference
to hookworm disease. Whether the observations with reference to D.
latum are applicable to other parasitic worms, especially to nematodes,
can not in the light of our present knowledge be stated with any degree
of certainty. That parasites may die in the intestine or other location
and disintegrate before they are eliminated from the body of the host
is by no means improbable. Cultures of larvae of parasitic worms in
vitro show that bacteria may kill the worms, and that the latter undergo
degenerative changes,- such as complete internal disorganization, quite
rapidly. That worms may be attacked by bacteria and other organisms
in the body of the host is by no means improbable. Weinberg has in
fact described what appears to be a disease in worms belonging to the
genus Ascaris, which is characterized by the presence of certain pig-
mented spots that are clearly visible through the cuticle. The present
writer has observed this condition in specimens of Ascaris lumhricoides
on several occasions.

Whether parasitic worms liberate their toxic secretions during life or
whether these substances partake of the nature of endotoxins and are
not liberated from the bodies of the worms unless the latter disintegrate

-T^ov. 19, 1921 Hemotoxins from Parasitic Worms 427

is still open to speculation, but the view that toxic substances from
parasitic worms are of etiological significance in parasitic diseases i^
supported by convincing evidence.

XIV. SUMMARY

Extracts of A scar is lumbricoides contain active substances that affect
blood deleteriously. The hemolysin which these extracts contain is a
thermostabile, nonspecific, alcohol-soluble substance which appears to be
rather firmly bound to the cells of the parasite, presumably to the cells
of the intestine in which it is elaborated. The hemolytic potency of
extracts of A. lumbricoides is not due solely to fatty acids, since chemical
fractions of the worms from which the fatty acids have been removed
by ether extraction are hemolytic. The hemolysin is neutralized by
normal blood serum.

The body fluid of A. lumbricoides shortly after removal from the host
contains oxyhemoglobin and is nonhemolytic. It acquires hemolytic
powers, however, as the worms are kept alive in vitro for a few days,
and loses at the same time its oxyhemoglobin content.

Body fluid from fresh specimens of Ascaris lumbricoides does not acti-
vate a hemolytic system, and alcohol-soluble fractions of the worms
from which ether-soluble substances have been removed does not act
as complement in combination with inactivated normal guinea-pig serum.

The hemagglutinin from Ascaris lumbricoides is a salt-solution-soluble
substance and has special affinities for rabbit blood cells. Unlike the
hemolysin, its action is not hindered by low temperatures (6° to 8° C).

Ascaris lumbricoides secretes a substance that inhibits the coagulation
of blood. This substance is present in the body fluid of the worm and
iias but a comparatively slight potency.

Ancylostom,a caninum secretes a nonspecific hemolysin, soluble in salt
solution, relatively thermolabile and inactive at low temperatures.
Normal blood serum inhibits the action of the hookworm hemolysin.

Bustomum phlebotomum secretes a hemolysin having properties similar
to that of Ancylostoma caninum. This hemolysin is completely soluble
in alcohol.

Salt-solution extracts of Haemonchus contortus have but a feeble
hemolytic action.

Salt-solution extracts of Ancylostoma caninum and of Bustomum
phlebotomum do not inhibit the coagulation of rabbit blood to any marked
degree.

A weak hemolytic substance is present in extracts of Trichuris depressi-
uscula.

Thysanosoma actinioides contains an alcohol-soluble hemolysin. Alco-
hol-soluble fractions of T. actinioides from which the ether-soluble fraction
has been removed are hemolytic, showing that substances other than fatty
acids are involved. The hemolysin from this cestode is active at 8° C.
70495°— 21 4

428 Journal of Agricultural Research voi. xxii. no. s

and is neutralized by normal blood serum. Extracts of a species of
Moniezia similar to those of Thysanosoma actinioides axe nonhemolytic.
The view that hemolysins and other hemotoxic secretions of parasitic
worms are of etiological importance in parasitic diseases appears to
be well founded.

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ASH CONTENT OF THE AWN, RACHIS PAI.EA, AND
KERNEL OF BARLEY DURING GROWTH AND MA-
TURATION

By Harry V. Harlan, Agronomist in Charge of Barley Investigations, and Merritt
N. Pope, Assistant Agronomist in Barley Investigations, Office of Cereal Investiga-
tions, Bureau of Plant Industry, United States Department of Agriculture

INTRODUCTION

The ash determinations here assembled were made with two primary
objects in view. Earlier studies had shown that in the varieties under
observation the awns possessed a physiological function. When they
were removed the kernel development was retarded and the spike became
brittle through the greater ash deposit in the rachis. In order to see
if usable variations existed in the amount of ash deposited in the rachises
and awns, a considerable number of varieties were studied.

The previous experiments were not as complete as was desired.
Mechanical difficulties had prevented the taking of samples to the point
of absolute maturity. There thus existed a possible doubt as to the
nature of the changes in the days immediately following the date when
kernel sampling became impossible. The determination of ash in the
awns and rachises was, therefore, continued for some time after maturity
in one series of varieties at Chico, Calif.

The results point a possible way to the securing of desirable non-
shattering awnless and hooded varieties. They also throw some light
on the ash content of the kernel during growth.

MATERIAL USED

Material for the study of ash in the barley spike was collected from
several sources. Two series of samples originated at Aberdeen, Idaho.
The awns, rachises, and paleas were obtained from the irrigation plots,
the kernel studies of which were previously reported. In this series
and the one from Minnesota the glumes were forcibly removed from the
kernels. To eliminate the possible effect of imperfect separation when
the glumes were thus removed, the kernels from a naked barley grown
at Aberdeen were included for comparison.

Two lots of samples were grown at Chico, Calif. The first of these
consisted of a collection of varieties embracing a wide range of botanical
characters. The second consisted of a lesser number of varieties, which
were allowed to stand in the field for a time after ripening. Frequent
samples were taken, and the change of ash after maturity was determined.

Further use also was made of the data from an experiment carried
on at St. Paul, Minn. Ash determinations were made on a number of
varieties grown at Arlington, Va., the detailed results of which are not
included.

Journal of Agricultural Research. Vol. XXII, No. 8

Washington, D. C. Nov. 19, 1921

aao (433) KeyNo. G-2SS

434

Journal of Agricultural Research voi. xxn, no. a

ASH OF THE AWNS

The awns of barley contain a very high proportion of ash. One of the
most finely divided carbons known has been secured from barley awns.
This extremely fine division is probably caused by the high percentage of
ash. The ash is deposited during the time the kernel is developing. At the
time of their emergence the awns contain little ash and are very flexible.

,i<

"*•

(

1. -

.*"

»*

\ao

,»•

•

,0*

.''

'••

_^.

.m.

• —1

./»

/•

—

02.

'3Z

■Bi

9.3

1 1

?<3

? ^

^ t

"it

7 /

' S

FlQ. I.— Percentage of ash, by progressive three-daj' averages, in the awns of Hannchen barley grown on
plots variously irrigated at Aberdeen, Idaho, in 191 7.

The ash at flowering time and for a few days immediately following
usually runs from 4 to 8 per cent of the dry matter. As may be seen
in Table I, the awns of the Hannchen variety may sometimes contain a
slightly higher percentage at flowering time. Varieties of this type, how-
ever, contain more ash than do those of most other types of barley. The
increase in the ash content after flowering is very regular. The daily

Nov. 15, i94i Ash Content of Awn, Rachis, and Palea

435

increment is quite uniform. In figure i it will be seen that within the
variety the rate of deposit has a direct relationship with the amount of
moisture in the soil and probably with the amount of water transpired.
On plot 8, which was not irrigated after flowering, the plants were suffer-
ing from lack of water during most of the time the kernels were devel-
oping. This lack of water is reflected in the percentage of ash in the
awns. The awns on this plot never contained as high as i6 per cent of
ash. The uniformity of the deposit was in no wise affected. The daily
increment, however, was less than on plot i, which received ample irriga-
tion. The normal ash content of tlie awn of Hannchen barley when grown
in the western United States is over 30 per cent at maturity. As will be
seen, the awns on the spikes from plot i reached this percentage several
days before matturity. Plots 2 to 7 received the same treatment as plot 8
until the time of their final irrigation. Only one irrigation after flowering
was given to any plot except plot i . The irrigation occurred on the day
the first ash determination was reported. Following the application of
water there was an acceleration in the rate of deposit of ash in plots 2 to
6, inclusive. The barley on plot 7 was maturing when the water was
applied. The leaves had begun to wither and the awns were almost
color-free at the time of the irrigation. The rate of deposit was not
materially increased over the rate on plot 8. The maturation was, how-
ever, delayed, and the final ash content of the awns was decidedly greater
than in plot 8.

Tabls I. — Percentage of ash in
plots variously

the awni
irrigated,

of Hannchen barley grown
at Aberdeen, Idaho, in igij

on eight different

Date.

Plot. I

Plot 2.

Plot 3.

Plot 4.

Plot 5.

Plot 6.

Plot 7.

Plot 8.

July 16

9.4
10.8
II. 6

13-9
14. 0
16.5

19. 6

20. 0
23.8
23-9
27-5
28.6

25-4
28.4
30.0
31.2
30.7
32-7

16. 2

12.5
14.2
16. I

14.7
16.5
17.7

19-3
20. 2
19.8

20. 6

21. 6
20. 4

20. 4

21-5

21. I

22. 4
21. 2

6.0

7
7
9
8

8.7
14. 0

15-7
18.4
18.8
19.8
21. 9
18. I
19.9
21. 9
21. 9

3
?

7.8
7-9
9-7
15-1
16.3
15-0
18.7

Aug. I

■2

13.8
13-4
16.5
13-4
17.2
17.7
17. 0
18.9
22. I

21. 7
19.9
21.5

22. I

24-5

25-4

24. 0

26. 2
22.7

9
12
10

13
II
10

13
II
12
13
13
15
15
13
13

7
2
8

S
0

10.3
13.2
16. 0
12. 2

15-4
18.4
18.3

21.5
23.2
18.0
21.7
22. 5
26. 2
22. 7
32.1

11. I

12. 6
13-9
13-5
13- I
13.2

13-8
16.6
ig. 0
21. 2
17.2
21.5

26. 5
24. 2
26.2
26.3

32-9
30.1
27.8
23. 2
32.3

20.5
23.1

23-9
22. 6
20. 2
20. 4
19.7
20. 0

25-7

28.2
33-^

29. I

7
8

29. I
33-1

28.2

436

Journal of Agricultural Research voi. xxn.No. 8

Table II. — Percentage of ash in awns, rachises, atid kernels of eight varieties of barley
grown at Chico, Calif., and sampled on lo different dates

PERCENTAGE OF ASH IN AWNS

C.I.

No.

Variety.

June

i6.

June

Jime

2S.

June

27.

June

30.

July

July
14.

531
234
257

916
261

19s
690
652

Hannchen
Nepal ' . .
Tennessee
Winter.
Odessa. ..
Mariout. .
Smyrna. .
Coast,.
Poda

36.7
16.6

29. I
34-3
34- I
32.0

31- 9
27-3

36.

34-4

31.8

35- 5 34- 6

32. I
35-6
34-5
35-5
26.6
28.4

33-2
34-6
36.7
29-5
32.8
31.0

34-4

34-

33-1

35-

35-2

37-

35- 0

29.

33- 0

30.

33- <i

32-

31-5
16. 4

34-1
36. I

33- S
32-7
32-5
32-4

33-2
13.8

35- 3
37-4
32.6

29- 5
32.2

33-4

32.8
15-5

34-3
37-2
34- o
31. 2
31-7
32.5

35-8
16.3

PERCENTAGE OP ASH IN RACHISES

531

234
257

916
261

195
690
652

Hannchen
Nepal ...
Tennessee
Winter
Odessa. .
Mariout.
Smyrna.
Coast. .. .
Poda

7.8
7-7
5-4
5-4

II. 7

9.6

8-5
7.6
8.2
S-o
S-4

10. 7

9.»

7-5
9. I
6.8
6.2
5-7

9-5

9.8

8-5
7-1
6.8
5-8

10. o
9.9

».9

8.7
7.8

7-7
6.4
6.0

II. 4
10. 5

8.3
9.6
8.6
6.8
6.9
6.0

"•S

». o
8.4
7.8
6.0
6.1

PERCENTAGE OF ASH IN KERNELS

531

234
257

916
261

195

690

652

Hannchen
Nepal ^ . .
Tennessee
Winter
Odessa. .
Mariout .
Smyrna .
Coast. .. .
Poda. . . .

3-3
1,8

3-2
3-6

3-0
2.7

30
2.8

3-4
3-6

31
2.7

3-2
2.9

3-6

3-5
3-4
2.9

3-1
3-0
2.8

3-4

3-1
3-4
3-2
2.8

2.9

3-4

3-5

3-2
3-6
3-2
3-8
2.9
2.9

3-3
1.9

2.8

3-1

3-6

1.8

3-3
2. I

3-6

3-1
2.8

3-5
2.8

3-4
2.0

3-0
3-6

3-1
31

2.9

1 Hoods.

' Naked kernels.

Since the studies on irrigation were not carried beyond the stage of
actual maturity, there was some question as to whether the awn had
ceased to accumulate ash when the studies were terminated. It was
impossible to carry these particular samples further, as this was a study
of kernel growth and the paleas could not be stripped from the kernels
after mechanical loss of water had commenced. In order to determine
whether there was a later transfer of ash a series of varieties was allowed
to stand in the field for six weeks after maturity, at Chico, Calif. Samples
were taken, commencing at about the stage where they were discontinued
at Aberdeen. These results are reported in Table II. It will be seen
that there was very little change of ash content after the growth of the

Nov. 19. 1921 Ash Content of Awn, Rachis, and Palea 437

kernels had been completed. The changes indicated in the table are
probable variations of individual samples, inasmuch as the average of all
the samples showed no consistant change.

Table III. — Percentage of ash in the rachises, awns, and kernels of SQ varieties of barley
grown at Chico, Calif., igij

C. I. No.

1079A
1289
678

1045
1097
1449
1041
1094
1284

679
1 046 A
1236
1061

914
1072

145 1

187

927
1060

1450

1281

1296

10S9A

1038

1121

669B

957

973

972
1076B

190
1074B

996
1058
1098B
1297
1283
1307

Variety.

Chinerne

Horsford

Hanna

Envoy

Black Hull-less

Hadaka

Thomas

Crocket

Feline

Franconian ....

Temple

Abyssinian ....

Consul

Italian

Squiers

Carrol

Svanhals

Odessa

Coolie

Mochi

Welch

Kitchin

Filer

Judith

Hanchamont ...

Abyssinian

Oderbrucker ...

Red River

Luth

Venezuela

Beldi

Algeria

Rasput

Gobi

Kurof

Claudia

Catto

Cheddar

Description.

Black awnless 6-rowed

Hooded 6-rowed

Lax 2-rowed

Dense 6-rowed

Naked 6-rowed

Short-awned naked 6-rowed

Naked 6-rowed

Shattering 6-rowed

Smooth-awned 6-rowed ....

Lax 2-rowed

Dense naked 6-rowed

Dense deficient 2-rowed . . .

Lax 6-rowed

Lax 2-rowed

Lax purple 6-rowed

Dense 2-rowed

Dense 6-rowed

Long-awned naked 6-rowed .
Smooth-awned 6-rowed ....

Dense 6-rowed

Lax 6-rowed

Lax 2-rowed

....do

Pvu-ple deficient

Lax 6-rowed

....do

Lax 2-rowed

Smooth-awned 6-rowed ....

Percentage of ash.

13.6

13- I
13.0
II. 4
10. 2
10. I
10. I

9-5
9.4

9-3

9.2
9.1
8.7
8.4
8.4

7.8

7-7
7.6
7.2
7.2
7.2

7-1
7.0

6.9
6.8
6.8

6-5
6.1
6.1

S-7
5-6
5-5
5-2
4.8

Awn.

Grain.

3-5

35-3

20. 9

21. 2

28.9

23-5
27. 6

2
3

4
8

27.9

28. 7

3
2

0
6

22. 5

24.7

24. 2

23-9
18.9

3
3

3
4

20. 4

20. I

23-9

20. I

20. 4

31-5

31-1

17. 2

19-3

23.2

20. 4

23. I

26.3

19- .5

20.8

25. 2

33-9
26. 7

3
2

0
8

28.7

17. 2

13-4

30.2

34- 0

30.1

17.4

Single samples from a larger number of varieties were taken at Chico
the same year. These samples were not taken until it was evident that
all growth in the plant had ceased. The results are reported in Table III.
The list of varieties included almost all the major botanical variations of
barley. The table is arranged in order of the ash content of the rachis,
the ash of the awns being given in the second column. It will be noticed
that the greater number of varieties have an ash content very much
lower than the Hannchen at Aberdeen. It is not thought that much of

438

Journal of Agricultural Research voi. xxn.Nas

this is due to environment, although part of it probably is. The water
available for the plants at Chico was less than at Aberdeen, since the
plots at Chico were not irrigated. Although the Hannchen variety was
not included in this nursery series, C. I. 679, Franconian, is of the same
general type as Hannchen, and Hanna 678 probably is even more closely
related. In Table II samples of Hannchen from a neighboring plot are
reported, and these do not differ materially from those grown at Aber-
deen. The agreement between the results at Chico and Aberdeen is
close when it is realized that varieties do vary a great deal according to
their environment, as was evident in the results from the irrigation
experiments. Varieties grown in the Bast, under humid conditions
where the ash content of the soil is very low, have a much lower percent-
age of ash than do those from the West. The determinations from
Arlington, Va., are not reported, but they show far less ash than those
from either Chico or Aberdeen. Despite the variation in the ash content,
the awns of different varieties seem to maintain the same relationship.
The varieties which are high in ash under the arid conditions of the
West are also the ones which are highest in ash at Arlington, even though
the ash content at Arlington may be only half that of the western-grown
samples.

Varieties which have a low ash content in the rachis do not necessarily
have a low ash content in the awns. The awn itself does not have the
same ash content throughout its length. Variation in individual samples
can easily come about through the loss of the tips of the awns in the field.
In Table IV are given the results of determinations made on the basal,
middle, and apical portions of the awns of three barleys from Chico,
Calif. The ash content of the tip is much greater than that of the base.
In the Hannchen and Tennessee Winter varieties, the ash reaches 40
per cent of the dry weight in the tips of the a^vns. The bases of the
awns in the Coast variety were low in ash as compared with those of
the Hannchen and Tennessee Winter varieties. This may have some
connection with the fact that the awns of the Coast variety do not break
cleanly from the grain in thrashing.

Table IV. — Percentage of ash in the tip, middle, and basal portions of awns in three
varieties of barley grown at Chico, Calif., in igiy

C. I. No.

Variety.

Date taken.

Percentage of ash.

Tip.

Middle.

Base.

257
690

531

Tennessee Winter

fJune 6
I July 14
("June 6
Ijuly 14
fJune 6
\July 14

34.3
37-8
29. 6

34-5
41.4

40.3

35-7
36.5
28.4
32.2
39-6
37-9

29.7

Coast

31-3
23.8

Hannchen

26. 4
33-2

32-5

Nov. 19. 1921 Ash Content of Awn, Rachis, and Palea 439

ASH IN THE RACHIS

The deposit of ash in the rachis of the barley spike is less easily inter-
preted than is the ash in the awns. The awns serve as a place of de-
posit, probably for ash excluded from the cell sap. The rachis, on the
other hand, is a conductive organ through which passes the nourish-
ment of the various kernels and the water which is transpired from the
awns. The daily deposit of ash in the rachis is confusing. Although
a large number of analyses were made they are not reported, as no
plausible explanation could be offered for the fluctuations. The gen-
eral trend of the results is indicated in figure 2.

In 191 7, in plot 8, which received no irrigation after flowering, the
ash gradually increased from about 2 per cent at flowering time to
about 7 per cent at maturity. In this case there were no large fluctua-
tions. Where irrigation water was applied, the ash content was con-
siderably increased. Although this increase was exhibited on all plots,
in many cases the increases were irregular, fluctuating and not easily
explained. The results in 19 16 were more uniform and showed a gradual
increase from flowering to maturity, the content reaching 11 to 14 per
cent at that time. In 191 7, on the irrigated plots, the content at ma-
turity ranged from 12 to 18 per cent.

While the drop in ash in plot i is doubtless exaggerated by the acci-
dent of sampling, most of the large fluctuations in the daily samples of
191 7 are not thought to be errors of determination. On the plots where
the water content was low the fluctuations either did not occur or were
small. The analyses of the awns and rachises were made from the
same samples at the same time and in the same way. Those of the awns
were satisfactory. It is probable that the variations in the ash of the
rachises were due to some relationship of soil water and the rate of
transpiration.

In a previous paper ^ it was shown that the removal of the awns re-
sulted in an increase of the ash content in the rachis of an awned barley.
From this it was inferred that the rachises of awnless barleys were likely
to be high' in ash. It was known that awnless and hooded varieties
shattered badly in the field. It was to discover varietal differences, if
such existed, that the samples were taken which are reported in Table
III. As previously stated, the experiment included not only varieties
which differed in the character of the awns but in many other taxo-
nomic characters as well. It was found that the ash content varied
greatly with the variety. As these samples were grown in California,
the percentage of ash is higher than if the samples had been grown in
the more humid districts. In all determinations made on barleys grown
in Minnesota and at Arlington, Va., under humid conditions and where

1 Hari,an, Harry V., and Anthony, Stephen, development OF barley kernels in normal and

CLIPPED SPIKES AND THE LIMITATIONS OP AWNLESS AND HOODED VARIETIES. In JOUf. Agr. Research,

V. 19, no. 9, p. 431-473, 13 fig. 1930.

440

Journal of Agricultural Research voi.xxu.no.;

17 /S 13 2021 ii232^ZffZ6Z7£d29^^l I 2^^6678

Fig. a.-Percentage of ash, by progressive three-day averages, in the rachis of Hannchen barley grown on
plots variously irrigated at Aberdeen, Idaho, in 191 7.

the ash content of the soil is low, there was a much lower percentage of
ash than in the western-grown samples. The analyses from the West,
are, however, more significant in this connection, as shattering occm-s
much more commonly in the arid regions than in the humid regions.

Nov. 19.1931 Ash Content of Awn, Rachis, and Palea 441

In Table III it will be seen that the variety containing the highest
percentage of ash in the rachis was an awnless variety. The second
highest was a hooded sort. Among those varieties having an ash con-
tent over 9 per cent in the rachis were the awnless and hooded varieties
referred to above, a variety from north Europe which was known to
shatter badly, and C. I. No. 1449, a short-awned variety from Japan.
In the original importation from which this last variety was obtained,
there were two types of barley, differing only in the length of awn. CI.
1449, which was short-awned, contained 10. i per cent of ash in the rachis,
while C. I. 1450, the long-awned strain, contained only 8 per cent.

The rachises of most of the common 2-rowed varieties are rather high
in ash, many of them containing from 7 to 9 per cent when grown at
Chico. The two samples of C. I. 1121 were taken from different parts
of the nursery. The analyses show that there was very little variation
due to location. C. I. No. 957, 973, and 972 are all of the Manchuria
type. They contain less ash in their rachises than do most of the 2-rowed,
but distinctly more than do the Coast types, C. I. No. 1076, 190, and
1074, which follow them in the table.

A number of smooth-awned varieties of hybrid origin are found in the
table. These were included because of the potential economic impor-
tance of smooth-awned strains. The awn of the common barley is ex-
tremely harsh and is very objectionable to farmers and feeders. The
annual acreage of barley is undoubtedly reduced because of the discom-
fort in handling the crop. On the other hand, it is known that the awn
possesses a physiological function and it is improbable that maximum
yields can be obtained from awnless and hooded varieties. In order
to retain the functional value of the awn and at the same time to remove
its objectionable features, the smooth-awned strains have been pro-
duced.

From the analyses given it appears that the smoothness of the awn has
in no wise limited its function. One smooth-awned strain is included
which has an ash content in the rachis of over 9 per cent. There are
two strains with ash contents of nearly 8 per cent. Three others are
found at the very bottom of the table with an ash content in the rachis
of about 5 per cent. It is evident that in the latter varieties the low ash
content is not due to any inactivity of the awn, as the awns themselves
contain over 30 per cent of ash, indicating that they have been very
active in transpiration. As can be seen in Table II, the Hannchen
variety would come in the upper part of the list given in Table III.

ASH OF THE PALEAS

Ash determinations were made on the paleas of the samples reported
in Table I. These determinations are found in Table V. The ash
content of the paleas is quite comparable with that of the awn as far as
the nature of the daily deposits are concerned. While the total per-

442

Journal of Agricultural Research voi. xxu.no. 8

centage at maturity is much less, there is the same uniform increment
from flowering until maturity. As with the a^vns, the daily increase on
plot 8, which received no irrigation after flowering, was less than on the
other plots which received one or more irrigations. UnUke the case of
the awn, however, the maximum percentage of ash was reached on plots
which suffered to a considerable degree from lack of water. The ash
content showed a response to irrigation even on plot 7.

Table V. — Percentage of ash in the paleas of Hannchen barley from variously irrigated
plots at Aberdeen, Idaho, in igiy

Date. Plot I.

Plot 2.

Plot 3.

Plot 4.

Plot s.

Plot 6.

Plot 7.

Plot 8.

July 16

4.9
8.0

6.4
9-9

9.4

9-5
10.4
12.6
12.7
12.6
12.6

13-9
13.2

13-4
13-8

13-9
14.6

11. 4
12.5

12. I
12.9

n-z

16.2

10. 7
7-3

6.4

7-3
7.2
8.0
8.2
8.4
8.7
9.0
8.2
10.7
9-7

10. 2
13-7
13-5
13-9

10.8
10.7
10.7

4. 0

5-
5
5
6
6
7
7
7
7
7
7
7
8

7
8
8
9
9
8

4
3
3

7-1

8.2

10. 1

9-9

10. 7

"•5
l-^

\-^
8-5
9.2

8.4
9.4

11. I
12.3

13-3

12. I
12.5

14. 0
10. 0
14. 0
15-3

8.6
8.3

6.4
7.2

7-1
11. 0

"li.'e"

12.3

II. 7

9.8

8.9

9-5
9-7
9.4
9.1
15-4

. 31

Aug. I

10. I

9.9

10. 2

10. 6
10.7
12. 0

7-7
8.7

9-3
8.0
10. 1
8.8
12. 2
13-8
13.0
13-4

12.7

3
0

6.9

7.6

10. 9

11. 2
12.5

II. 9
12.7

9-5

12.7
10.7
13.0
IO-3
8.5
II. 7

7-9
8.1
9.0

8.2

II. 8

11.6

12.7

9-7
9.9
9.6

5
2

7
6
I
9
3
4
I
a
8

No determinations were made which would show the variations in the
ash content of the paleas of different varieties. With mature samples,
such as those discussed in Table III, it is impossible to strip the paleas
from the kernels. For the same reason the analyses of the kernels in
Table III are not particularly valuable. The ash content of the caryopsis
is much lower than that of the inclosing glumes, so that any variations
in the ash of the glumes, or in the proportion of caryopsis to glumes,
appear in the table as a difference of the ash content of the kernels.

ASH IN THE KERNELS

In the previous papers pubUshed on kernel development,^ the ash in
the kernel was computed as a percentage of the dry matter. In the case

• Harlan, Harry V. daily developmsnt op kernels op hannchen barley from flowering to
MATURITY AT ABERDEEN, mAHO. In Jour. Agr. Research, v. 19, no. 9, p. 393-430, 17 fig., pi. 83-91. 19J0.
Literature cited, p. 429.

Harlan, Harry V., and Anthony, Stephen, op. ax.

Nov. 19, 1921 Ash Content of Awn, Rachis, and Palea

443

of the awns, paleas, and rachises, this is probably the best method of
comparison. These organs do not increase perceptibly in size during
the time the deposit of ash is taking place. In the awns the deposit
probably consists of ash eliminated from the cell sap. In consequence
of this very heavy deposit, the ash in the awn reaches a percentage of the

6,0

7.0

6,0

^.0

2.0

)

*-*.

«^

•««.

"iH

*"*'

N..

"«»*

0^'

».

—

*5;

r"^

..«

15. W7 /<? /SaOZI^JS^ Z^Z5g6Zr,Zd.&^^^ I ;S 3 ^ 6 6 7 6

Fig. 3.— Percentage of ash in barley kernels, computed on the basis of dry matter (solid line), water (broken

line), and wet weight (dots and dashes), from fiowering to maturity, at Aberdeen, Idaho, 1917.

total weight which overshadows any variation of sample or defect of
method in calculation. In the case of the kernel it is not thought that
the dry matter is a desirable basis of computing ash. When computed
on this basis, as will be seen in figure 3, the ash content at flowering time
is very high. In most determinations it has been around 8 per cent at
this period of growth.
07495°— 21 5

444 Journal of Agricultural Research voi. xxu, no. s

Shortly after fertilization the percentage of ash commences to drop,
falling very rapidly for a few days and then more gradually until com-
plete maturity. This is obviously not a clear statement of what occurs.
The percentage of ash on the dry-matter basis is a perfectly accurate
statement, but the plotted curve of such percentage does not give a
graphic idea of what is taking place in the kernel. There is a daily
increase in total ash. This increase is almost uniform. The total ash
content of the kernel when plotted is an ascending nearly straight line.
Whether more of this ash is contained in one part of the kernel than
another is not apparent. The ash at flowering time must be in solution
and in the protoplasm. There has not been time for any deposit in the
newly formed cell walls. Until several days after flowering the ash
content must be in the cell sap, the proteids, and such penetration of cell
walls as probably would occur if the tissue were not living.

As about So per cent of the content of a newly formed kernel is water,
it was thought at first that calculating the percentage of ash on the basis
of water would be the best method of comparison. In the very early
stages, before any deposit could occur in the cell walls, this might be
true. However, as the development of the kernel proceeds, the water
occupies a smaller and smaller percentage of the kernel. Not only does
the proportion of cell walls increase, but the proportion of the proteid
matter in the active tissue probably is increased by the growth of starch
grains. These starch grains, being formed in the cells, must occupy
space previously largely occupied by cell sap.

If the ash is to be accounted for entirely on the basis of cell sap, the
concentration of the cell sap must show a progressive increase to account
for the total ash. This is highly improbable. The curve of percentage
of ash based on water content is, however, more regular than the one
based on dry matter and is in the direction of the actual ash deposit.

The ash was finally computed on the basis of the wet weight of the
growing kernel. By computing it on this basis, allowance was made for
both the ash in the cell sap and that in the organized components of the
cell. The use of such a method assumes that the ash in the dr}^ matter
would be a mechanical infiltration from the cell sap which would eventu-
ally show the same percentage throughout the cell. When computed in
this way a striking uniformity is revealed (fig. 3). Although the pro-
portion of water and dry matter varies over a range of 40 per cent during
the growing period, the percentage of ash on the basis of wet weight is
almost constant. In Table VI are given the analyses of kernels from
various plots. These plots differ in irrigation, in the years grown, and
in the variety used. The awns from the same samples from which the
kernels were taken show a variation of 15 per cent under the radical
changes of conditions of growth. The variation in the percentage of
ash on the basis of wet weight of kernel is a matter of tenths of a per cent.
Many of the apparent fluctuations have plausible explanations. At

Nov. 19, 1921

Ash Content of Awn, Rachis, and Palea

445

Minnesota, for instance, the grain was badly lodged and ripened very
unevenly. There was also considerable rain at ripening time which
delayed the ripening of part of the spikes. That many of these irregular-
ities were due to the stage of ripening was apparent in a table published
in a previous paper.* In this table the kernels with high ash content
are the kernels which weighed less than 50 mgm. In other words,
they were kernels in which maturation had been carried to the point
where the mechanical loss of water had reduced the wet weight below
50 mgm.

Table VI. — Percentage of ash in kernels 0/ barley from flowering to maturity, compile
on the basis of the wet weight

Days from
flowering.

13-
14.

15-
16.

17-
18.
19.
20.
21.
22.

23-

24.

25-

Plot I,

1917.

I. 41

1-39
I. 17
.90
.91
.82
.90
1.03
.90
•94

•97
I. 01

•97
I. 01
I. 01
I. 06
I. 10

•99

04
08

1.07

Plot 3.
1917.

i^3i
.88

.87

.76

I. 14

•85
I. 00
I. 14
I. 21

I- 55
.82

•95
.92

I. 04
.96
.98
I. 06
I. 04
I. 02
1.08
1.08
I. 00

Plot 4,
1917.

0. 96

1. 18

•94
•94
•97
I. 27
I. 12
I. 00
I. 07
I. 25

•95
I. 00

•94
I. 10

•99
I. II

•97
1.36
I. 14
I. 00

Plot 5.
1917.

I. II
.87

•79

.78

I. 12

•95
1.08

.96
I. 04
I. 06
I. 04
1.03
I. 12

.91
I. 40
I. 06
I. 04

Plot 6,
1917-

0.81

.76
.78

1.08
.90
.92
.82

1.49

I- IS

1.32

.90

•93

Plot 7.
1917.

.96

.86

I. 01

.94

.85

I. i»

I- 15

Plot 8,
1917.

2.23

•99

I. 61

I. 07

.96

.80

•94
.91

•73
1.03

•97

.91

I. 06

I. 00

I. 14

I. 04

I. 00

I. 06

.91

.89

I. 17

1.23

Hann-
chen,
1916.

0. 62
.81

1. 04
•93

•85

.87
I. 00
I. 14
I. 04
I. 04
I. 02

I. 06
I. 22

1.08

1-34
I. 19
I. 19

1-35
1^34

r-5i
I. 62

Hann-

chen,

clipped

1916.

.92
.96
•93

94
06

15
15

I. 17
I. 20
1.32
I. 26
I. 26
^•3i

I. 21
I. 42
1-57

1-75

Man-
churia,
1915.

^•95

I. 02

.90

.84

.96

•77

.91

I. 02

1.08

I. 12
1-13
1-13
I- 13
I. 12
I. 17

I. 16
I. II
I. 29
I. 26
I. 20
1-25

1-25
I. 02
1.08
I. 41
1.79

Man-
churia,
clipped,

1915-

37
30
26

At final maturity, where the base of calculation was reduced by the
rapid mechanical loss of water, there was sudden rise in the percentage
of ash. This increase is taken to indicate maturity. The taking of
samples usually ceased just before the final rapid fall of water content.
The glumes began to adhere to the caryopsis several days before maturity.
After they commenced to adhere the separation of glumes and caryopsis

* Haki,an, Harry V., and Ajnthony, Stephen, op. cit.

446

Journal of Agricultural Research

Vol. xxn, No. 8

was imperfect. Fragments of the inner tissues of the glumes frequently
remained clinging to the carj'opsis and pieces of the outer layers of the
pericarp were as often removed with the glumes. It was thought that
this small interchange of tissue did not affect the results, but to be certain
a comparable series of kernels from a naked barley was studied. The
results were added to figure 4. The curv^e of the percentage of ash based
on wet weight is essentially the same as in the hulled varieties. In this
figure it is apparent that neither the application of irrigation water nor
the difference in the character of the barley influenced the percentage of
ash when computed on the basis of wet weight. The analyses of a num-
ber of mature samples of commercial naked varieties were also available.
When the ash was recalculated on a wet basis of 45 per cent water the
ash content was about the same as that obtained in the field.

/.6

.e
,7

—

1
\

■'

' — J
1

^•

/it

^-i

/>1

1*^

-— '

f»^

■~~

I Z 3 9-^6

7 8 9 10 II 12 J3 /^ iS 16 17 18- 19 20 Zl Z2232^2S

Fig. 4.— Graph showing percentage of ash on the basis of wet weight in the kernels of Hannchen barley on
2 plots differently irrigated, in 1917, and of Baku barley grown in another year at Aberdeen; Idaho.

DISCUSvSION OF RESULTS

The extremely heavy deposit of ash in the awns of barley indicates that
the awn, or parts of the awn, are used as a depository for the excess ash
absorbed by the roots. The fact that some varieties contain much more
ash in the awns and rachises than others is due probably to two causes.
There most probably is a difference between varieties in the amount of
water transpired. As was shown in the irrigation plots at Aberdeen,
this results in a marked variation of ash deposit. There may also be a
difference in the selective functions of the roots of different varieties.
Some varieties may absorb more ash from the soil than do others. This
is strongly indicated in the ash content of the rachises. It is also of
greatest importance in this connection. Varieties of the Coast type are
characterized by a low ash content of the rachis. In most of the shatter-
ing varieties the rachises are high in ash content. The hooded varieties
have long been known to shatter badly. From results previously re-
ported it would seem that much of this is due to the loss of the awn as an
organ partially utilized for the elimination of ash. On the other hand.

Nov. 19, 1921 Ash Content of Awn, Rachis, and Palea

447

v</

«35

■--

_/*

^fVA/

— — — -

/''

^^^

1
1
1

'*«•

iH^

■ \

•

1
1
1

1
i
1

/?/9C///S

'~'

_,^^i»*

^*»— n_^

.''

**•■

**—•*.

<y s

2527 i30

7
UULY

Tig. 5. — Percentage of ash in the awn and rachis of Hannchen (solid line), Tennessee Winter (dots and
dashes), and Coast (broken line) barleys sampled on 10 different dates, at Chlco, Calif., in 1917.

the hooded varieties most largely grown have come from hybrids whose
parents were both from humid districts. The resulting hybrids might
be less brittle if parents adapted to arid conditions were used.

448 Journal of Agricultural Research voi. xxii. no. s

On account of the low ash content of the rachis of the Coast barley
(fig. 5), varieties of this type may be useful in the production of non-
shattering awnless and hooded sorts. Barleys of the Coast group prob-
ably take less ash from the soil than do most others. There certainly is
less deposited in the awns and rachises than in those of the other common
varieties. Crosses of hooded varieties with varieties of the Coast type
should give hooded segregates which are less brittle than the common
hooded forms. Indeed the Meloy, one of the best hooded varieties under
cultivation, is probably the result of such a cross. In a more complex
cross it might be possible to use some of the characters of the Hanna
variety. The Hanna is not classified as a shattering variety, yet its
rachis contains a high percentage of ash. In this case the rachis is able
to withstand a heavy deposit. It is possible that this resistance might
also be of use, although hooded crosses of this sort, when not combined
with the Coast, have not been very promising. In this connection it is
desired not to overemphasize the relation of ash content to shattering.
There is an obvious relation, but the ash content is only one of a number
of factors. The tenacity of the vascular bundles, the character of the
cell walls, and the size of the rachis, all have a bearing on shattering.
There is also more than one type of shattering. In the Manchuria
barley, for instance, when grown in Idaho under irrigation, the kernels
become loosened from the spike without the rachis itself being affected.
In this case the ash content of the paleas may have some bearing on
deciduousness.

The ash of the kernel is of particular interest. In this case all of the
ash is contained within cells which are engaged in highly active meta-
bolism. The ash is either in the cell sap itself, the active proteid content,
or the cell walls. When the ash is computed on total wet weight a very
uniform percentage is maintained. It is obvious that at no time is any
part of the kernel set aside as a repository for ash. There is very little
difference between the kernels of plants which are dying from drouth and
those which are growing under an ample supply of water. Why the ash
content of the active kernel is maintained at a nearly constant precentage
and whether a higher percentage of ash than that exhibited interferes
with normal metabolism is not indicated from these analyses. That the
uniform percentage of ash in some way is connected with the fundamental
processes of growth is indicated further by the fact that the percentage
coincides with that found in roots, tubers and fruits, all storage organs,
and even with that of meat and eggs.

The percentage of ash based on the wet weight of kernel is not quite
constant. There is a loss in percentage immediately following fertiliza-
tion and then a gradual increase until full maturity. This behavior can
not be adequately interpreted. It appears that at the time of fertiliza-
tion the ash content of the ovary is very high. Immediately after fer-
tilization there is a decided distention, partially due to the turgidity of

Nov. 19, I92X Ash Content of Awn, Rachis, and Palea 449

a high water content. The tissues arising from the fertiUzed egg cell
occupy a very small part of the growing kernel for several days after
fertilization. The ovary wall increases very rapidly. A tissue develops
at the end of the kernel arising from the ovary walls which persists for a
considerable time and which grows very rapidly for the first few days
after flowering. Histological sections of this tissue indicate that very
little is concerned in its growth except the addition of cell walls, the
enlarging of cells, and the increase of the watery cell content. A small
starch deposit is found in the cells, but it is negligible. This high pro-
portion of watery tissue might result in the drop of ash content immedi-
ately following fertilization. The gradual increase from then to maturity
may be due to the fact that the proteids contain a greater percentage of
ash than does the cell sap, or it may come about from a light deposit in
some limited tissue of the caryopsis.

SUMMARY

The awn of barley receives a very large deposit of ash, comprising over
30 per cent of the dry weight in some varieties. Barleys differ in the
amount of ash deposited in the awn and probably in the selective func-
tion of the absorbing roots. Within a variety the amount of ash in the
awn is correlated with the supply of soil water and probably with the
amount of water transpired.

There are varietal differences in the amount of ash deposited in the
rachis. The rachises of hooded and awnless varieties are usually high in
ash and usually brittle. The tendency to shatter may possibly be over-
come in hooded varieties by crossing them with barleys of the Coast
type, which have little ash in their rachises.

No part of the kernel proper is used as a repository for ash. The ash
of the kernel is the ash of cell sap and of highly active protoplasm.
When computed on the basis of the wet weight, the wet weight being a
measure of the organ when active, there is almost no variation in the
proportion of ash. During most of the period of growth the variation
is only 0.3 of i per cent, the content increasing gradually from slightly
less than 1 per cent in early growth to slightly more than i per cent at
maturity.

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"Vol. XXII NOVE^^BER 26, 1921 No. 9

JOURNAL OF

AGRICULTURAIy
RESEARCH

CONXENXS

Page

Temperature Relations of .Stone Fruit Fungi - - - 4S1

CHARLES BROOKS and J. S. COOLEY

(Contribution from Bureau of Plant Industry )

Transportation Rots of Stone Fruits as Influenced by
Orchard Spraying - - --- - - " 467

CHARLES BROOKS and D. F. FISHER

( Contribution from Bureau of Plant Industry)

Storage of Coniferous Tree Seed - - - - - 479

C. R. TILLOTSON

(Contribution from Forest Service)

Susceptibility of the Different Varieties of Sweet Potatoes
to Decay by Rhizopus nigricans and Rhizopus tritici - 511

L. L. HARTER and J. L. WEIMER

(Contribution froiti Bureau of Plant Industry)

PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE,

WITH THE COOPERATION OF THE ASSOCIATION OF

LAND-GRANT COLLEGES

WASHINGTON

GOVERP Mr NT PRINTING OFFICE

1922

EDITORIAL COMMITTEE OF THE

UNITED STATES DEPARTMENT OF AGRICULTURE AND

THE ASSOCIATION OF LAND-GRANT COLLEGES

FOR THE DEPARTMENT

KARL F. KELLERMAN, Chairman

Physiologist and Associate Chief, Bureau
of Plant Industry

EDWIN W. ALLEN

Chief, Office of Experiment Stations

CHARLES L. MARLATT

Entomologist and Assistant Chief, Bureau
of Entomology

FOR THE ASSOCIATION

J. G. LIPMAN

Dean, State College of Agriculture, and
Director, New Jersey Agricultural Expert-
tnent Station, Rutgers College

W. A. RILEY

Entomologist and Chief, Division of Enlo-
tnology and Economic Zoology, Agrtcul-
iural Experiment Station of the University
of Minnesota

R. L. WATTS

Dean, School of Agriculture, and Director;
Agricultural Experiment Station, The
Pennsylvania State College

All correspondence regarding articles from the Department of Agriculture should be
addressed to Karl F. Kellerman, Journal of Agricultural Research, Washington, D. C.

All correspondence regarding articles from State Experiment Stations should be
addressed to J. G. Lipman, New Jersey Agricultural Experiment Station, Ntv?
Brunswick, N. J.

DRNAL OF AGEICrailML ffiSEARi

Vol.. XXII Washington, D. C, November 26, 1921 No. 9

TEMPERATURE RELATIONS OF STONE FRUIT FUNGI

By Charles Brooks and J. S. CoolEY, Pathologists, Fruit Disease Investigations ,
Bureau of Plant Industry, United States Department of Agriculture

The two fungi that cause the heaviest market losses on peaches and
other stone fruits are Sclerotinia cinerea (Bon.) Wor. and Rhizopus
nigricans Ehr. The former is often referred to under its conidial name
of Monilia and is the cause of brown rot, while the latter is the cause of
black mold rot.

The present paper gives the results of investigations in regard to the
temperature responses of these two fungi under various conditions of
growth.

In all the experiments except that reported in figure i the spores
were inoculated into the fruit from pure cultures. Except where other-
wise stated the cultures were obtained from the host into which the
inoculations were made. The fruit was warm when inoculated but after
inoculation was placed in moist chambers and stored at once at the
temperature indicated. Five or more peaches or prunes were used at
each temperature in each test. The fruit was at the proper maturity for
picking and shipping and was carefully selected for quality and sound-
ness. In dividing the fruit into lots for distribution at the various
temperatures uniformity was secured by selecting seven peaches or
prunes (or as many as there were temperatures) that were similar in size,
color and degree of maturity and distributing these one each in seven
moist chambers and repeating the process till the desired amount of fruit
was obtained.

Records were made of the diameters of the rots at intervals of one or
two days, and the average of all the rots at a given temperature on a
particular date was taken as a basis for plotting the curves in the accom-
panying figures. The equipment used in securing the various tempera-
tures has been described in an earlier publication.^

SWEET CHERRIES

In 19 19 a temperature experiment was made on Governor Wood
cherries. These had been shipped by express from Wallingford, Conn.,
to Washington, D. C, and arrived somewhat bruised and with considera-
ble brown rot. The specked and rotten cherries were discarded, but the
slightly bruised ones were included in the experiment. The cherries
were divided into five equal lots and distributed without inoculation at

' Brooks, Charles, and Cooley, J. S. temperature relations of applb-ROT fungi. In Jour. Agr.
Research, v. 8, no. 4, p. 139-164, as fig., pi- 1-3- 1917-

Journal of Agriculture Research, Vol. XXII, No. 9

Washington, D. C. Nov. a6, 1921

aap Key No. 0-256

75308—22 1 (451)

452 Journal of Agricultural Research voi. xxii. No. 9

five different temperatures.^ After 10 days' storage notes were taken
and results obtained as shown in figure i.

All of the cherries at 15° and 20° C. were partially or entirely rotten
and nearly all of those at 10°. At 5° sixty-six per cent were affected, and
at 0° thirty-four per cent. The results show the great inhibiting effect of
low temperatures but perhaps give greater emphasis to the extreme diffi-
culty of controlling Monilia rot at any temperature when the fruit has
already received bad treatment and an opportunity has been given for
the rot to pass through its initial stages while the fruit was warm.

PRUNES

But one temperature test has been made on prunes. The fruit was
from Wenatchee, Wash., and was shipped from that point in a pony
refrigerator August 31, 1920, arriving in Washington, D. C, in good
condition 13 days later. Inoculations were made with Monilia and
Rhizopus, and the fruit was distributed at once to the various tempera-
tures. Figure 2 shows the development of the rots 5 days after inocula-
tion.

PEACHES

A large number of temperature experiments have been made with
Monilia and Rhizopus on peaches. The Carman, Belle, and Elberta
peaches used in the 191 8 experiments were purchased in the Washington
market. The Belle and Elberta used in 19 19 were from Rockville, Md.,
and the experiment was started the day after they w^ere picked. The
Carman and Belle peaches used in 1920 were from Vienna, Va., and were
inoculated the day after they were picked. These peaches were slightly
greener than those of the other experiments.

The curves of the various figures show very great uniformity. The
Rhizopus cultures from peaches gave results similar to the cultures from
cherries and strawberries, both in temperature response and in rapidity
of rotting.

An interesting contrast is seen between the behavior of the fungi on
peaches and on dextrose potato agar. A comparison of figiu-es 3, 4, 5,
and 6 with figure 7 shows that Monilia has grown just as freely at the
higher temperatures and much earlier and more rapidly at the lower
temperatures when grown on peaches than when on agar. At 10° C. rots
usually became evident on the fruit within 3 days, while on the agar there
was practically no growth at the end of 7 days. At 5° the rots were well
started in 6 days, while the agar colony had scarcely made an equivalent
growth at the end of 14 days. At 2>^° the rots made a start in 8 to 12
days, but there was no evidence of growth on the agar at the end of 20
days. A comparison of figures 8, 10, 12, 14, 15, 16, and 17 with figures 9,
1 1 , and 1 3 show^s that the reverse condition holds with Rhizopus. This
fungus made a more rapid growth and developed at lower temperatures
on the culture media than it did on the fruit. On both food materials it
had its most rapid growth at 30°. With the culture media tlie growth at
20° and 25° was but little slower than at 30°, but on the peaches the
growth at 20° fell far behind that at 30°. At 15° and also at 10° the

1 Temperature equivalents:

°C. °F. 'C. "F.

20 68 5 41

IS 59 o 33

10 50

Nov. 26,1921 Temperature Relations of Stone Fruit Fungi

453

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i5" /o /s

£0

F:g. 1. — Xatural infections of brown rot on Governor Wood cherries. The base line shows the temperatures
and the perpendicular the percentage of cherries affected with brown rot.

% ^£:<^/?£-£>S C£/\/T/<3/?/9£>£r

Vv~;'2. — Monilia (solid line) and Rhizopus (broken line) on Italian prunes. Temperature is indicated'on
the base line and diameter of rot on the perpendicular.

'^£>/9yS

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Fig. 3,— Monilia on Elberta peaches. Experiment started August 2c, 1519.

454

Journal of Agricultural Research voi. xxii, No. 9

•?ii>/?K?

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Fig. 4. — Monilia on Belle peaches. Experiment started August 27, 1919.

'4-P/?yS

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Fig. 5. —Monilia on Carman peaches. Experiment started August 5, 1923.

<5 O

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Fig. 6.— Monilia on Belle peaches. Experiment started August aS, 1920

Nov. 26, 1921 Temperature Relations of Stone Fruit Fungi

455

c 2.

'O ,'d' :so as

Fig. 7. — Peach Monilia on potato agar with 3 per cent dextrose added. In Petri plates. Experiment

started November 22, 1918.

Fig. 8. — Peach Rhiiopus on Eiberta peaches.! Experiment started Augiist 23, 1918.

456

Journal of Agricultural Research voi. xxii, No. 9

/oo

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I

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yS£>l^y^ ^

■n

jyatusK

7 /

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Fig. 9. — Peach Rhizopus on potato agar with 2 per cent dextrose added. Experiment started November

32, 191S.

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Fig. 10. — Cherry Rhizopus on Elberta peaches. Experiment started August 23, 1918.

Nov. 26.1921 Temperature Relations of Stone Fruit Fungi 457

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Fig. II. — Cherry Rhizopus on potato agar with 2 per cent dextrose added. Experiment started November

22, 1918.

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/?£0/?££:<5 c£-Arr/<^/?/9^£:

Fig. 12.— Strawberry Rhiropus on Elberta peaches. Experiment started August 23, 1918

458

Journal of Agricultural Research voi xxii. no. ?

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Fig. ij — Strawberry Rhizopus on potato agar with 2 per cent dextrose added. Experiment started

November 33, 1918.

O Z.5 S /O /6

Fig. 14 — Peach Rhizopus on Carman peaches. Experiment started August i, 1918.

Nov. a6, I9SI Temperature Relations of Stofie Fruit Fungi 459

—

1 ^/

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Fig. 15.— Peach Rhizopus on Belle peaches. Experiment started August 37 1919.

za/}y,S

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Fig. 16.— Peach Rhizopus on Carman peaches. Experiment started August s, 1920.

'SDi^YS

^5" 7.S /O /S

Fig 17.— Peach Rhizopus on Belle peaches. Experiment sUrted August 28, 1920.

460 Journal of Agricultural Research voi. xxii. no. 9

growth started earlier and proceeded more rapidly on the agar than on the
peaches. The fungus finally made a slow growth on the agar at 5°, but
on peaches it made no growth at this temperature and with the exception
of a few overripe peaches none at 7K°-

These contrasts in the behavior of the two fungi can probably be partly
explained by the fact that Monilia is a parasite and adapted to growth on
living material, while Rhizopus is a saprophyte and suited to growth on
dead material, like the agar or inactive living material such as overripe
fruit. It is interesting to note that with both fungi unfavorable
food material and unfavorable temperatures work together in delaying
growth, one unfavorable factor adding to the other in delaying or inhibit-
ing activity.

A study of figures 3, 4, 5, 6, 18, and 20 gives a detailed idea of what
can be expected of Monilia rot at any transportation or storage tempera-
ture. The results in the last two figures have been obtained by averag-
ing those of the first four figures and therefore stand as a summary of
the various experiments. With fruit that is infected with brown rot
(Monilia) 3 days at 15° C. would result in heavy losses, 3 days at 10°
would mean badly specked fruit that would go down rapidly at that
temperature and that would be entirely destroyed by a day at "a higher
temperature. Brown rot cannot get started in 3 days' time at 7K°. but
by the end of the fourth day fruit at that temperature may be specked with
rot. In 6 days the fruit at 5° may be spotted, in 9 to 12 days growth
may be evident at 2}4°, and at the end of 3 weeks rots may have started
at 0°. Brown rot does not develop rapidly at the lower temperatures
even when well started, yet its later growth is inhibited far less than its
initial stages.

A study of figures 8, 10, 12, 14, 15, 16, 17, 19, and 21 shows that
Rhizopus has more decided temperature limitations than those that
have been pointed out for Monilia. The results in the last two figures
have been obtained by averaging those of the first seven and therefore
stand as a summary of the various Rhizopus experiments. At 15° and
20° C. the growth rate of Rhizopus rot is practically the same as that of
Monilia rot, Rhizopus being a trifle more rapid at 20° and Monilia just a
little more rapid at 15°. At 10° Monilia rot develops more than twice
as fast as Rhizopus rot, and at 7^° Rhizopus is practically eliminated.

Whether Rhizopus could make any start whatever at 7^° C. seemed
to be determined mainly by the maturity of the fruit. The curves of
figure 15 show that Rhizopus had not made a start at 7^2° in 11 days.
The peaches at that temperature were still free from rot at the end of 14
days, were removed to a warm room at that time, and were entirely
rotted with Rhizopus 2 days later. The results show that the fungus
was held completely in check at j}4° but was alive and ready for rapid
development when given a more favorable temperature. With the ex-
periments reported in figure 17, Rhizopus had produced evident rotting
at 7^° in 6 days. At that time only the ripest peaches were affected,
but at the end of 12 days rots began to develop on the greener peaches.
When once started at this temperature Rhizopus rot made a fairly rapid
growth. The results as a whole show that with the usual number of
days in transit for most peach shipments Rhizopus can produce little or
no damage at 10° and none at 7/^°.

Figures 3 to 21, inclusive, show the development of the rots when the
fruit is stored at the given temperatures immediately after inoculation.
Figures 22, 23, and 24 show the effect of i day's delay at a higher tem-

Nov. 26, 1921 Temperature Relations of Stone Fruit Fungi

461

perature than that at which the fruit was finally held. A study of the
figures brings out the facts that with Monilia i day at 25° C. followed by
I day at 10° results in as large spots as 5 days at 10°; i day at 20^
followed by i at 10° results in as large spots as 4 days at 10°; i day at
25° followed by i at 7^2°, in as large spots as 6 days at y}4°', i day at

Fig. 18.— Monilia on peaches. A summary of the experiments on brown rot obtained by averaging the
percentages of figures 3, 4, 5. and 6.

25° followed by i day at 5°, in as large spots as 10 days at 5°; i day at
25° followed by i day at 2X°, in as large spots as 12 days at 2^2°. It
will also be seen that with Rhizopus i day at 25° followed by i day at
10°, or I day at 15° followed by 4 at 10°, results in larger spots than 7
days continuously at 10°.

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Fig. 19. — Rhizopus on peaches. A summary of the experiments on Rhizopus rot obtained by averaging
the percentages of figures 8, lo, 13, 14, 15, i6, and 17.

Peaches inoculated with Monilia and promptly cooled to 2^°, 5°, 7>^°,
or even 10° C. have had but little or no rot at the end of 3 or 4 days, but
similarly inoculated peaches delayed at 25° for i day» before storing at
these lower temperatures have developed so much rot by the end of the
fourth day after inoculation that they were commercially worthless.
Peaches inoculated with Rhizopus and promptly cooled to 10° have been

462

Journal of Agricultural Research

Vol. XXII, No. 9

3 DAYS

6 DAYS

12 DAYS

10
DEGREES

7.5 S

CENTIGRADE

Fio. 30. — Monilia on peaches. The drawings represent the average of the results from the various experl -
ments. The shaded portions indicate the extent ot the decay. The upper series shows the size of the
rots at the various temperatures after 3 days, the second series the size after 6 days, and the third the
size after 12 days.

3 DAYS

6 DAYS

DEGREES CENTIGRADE

Fig. ai.— Rhizopus on peaches. The drawings represent the average of the results from the various experi-
ments. The shaded portions indicate the extent of the decay. The upper series shows the size of the
rots at the various temperatures after three days and the second series the size after six days.

Nov. 26, 1921 Temperature Relations of Stone Fruit Fungi

463

J^Z.5'

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/?/9y^ /^^72£7P /A/0CaL/9T/0/Y

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Fig. 22. — Effect on Monilia rot of one day's delay at 25° C. The base line shows the number of days after
inoculation and the perpendicular the diameter of the rots. The curves show the rate of development
of the rots at the temperatures indicated at the end of the lines. The dotted lines give the results on the
fruit placed at once at the given temperatures and the solid lines the results on similar fruit delayed one
day at 25° before placing at these temperatures. Elberta peaches, August 20, 1919.

Z 3 <?■ <5' & 7 8

Fig. 23.— Effects on MoniUa rot of one day's delay at 25° or 20° C. before storing at lo". The base line gives
the number of days after inoculation and the perpendicular the diameter of the rots. The curves show
the development of the rots under the particular storage treatment. Experiment on Carman peaches,
August 5, 1920.

464

Journal of Agricultural Research

Vol. XXII, No. 9

entirely free from rot at the end of 6 days, while similarly inoculated
peaches .delayed for i day at 25° before storing at 10° have been almost
entirely rotten at the end or 6 days, and those held at 15° before storing
at 10° have been considerably damaged by the end of 6 days.

The results show the great value of low temperatures in controlling
peach rots and the extreme importance of securing these temperatures
promptly. It is evident that in unfavorable weather success with
long-distance shipments requires not only a low car temperature upon
arrival at destination but a low temperature from the time the peaches
are packed and as much coolness as possible from the time they are
picked. It is not an unusual thing for peaches to remain at the pre-
vailing seasonal temperature for a day or more before being loaded into

Fig. 34.— Effects on Rhizopus rot ot one day's delay at as° or 15° C. before storing at 10°. The base line
gives the number of days after inoculation and the perpendicular the diameter of the rots. The curves
show the development of the rots under the particular storage treatment.

the car, and with most makes of refrigerator cars and the usual methods
of icing it is likely to be one or two more days before a really protecting
temperature is secured. The fruit in the top layers is often still above
10° C. after several days in transit. Prompt loading, better refrigerator
cars and heavier icing, particularly during the first part of a trip, would
contribute greatly to lengthening the life of stone fruits; but the experi-
ments that have been reported show that there would still be a gap that
would sometimes result in heavy losses of fruit and that could only be
filled by some method of precooling.

One of the unfortunate things in regard to delayed cooling is that
its harmful effects may not be immediately evident. Peaches may ap-
pear practically sound after a delay before loading and cooling and yet
that delay may have allowed the rots to make a start that will require

Nov. 26, 1921 Temperature Relations of Stone Fruit Fungi 465

that the temperature be held several degrees lower or that the destina-
tion be selected several days nearer in order to insure the delivery of
sound fruit.

SUMMARY

(i) A temperature of 10° C. (50° F.) has held Monilia in check for one
or two days and Rhizopus in check for three days. A temperature of
7^12° C. (45K° F.) has held Monilia in check for three days and Rizopus in
check for six or more days. A temperature of 5° C. (41° F.) has held
Monilia entirely in check for four days, and 2%° C. (36K° F.) has held
it in check for six days.

(2) Low temperatures have resulted in relatively less inhibition of
growth with Monilia when grown on peaches than when gro^rn on potato-
dextrose agar, and a relatively greater inhibition with Rhizopus when
grown on peaches than when grown on potato-dextrose agar. Both
fungi have grown at lower temperatures on ripe fruit than on green fruit.

(3) Peaches stored at 10° C. (50° F.) immediately after inoculation
have been three to five days slower in developing rot than those delayed
one day at 25° C. (77° F.) before storing at 10° C. (50° F.). Peaches
stored immediately at 'j%° C. (45 K° F.) have been five days slower in
developing brown rot than those delayed one day at 25° C. (77° F.
before storing at 7^° C. (45^° F.).

TRANSPORTATION ROTS OF STONE FRUITS AS
INFLUENCED BY ORCHARD SPRAYING

By Charles Brooks and D. F. Fisher, Pathologists, Fruit Disease Investigations,
Bureau of Plant Industry, United States Department of Agriculture

The present paper reports the pathological results of five years' ship-
ping and storage experiments with green prunes and sweet cherries and
is an attempt to demonstrate certain underlying facts that help to place
the responsibility for transportation and market losses in perishable
fruit shipments. Sprayed fruit and dusted fruit have been compared
with untreated fruit from the same orchards under various transportation
and storage conditions.

Spoilage of fruit has been almost entirely due to Monilia or brown rot
[Sderotinia cinerea (Bon.) Wor.], blue mold rot [Penicillium expayisum
(Lk.) Thom], and black mold rot [Rhizopus nigricans Ehr.]. Monilia
attacks the fruit both in the orchard and on the market, but Penicillium
and Rhizopus are able to develop only on the harvested fruit.

SPRAYED AND UNSPRAYED SWEET CHERRIES IN TRANSIT AND IN

STORAGE

The shipping experiments on cherries were made from the orchard of
L. T. Reynolds of Salem, Greg. The varieties used were Napoleon
(Royal Ann), Black Republican, and Lambert. Various standard spray
materials were used on the different orchard plots, including 2-4-50
Bordeaux plus 2 pounds of rosin fish-oil soap, 8-8-50 self-boiled lime-
sulphur plus 2 pounds of rosin fish-oil soap, and commercial lime sulphur
diluted I to 50. In the 1919 experiments a neutral Bordeaux (4 pounds
copper sulphate in 100 gallons water neutralized with lime) was substi-
tuted for the 2-4-50 Bordeaux, a casein spreader ^ was added to the lime
sulphur solution, and one plot was treated with 85-0-15 ^ sulphur dust.

In 19 15 sprayings were made May 7 and 8 and June i ; in 19 16, April i,
April 21, May 12, and June 15; in 1917, April 25, May 14, May 31, and
June 22; in 1918, April 15, May 2, May 17, and June 18; and in 1919,
June 7 and June 16. The earlier applications (before May 10) were for
the control of blossom infection and probably had little effect upon the
occurrence of rot on the ripe fruit.

In 1915 and 1916 there was practically no foliage injury from any of
the spray materials used, but in each of the following three years very
definite injury occurred on particular plots. In 1917 and 1918 lime
sulphur caused heavy defoliation, and in 19 19 Bordeaux produced con-
siderable foliage injury. All of the spray materials, with the possible
exception of neutral Bordeaux, reduced the size of the cherries. In most
years this was scarcely perceptible, but in 19 17 the dwarfing effect was
sufficient to cause considerable loss.

The cherries of the Willamette Valley are often seriously damaged with
brown rot, yet during the five years' work at Salem there was not a
season in which the experimental orchard had as much as i per cent of
rot at picking time on either the sprayed or unsprayed fruit.

Shipping experiments were made each year to determine the effect of
the orchard treatment upon the carrying quality of the cherries. One

'Fisher, D. F. controi, op apple powdery mildew. U. S. Dept. Agr. Farmers' Bui. 1120, 14 p.,
8 fig. 1920.

• The formula is given in sulphur, lime, arsenic sequence: 85 parts sulphur, no lime, and 15 parts
arsenate of lead.

Journal of Agricultural Research, Vol. XXII, No. 9

Washington, D. C. Nov. a6, 193 1

aaq Key No. G-ijt

75308—22—2 (467)

468

Jourtial of Agricultural Research voi. xxn, no. 9

or more lo-pound boxes of sound cherries from each plot were included
in each shipping test, thus giving a minimum of approximately 1,000
cherries upon which to base any item of a count.

All the shipments were made by express, a part without ice and a part
in pony refrigerators. The refrigerators held sixteen lo-pound boxes of
fruit. They were well insulated and when properly iced maintained a
temperature of 10° to 13° C. (50° to 55.4° F.), usually bringing the warm
fruit do^^^l to this temperature in less than two days.

The results of the various shipping experiments are given in Tables I
to VI and a summary in figure 2.

Table I. — Effect of spraying Napoleon^ and Black Republican^ cherries, Salem,

Oreg,, 191 5

Condition of fruit after shipment and storage.

Percentage

Variety.

Orchard treatment.

Monilia at
picking
time.

Percentage of rot.

Percentage
of

sound

Monilia. Penicillium.

Rhizopus.

fruit.

Napoleon

Bordeaux [ 0. 2

10. 6

3-8

88. q

Self-boiled lime sulphur . 2

II. I

37-9

12. 9

38.1

Untreated

• 7

52. I

7-3

20. 2

20. 4

Black

Bordeaux . . . . :

•03

6.5

15-4

4.8

73-3

Repub-

Self-boiled lime sulphur

.07

2. 0

10. I ! I. 6

86.3

lican.

Commercial lime sul-
phur.

•05

7.8

12. I 1 . 03

80. I

Untreated

•03

17-3

"J. 7 T. •?

77-7

' The Napoleon cherries were picked June 17, stored at 5° C. (41° F.) on June 18, removed and shipped
by express without refrigeration June 27, received at Wenatchee, Wash., June 29, still practically free
from rot, and held in a -warm laboratory till July 2, when notes were taken.

" The Black Republican cherries were picked Jmie 24, stored at 5° C. (41° F.) June 25, removed and
shipped by express without refrigeration June 27, received at Wenatchee, Wash., June 29, still practically
free from rot, and held in a warm laboratory till July 6, when notes were taken.

Table II.

-Effect of spraying Napoleon ^ and Black Republican - cherries, Salem, Oreg.,
1916

Orchard treatment.

Percentage of rot after shipment
and storage.

Percentage

Variety.

Monilia.

Penicil-
lium.

Rhizopus.

of sound
fruit.

Napoleon

Bordeaux

12. 6

40. 5

21-5

64-5
80.8

19. 6

24.7
14. 2
36.9

35- 0

7-4
12. 0
16.2

4. I

16.7
4.0

•7

14. 2

. I

3-7

2.4

20. 0

°
I. I

•3
.6
. 2

80. 0

Self-boiled lime-sulphur ....
Commercial lime-sulphur. . .
Commercial lime - sulphur

(last spraying omitted).
Untreated

43-8

59-9
II. 4

2. ";

Black Repub-
lican.

Bordeaux

75. 3

Self-boiled lime-sulphur ....
Commercial lime-sulphur. . . .
Commercial lime - sulphur

(last spraying omitted).
Untreated

74-3
71.0
62. I

64.9

' The Napoleon cherries were picked July 3, stored at 5' C. (41' F.) July 4, removed and shipped by
express without refrigeration July 6, received at Wenatchee, Wash., July 8, and held in a warm laboratory
till July 20, when notes were taken.

' The Black Republican cherries were picked July 6 to 10, stored at a temperature of s° C. (41° F.) till
July 14, shipped by express without refrigeration to Wenatchee, Wash., received July 16, and held in a
warm laboratory till July 21, when notes were taken.

Nov. 26, 1921

Transportation Rots of Stone Fruits

469

Table lll.—Efect of spraying Napoleon ^ and Lambert ^ cherries, 1916

Orchard treatment.

Bordeaux

Self-boiled lime- sul-
phur ,

Commercial 1 i m e - s u 1-
phur

Commercial 1 i m e - s u 1-
phur (last spraying
omitted)

Untreated

Percentage of rot after shipment and storage,

On Napoleon.

On Lambert.

July 12.

July 13.

July 22.

Monilia.

Other rots.

Monilia.

Other rots.

Monilia.

Other rots.

2. 2

0. 2

6.0

0.8

5-6

4. a

II. 0

23.8

I. 2

5-7

I.-6

4.0

7-5

7.0

II. 6

I- 5
14.8

0
. I

9-5

37-7

I. S
1.6

3-7
21. 0

12. 0
3-6

' The Napoleon cherries were picked July 5 and shipped in pony refrigerators the same day, received in
Washington, D. C. , July 12, with ice pans empty and fruit warm, and held without cooling till July 13.

2 The Lambert cherries were picked July 14 and shipped in pony refrigerators the same day, received in
Washington, D. C, July 21, and notes taken on July 22.

Table IV. — Effect of spraying Napoleon} Black Republican.- and Lambert^ cherries,

1917

Percentage of rot after shipment and storage.

Percentage of rot after
shipment on Lambert.

Orchard treatment.

On Napoleon.

On Black Republican.

Mo-
nilia.

Penicil-
lium.

Rhizo-
pus.

Mo-
nilia.

Penicil-
lium.

Rhizo-
pus.

Mo-
nilia.

Penicil-
lium.

Rhizo-
pus.

Bordeatix

0
. 1

•3

0. 2
0

• 3

0
. I

1-3
•9

0. 2

. I

2.7

1 1., 8
2. 0

•4

•3
. I

2.4

9-3
I. I

. I
1.4

0. I
0

0-5
0

2.8

Self-boiled lime - sul-
phiu'

2. 3

As above, but last ap-
plication omitted. . . .

Commercial lime - sul-
phur

5-0

•3
. I

4. 0

Untreated

II. 9

' The Napoleon cherries were picked July 7 and shipped in pony refrigerators the same day, received in
Wenatchee, Wash., July 12, and held in a warm room till July 14, when notes were taken.

' The Black Republican cherries were picked July 15, packed in pony refrigerators the same day and
shipped to Wenatchee, Wash., received July 18, held under ice till July 19 and then at room temperature
till July 34, when notes were taken.

' The Lambert cherries were picked July 21, shipped in pony refrigerators the same day, received in
Washington, D. C, July 30, when notes were taken.

470

Journal of Agricultural Research voi. xxii, No. 9

Table V. — Effect of spraying Napoleon cherries, IQ18 '

Orchard treatment.

Percentage of rot.

Lot.

On fruit picked and packed in the On fruit picked and packed in the
middle of the day. cool of the morning.

Monilia.

"ir^"- ! Rh'-p-- ' ^'onm.. : ^^■

Rhizopus.

Bordeaux

1-5
1.9
1.9

4-3
.6
0

. 2

6.5
0

0
1-5

10. 0

9.4
7.0
6.0

9-3
2.9
1.6

2.7
3-6

I. 2

12. 0

2.9

• /
1.9

I. 0

4.0

•3

26. 9
39- 0

4.6
"•3

41. 0

Iv I

36.5

9.0

22. 0

33-7

Lime-sulphur

Lime-sulphur (last

application

omitted).

Untreated

Bordeatix

•

0 0. 8

8.7
52.3

67.4
42.9

24-5
21. 4

Bordeaux (last ap-
plication omit-
ted).

Lime-sulphur

Lime-sulphur (last
application
omitted).

Untreated

Bordeaux

I. 2

1. 2

2. I

. 2

•4
2.8

. 2

1.6
.6

•9
•9
.8

6.2
3-2

5.8

Bordeaux (last ap-
plication omit-
ted).

Lime-sulphur

Lime-sulphur (last
application
omitted).

Untreated

31-5

• 60. 4
19.0

64.8

' The cherries were picked June 26. Lot \ was held 12 days at Salem, Oreg. , in an open warehouse. Lot
B was shipped in pony refrigerators to Wenatchee, Wash. , held under ice till J uly 3 . and without ice one day,
notes being taken July 3. Lot C was shipped in pwny refrigerators to Washington, D. C, arriving July 3,
and held warm till July 5, when notes were taken.

Table VI. — Effect of spraying Napoleon cherries, igig ^

Plot

No.

Orchard treatment.

Bordeaux . . . .
Lime-sulphur
Sulphur dust.
Untreated

Percentage of rot.

July 3, after 6
days in transit.

Mo-
nilia.

Pent
cillium.

14.7

22. 4

I. O

I. 2

22.8

1-5

July IS. after storage at
15° C. (s9° F-) for ij
days.

Mo-

Peni-

nilia.

cillium.

II- 5

3-5

13- «

4-5

9-3

37- 0

57- 0

15-2

Rhizo-
pus.

35-2

July 15, after storage at
5° C. (41° F.) for 13
days.

Mo-

Peni-

nilia.

cillium.

10. 2

7-5

12.3

4.2

19.4

26.5

28.0

•7

Rhizo-
pus.

' The cherries were picked from plots i and a on June 25. A rain followed on June 26, and the cherries
from plots 3 and 4 were picked on June 27. The rain probably resulted in there being relatively less pro-
tective material left on the dusted than on the sprayed fruit. The picked fruit from plots i and 2 was not
placed under ice till June 27. and this delay in cooling may have partly or entirely offset any harmful
effects from the rains received by plots 3 and 4 Two lo-pound boxes from each plot were shipped in a pony
refrigerator to Washington, D. C, were received warm July 2, were held overnight at a temperature of
approximately 7° C. (44.6° F.), andnotes were taken July 3. The sound fruit was saved, and half of the cher-
ries from each plot were stored at a constant temperature of s° C. (41° F.) and half at a constant tempera-
ture of is" C. (59° F.). On July 15 notes were taken on the amount of rot that had developed in storage.

Nov. 26, 1921

Transportation Rots of Stone Fruits

471

A discussion of the results from the cherry experiments is given on
pages 474-477-

SPRAYED AND UNSPRAYED ITALIAN AND AGEN PRUNES IN TRANSIT

AND IN STORAGE

The prune is best known as a dried product, but a considerable part of
the western crop, especially from the irrigated districts, is shipped to
the eastern markets as "green" or fresh prunes. The question of the
development of rots is a very important one in such shipments, and it is
also an important consideration when delays occur at the drying plants.

The spraying experiments were made in the orchards of A. W. Moody,
Felida, Wash., and L. T. Reynolds, Salem, Oreg. Both Italian and Agen
(Petite or French) prunes were included in the tests. The spray materials
were similar to those described for the cherries. In 1915,1918, and 191 9 a
4-4-50 Bordeaux was used, and in 1916 and 1917 a 2-4-50 Bordeaux.
In 1916 to 1919, inclusive, a 50-35-15 sulphur dust ^ was used in the
earlier applications and a 50-50-0 in the last. In 19 19 a second sulphur
dust plot was given an 85-0-15 mixture in the earlier application and
an 85-15-0 mixture in the last, and two different brands of Bordeaux
dust were tested.

In 1915 sprayings were made March 24, April 8, May i, June 21, and
August 6 in the first orchard, and May 29, June 21, and August 6 in the
second orchard; in 19 16, April 8 to 12, April 25 to 27, May 30 and
August 30 at Felida, Wash., and April i, April 21, and June 16 at Salem,
Oreg., in 1917, April 28, May 18, June 15, and September 12; in 1918,
April II, April 29, May 27, and August 20; and in 1919, April 8, April
25, May 21, and August 25. The earlier applications (before May 10)
were for the control of blossom infection and probably had little effect
upon the occurrence of rot on the ripe fruit.

Table VII. — Effect of spraying Italian prunes, Felida, Wash., IQI5^

First orchard .

Second orchard.

Orchard treatment.

Bordeaux

Self-boiled lime

sulphur.
Commercial lime

sulphur.

Untreated

Bordeaux

Self-boiled lime

sulphur.
Commercial lime

sulphur.
Untreated

Percent-
age of

Monilia
rot at

harvest.

0.9

•7

3-4
4.2

Z-3,
4.8

5-4

Percentage of rot Sept. sr, after
shipment and storage.

Monilia.

2.7

7.0

9-3

30-3

35-7

20. 6
56.0

Peni-
cillium.

Rhizopus,

27.7
36-4

44.2

29.8
22.7 ]

25-4 !

32.6 j

I
14. 1 i

33-5
31-5

21-5

28.5
28.8
38.9

17-5
28.6

Percent-
age
sound
Sept. ai.

36. I
25-1

25.0

II. 4

12.8

7-9

29-3

1-3

' The trees in the first'orchard were 24 years old, and those in the second 15. The prunes were har-
vested Sept. 7 to 10, shipped to Wenatchee, Wash., by ordinary express, and held without refrigeration
till Sept. 21.

Fifty pounds sulphur dust. 35 pounds of lime, and is pounds arsenate of lead.

472

Journal of Agricultural Research

Vol. XXIt, No. 9

Although brown rot usually caused heavy losses in the neighboring
orchards, sometimes destroying more than 75 per cent of the crop, it was
never serious even on the control plots in the orchards in which the
experiments were made. Notes were taken on the amount of rot at
picking time and also on the amount developed in shipment and in
storage. The shipping tests were carried out as described for the
cherries. The prunes used in the experiments were carefully picked from
the tree several days before the drying season began. They were too
green for drying, yet riper than the average "green" prune shipments.

Table VIII. — Effect of spraying Italian prunes, Felida, Wash., IQ16 '

Orchard treatment.

"Bordeaux

•Self-boiled lirae-sul

phur

As above, but last ap

plication omitted.
Commercial lime-sul

phur

Sulphur dust ....,.,
Sulphur dust (lastap

plication omitted)
Untreated

Percent-
age of

Monilia-
rot at

harvest.

2.4
3-8

2. 2
1.6

4-7
8.0

Percentage of rot Sept. 19, after
shipment.

Monilia.

Peni-
cillium.

2.7

■3
1.9

o
.6

2.7

Rhizopus.

0-3

o
1-5

2.8

Percentage of rot Sept. 23, after
shipment and warm storage.

Monilia.

8.0

3-5
19.4

6-5
2.8

5-4

Peni-
cillium.

12. I
8.2
I. I

8.4

Rhizopus.

12.8

14.4

2.9

8.9
II. 4

» The prunes were harvested Sept. 13 and Sept. 33. Fruit from the first picking was shipped in pony
refrigerators to Washington, D. C, received in good condition Sept. 19, and held at room temperature
till Sept. 23.

Table IX. — Effect of spraying Italian prunes, Salem, Oreg., igi6 '

Orchard treatment.

Bordeaux

Self-boiled lime-sulphur.
Commercial lime-sulphur
Untreated

Percentage of rot after shipment and
warm storage.

Monilia.

7- I

7.6

1.9

12. 9

Penicilium. Rhizopus

3-8

1-7

2.6

' The prunes were practically free from rot in the orchard, were harvested Sept. 6. shipped by express
without refrigeration to Wenatchee, Wash., and held at room temperature till Sept. 16, when notes were
taken.

Nov. 26, 1921

Transportation Rots of Stone Fruits

473

Table X. — Effect of spraying Italian prunes, Felida, Wash., igiy '

Orchard treatment.

Bordeaux ■. . .

Self-boiled lime-sulphur .
As above but last omitted

Lime-sulphur 1-50

Sulphur dust

Untreated

Percentage of rot.

Monilia at picking
time.

First
picking.

3- I
1-5
4.0
4.6
6.9

14-5

Second
picking.

0.4

•3
•7
•4
.8

2-5

Total.

1.6
.6

1-5
2. o
3-8
6.5

After shipment to
Wenatchee, Wash.

MoniUa

2-3
I- 5
1.8

■ -3

2.8

18.2

Penicil-
lium.

15.6
10.8
48. I

31-7
36.2
15.6

Rhizo-
pus.

After shipment
to Washing-
ton. D. C.

10.5

12. 7
26. 4

Penicil-
lium.

82.9

62.6

' The prunes were harvested Sept. 32, to 26 and Oct. 2 to 5. The shipment to Washington, D. C, was
nude in a pony refrigerator, started Sept. 25, received Oct. n with no ice and in such bad condition that
several lots were discarded. The shipment to Wenatchee, Wash., was by express without refrigeration,
started Sept. 26, received Sept. 28 and allowed to stand in a warm room till Sept. jo, when notes were
taken.

Table XI. — Effect of spraying Italian prunes, Felida, Wash., iQi8^

Orchard treatment.

Monilia

picking

time.

Bordeaux

Self-boiled lime-sulphur.
As above but last omitted
Commercial lime-sulphur .
As above but last omitted

Sulphur dust

Untreated

Percentage of rot.

Monilia.

After shipment to We-
natchee, Wash., and
8 days' storage at 15°
C.

1-3

o
.6

o
•3
•4

1-5

Penicil-
lium.

1.6

Rhizo-
pus.

After shipment to Wash-
ington, D. C, under
ice and 2 days' delay
at 25° to 30° C.

Penicil-

lium.

0.4

1-7

•4

1-5

Rhizo-
pus.

31-
37-
9-
34-
IS-
39-
24.

' The prunes were harvested Sept. 6 and 11. The shipment to Washington, D. C, was made in a pony
refrigerator, started Sept. 6, received in good condition Sept. 14, and notes taken Sept. 16. The shipment
to Wenatchee, Wash., was without refrigeration, started Sept. 11, received Sept. 12, and held in cellar
•tcrage till Sept. 20, when notes were taken.

474

Journal of Agricultural Research

Vol. XXII, No. 9

Table XII. — Effect of spraying Italian and A gen (Petite or French) prunes, Salem

Oreg. , igiQ '

Percentage of rot.

After shipment

Total rot in the re-

to Wenatchee,

After shipment

frigerator shit>

Wash., without

to Wenatchee,

ment after the j

ice and 5 days'

Wash., under

days' cool stor-

storage with-

ice and 5 days'

age had been fol-

Variety.

Orchard treatment.

out ice, iS° C.

storage under

lowed by 3 days'

"o.

(64.4° F.).

ice.

warm storage.

a
1

■(J

.2
1

3
•3

(ti

Italian... .

Bordeaux

1.6

3- 2

4.6

9.6

2.1

As above but last omitted. . .
Commercial lime-sulphur

12.5

2.1

•9

■7

3-9

0.4

8.3
. I

71.8

1.3

1-3

17-5
.6

0-5

0
0

36.1
II. 0

1-3

A£en

Self-boiled lime-sulphur

1. 1

As above but last omitted. . ,

. 2

13-9

38. S

1.9
I. 2

7.6

I. I

•9

8.7
1.8

2.8
1.4
7.0

0
0

1-3

20. 3
34-6

.8
. 3

1.6

Untreated

1 The prunes were harvested Sept. 15, shipped to Wenatchee, Wash., Sept. it, received Sept. 18, and
notes taken Sept. 23.

2 No shipment.

The contrast in the amount of brown rot (MoniHa) on the fruit from
the different plots after shipment and storage is shown graphically in
figure I.

A study of the figure shows that the unsprayed fruit developed six
to nine times as much brown rot under transportation and market

si BO/?£>£/?UX

SULPf-/U/? OUST SO-3e-/S —

^i so/?£>£:/9ax otASr b

/o ;so .so '?c

Fig. I. — Brown rot on Italian and Agen prunes after shipment and storage. 1919.

conditions as the fruit from the plots receiving the best orchard treat-
ment. The sulphur dust was as efficient as the sprays, but the Bordeaux
dust was far less efficient.

The results show that orchard spraying may have great value on the
market even when the amount of disease in the orchard has been negli-
gible.

DISCUSSION OF RESULTS

In order to obtain the composite results from the various prune and
cherry experiments the data from the different spraying and shipping
tests have been brought together and averaged. The results are shown
in figures 2 to 6, inclusive.

Nov. 26, 19:1

Transportation Rots of Stone Fruits

475

Figure 2 shows the comparative efficacy of spraying and dusting as
determined by the average of the four years' results on prunes. Little
contrast is shown between the two methods of treatment, both sulphur
dust and self-boiled lime-sulphur having reduced the amount of brown
rot at picking time from 4 per cent to approximately i per cent and

<s /o /s

:so

SO-35-J5 <SULFNO/? £>i/,5T

/(VT/frT£/? ^MPAfS/^

a£LJ^-Bi>/^£P L/M£-SULPHU^
S0-35-i5 SULPHUR OUST

Fig. 2. — Comparative results from spraying and dusting in a four years' test on prunes.

reduced the amount developed in shipments from 16 per cent to 2.5
per cent.

It was pointed out earlier in the paper that the different spray appli-
cations were probably not of equal value in the control of brown rot on
the fruit. The great importance of the last application in this connec-
tion is shown graphically in figures 3 and 4. A reference to these figures

S^R/9y£D

/'^r/^CSA/T/fGE OrMO/V/L//? /?OT

o ^ /o /s

;so

Fig. 3.— Brown-rot control of cherries as influenced by a late spray application (about three weeks before
picking time). The average results obtained from 15 different shipping tests.

shows that with the prunes approximately one-half and with the cherries
approximately one-third the brown-rot control was due to this late
spraying.

The comparative results obtained with the different rots in the various
shipping experiments are shown in figures 5 and 6. The term sprayed

s^/?/9yEi?

i/Ms^^t9y£ri>— -" —

^/¥?t9y££f •

Sf^^y£ii/^Sr i^Pt/C/9T/CW 0/t?/TT£O

i/MS/'^/fy£0

/'£^^£/Vr/?0£ OFAfOMl//? /rtTT
O ^ /O /S

Fig. 4. — Brown rot control of prunes as influenced by a late application of spray or dust (three to five
weeks before picking time). The average results obtained from 7 orchard experiments and 11 shipping
tests.

as used in these two figures includes both dusting and spraying.
As has already been pointed out, there was no year in which there was a
serious epidemic of rot in the orchards under investigation. The amount
of rot on the untreated cherries at picking time never ran as high as i per
cent. The average amount of brown rot on the sprayed prunes at

476 Journal of A gricuUural Research voi. xxii. no 9

picking time, as shown in figure 6, was 1.6 per cent and the average
amount on the untreated prunes was 4.6 per cent. The orchard loss
from rot with either the prunes or the cherries would be considered of
very minor importance in practical operations, scarcely justifying the
expense of spraying; yet even under these conditions the orchard spray-
ing has shown decided beneficial effects in the carrying quality of the
fruit in transportation and storage. The good eft'ects, however, have
been largely if not entirely confined to the control of Monilia rot. With
both the prunes and cherries the unsprayed fruit has developed approxi-
mately four times as much of Monilia rot as the sprayed fruit,
but has shown .practically no greater susceptibility to Penicillium
and Rhizopus rots. These contrasting results are in harmony with
the nature of the different fungi. Monilia is a parasitic fungus and
able to penetrate the sound skin of both ripe and green fruit;
PenicilHum and Rhizopus are saprophytic fungi, able to attack only the
harvested fruit and dependent upon bruises and skin cracks for first
points of entrance. The Monilia spores come primarily from the orchard,
but Penicillium and Rhizopus have an almost universal distribution.

/?Or^L /9^/^ r ^ '^ ^^ /.^ ^ ZS-

\sp/f/?yi^£> "^ =— ■ ■

P£N/C/LUU»? ^SP/?/?yS£>

/>A/D ffH/20PLis\y/vs/'/?/9rep

Fig. s. — A comparison of the average development of rot on sprayed and unsprayed cherries in i8 different
shipping and storage experiments.

Under such circumstances it would be expected that orchard spraying
would furnish at least partial protection against brown rot in transpor-
tation and storage, since it would both decrease the supply of spores and
furnish a more or less complete protecting film on the fruit. On the
other hand, orchard spraying could not be expected to have any appre-
ciable effect upon the spore supply of fungi like PenicilHum and Rhizopus
that are of general occurrence, and a film of spray on the skin could
offer little protection against fungi that enter through breaks in the skin.
While these contrasts in the different fungi are of importance in con-
nection with the present studies, it should not be inferred from the fore-
going statements that skin punctures have no effect upon the occurrence
of Monilia rot, for it is well known that any abuse to the fruit is decidedly
favorable to the development of the disease; ^ nor should it be inferred
that Rhizopus and Penicillium are entirely unable to penetrate the sound
skin, for when these fungi are once well established in a crate they may
spread out from a center of infection without much regard to the sound-
ness of the adjacent fruit. This is particularly true of Rhizopus, and
especially where it is favored by a high temperature. Under such a
condition it often spreads through a package of stone fruit in a most
rapid and indiscriminate manner.

1 Ramsey, H. J. the h.\ndling and shipping op fresh cherries prom the wxi,i,.\mette valley
U. S. Dept. Agr. Bui. 331, 28 p., ii fig., 1916.

Nov. 26, 1921

Transportatio7i Rots of Stone Fruits

477

With all the rots temperature has been an extremely important factor.
In the 1 9 19 experiments (Table VI) part of the fruit was stored at 15° C.
and part at 5°, with striking contrasts in the results. Rhizopus was en-
tirely eliminated at the lower temperature, and Penicillium and Mon-
ilia were greatly reduced. Short shipments without refrigeration have
resulted in heavy losses, while fresh fruit, both sprayed and unsprayed,
has been shipped across the continent in pony refrigerators under ice
with no decay upon arrival. Fruit that was free from rot after seven
days in the refrigerators became badly decayed after standing one or
two days in a warm room, the unsprayed fruit always developing the
most rot but the sprayed fruit never remaining free from it. Refrigera-
tion is always valuable; but it is evident that its importance increases
with any decrease in orchard or packing-house care.

It is evident that there is a widely distributed responsibility for the
occurrence of stone fruit rots in transit and in storage. Orchard spraying
may be one of the important factors in the control of Monilia rot on the

/o /^ ieo

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^W/'/HEA/T \UJVSP/I9r£p

Fig. 6.— a comparison of the average development of rot on sprayed and misprayed Italian prunes in i:
different shipping and storage experiments.

harvested fruit, but as a protection against Penicillium and Rhizopus
rots it has little or no value.

SUMMARY

(i) Orchard spraying has reduced the amount of Monilia or brown rot
developed on sweet cherries in transportation and storage experiments
from 24.3 to 6.4 per cent. All the cherries were from orchards where
there was less than i per cent of rot on either the sprayed or unsprayed
fruit at picking time.

(2) In similar shipping and storage experiments with Italian prunes
there has been an average of 28 per cent of brown rot on the untreated
fruit and 7.1 per cent on the sprayed or dusted fruit. The amount of
rot on the unsprayed fruit at picking time was 4.6 per cent and on the
sprayed fruit 1.6 per cent.

(3) About half the brown rot control secured in the shipping tests
with prunes and about one-third of that secured with cherries was due
to the spray application made three or four weeks before picking time.

(4) There has been little contrast between the brown rot control
secured with sulphur dust and that secured with the standard spray
materials.

(5) Spraying and dusting have had little or no effect upon the develop-
ment of Penicillium and Rhizopus rots in transit and storage, their
occurrence apparently being much more influenced by the prevalence
of bruises and skin punctures.

(6) The unsprayed fruit has shown a greater need of refrigeration
than the sprayed, and the injured fruit a greater need than the sound.

STORAGE OF CONIFEROUS TREE SEED

By C. R. TiLLOTSor^

Forest Exartmier, Forest Service, United States Department of Agriculture

During the period from 1909 to 19 13 the United States Forest Ser\'-ice
was especially active in its reforestation program. There were large
areas of deforested land on the national forests, and there was a sincere
desire on the part of the organization to serve the public interest by
bringing these lands into a productive state as soon as possible. The
program involved the growing in nurseries and planting of many millions
of young trees each year and also the sowing of seed directly on extensive
areas of deforested land. To carry out the program large quantities of
seed were needed. In the year 19 10 alone, 63,000 pounds of seed were
collected. Foresters know that seed is not borne in the same abundance
upon trees each year. A good seed crop in any region is often followed
by one to several very poor or lean crops. It accordingly is desirable
to collect during years of plenty seed in large enough quantities to last
several years. The Forest Service did pursue this course and thereupon
became confronted with the problem of how to store the seed so that
it would not deteriorate greatly in germinative ability and energy before
it could be used. This problem was not a new one. European foresters
had been faced with it a good many years ago and had made substantial
progress in its solution for some species. One of the most intensive
sets of European experiments with coniferous seed was perhaps that of
Dr. Adolf Cieslar ^ which was begun in the spring of 1886 and continued
over a period of 1 1 years. Dr. Cieslar attempted to determine not only
the efifect of air-tight storage on seeds of Norway spruce, black (Austrian)
and white pine, but also the effect of heating these seeds before placing
them in storage. He came to the conclusions that (i) storing under
air-tight covering lengthens the life of these species of seed so that when
stored in this way they often show a considerably higher germination
percentage, especially in the later years of storing, than seeds of the same
origin stored in the air, this difference in favor of air-tight storing of
seed amounting to 33 per cent in the case of 6-year-old Norway spruce
seed ; (2) storing seed away from the air also results in higher germinat-
ing power; (3) tbe application of heat at 45° to 55° C. to seeds of white
and black pine at the beginning of storing injures the viability of these
seeds and also their germinating power to a considerable extent, but Nor-
way spruce is injured less by this means, and its germinating power is
even kept at a high point by strong heating; (4) heating at 30° to 40° C.
for one hour has a not unfavorable effect on the seed of these three species.
When stored away from the air, such seed maintains both viability and
germinating energy at as high a point as that of unheated seed; in fact,
the slightly heated seed shows in later years of storing, a tendency to
germinate in a very rapidly rising curve.

1 Cieslar, Adolf, versuche uber aufbbw.\hruno vo^f NADELHOLWAME>f unter luftdichtem
VBRSCHLussE. /» Centlbl. Gesam. Forstw., Bd. 23, Heft 4, p. 167-174- i^97-

Journal of Agricultural Research, Vol. XXII, No. 9

Washington, D. C. Nov. 26, 1921

aar Key No. F-7

(479)

^.So Journal of Agricultural Research voi. xxii, no. 9

Experiments conducted by the German Chief Forester Haack ^ at the
Eberswald Forest Academy from 1906 to 1909 confirm some of the results
of Dr. Cieslar and give additional information on the subject of seed
storage. His experiments were confined to seed of Scotch pine. He
found that exclusion of air can not wholly prevent a loss of germinating
power with increasing age of the seed. This is manifested less by a final
decrease of germinating percentage than by a falling off in germinating
energy. But compared with that of seed stored in the air, this decrease
of germinating power is extremely slight. After three years the air-
tight seed had a germinating power of nearly 90 per cent as against 22
to 70 per cent for seed stored in the open air in the same room.

Another conclusion of Haack's was that in no case should seed that
has been shut up in air-tight containers without previous thorough
drying be placed in a storeroom in which the temperature is likely to
increase, even if only occasionally (for instance, in attics). This con-
clusion was based on two experiments. In one, two air-tight bottles
were placed on thermostats heated to 36° C. and left for eight weeks.
One of the bottles contained air-dried seed, the other seed from which
5 per cent of its weight in moisture had been removed in an exsiccator.
In the test, the former germinated only a little over i per cent, the latter
96 per cent. In a similar experiment at a temperature of 30° C, the
germination test at the end of four months gave 40 per cent and 9^
per cent, respectively. It might have been just as well for Chief Forester
Haack to emphasize the necessity of thorough but not excessive drying
of the seed before placing them in air-tight containers rather than stress
the point of storing in a fairly cool room not subject to a rise in tem-
perature. Such rooms are to be had only by some special arrangement.
Later in the same article, Haack does state that in storing pine seed
air-tight, care should be taken that the seed is neither moist nor over-
dr)^ He decided that the degree of dryness which the seed has when
it comes from the kiln or when spread out in a well-heated room or
dried in the sun would probably be best — about i to 2 per cent lighter
than its average weight in the ordinary seed bin. His experiments
showed that long-continued drying is harmful to pine seed. The ger-
minating power of Scotch pine seed left in the exsiccator for four years
fell to 16 per cent at the end of that time, while the same seed not dried
in this way still show-ed a germination of 80 per cent.

Haack conducted one experiment to determine the eft'ect of different
temperatures of the storeroom upon seed in air-tight containers. Scotch
pine was stored for three years in a room heated to a temperature oi
20° to 25° C, in an unheated room, and in a cellar i meter in depth
Both cellar and unheated room were free from frost in winter; in sum-
mer the unheated room was somewhat warmer than the cellar. The
results indicated that a temperature of 20° to 25° C. in the heated room
was injurious to the seed. The germination percentage dropped from
96 to 82 in the three years. With strong seed there was very little
decrease in germination of seed stored either in the basement or in the
unheated room. With weak seed, however, not thoroughly dried before
being placed in the container, there was, after three years, a difference
in germination of 20 per cent in favor of the seed stored in the basement.

These experiments of Dr. Cieslar and Chief Forester Haack, while
thorough in themselves, w^ere confined to only four species, three of

1 Haack [Otto H. A.], der kiefersamen. vErhaltnis zwischen keimprozent und praktischem
WERT, mehrjahrige /ufbewahrung ohne verminderung des keimprozents. In Ztschr. Forst. u.
Jagdw., Jahrg. 41, Hett 6, p. 353-381, i fig. 1909.

Nov. ; o, 1921 Storage of Coniferous Tree Seed 48 1

them European. In order to meet an immediate need for information,
it seemed desirable to extend and expand upon them somewhat to
those American species most used in reforestation operations on the
national forests. These species were western yellow pine {Pinus pondc-
rosa Law.), western white pine {Pinus monticola Dougl.), white pine
(Pinus sirobus Linn.), Engelmann spruce (Picea engelmanni Engelm.),
Douglas fir {Pseudotsuga taxifolia (Law.) Britton), and lodgepole pine
(Pinus contorta Loud). The study should now be followed up with
those more sensitive coniferous seeds, the true firs, the cedars, arbor-
vitas, redwoods, and the numerous species of American hardwoods of
which so little is known.

The study brings a realization of the fact that it is a mistake to deal
with so many variables in an intensive project of this nature. It is
difficult if not impossible to be sure of the cause or causes for any particu-
lar result. There is now a need for further investigations of slightly
smaller scope, in which the variables will be reduced to a minimum and
in which by laboratory methods, the exact physiological, chemical, and
any other changes which the seeds undergo can be followed closely.
These changes almost surely will throw light upon the behavior of seed
in storage.

FACTORS AFFECTING EXPERIMENTS

Some of the conditions under which this study was carried out should
be stated. Because of a shifting in personnel the study in various stages
has come under the direction of several men. This has not been con-
ducive to the best development of the project, and it may be that the
analyses of the results are not so thorough as though made by the one
who conceived the study. Apparently through oversight, no tests of
the seed were made before they were put in storage. It is not known,
accordingly, to what extent deterioration progressed in the seed during
its first year of storage. That there was deterioration in the case of most
containers is shown by the very general superiority after one year of the
seed stored in air-tight bottles. Because of this lack of an original test,
it has, in making analyses of the results, been necessary to use as the
basis of comparison, the germination of seed after storage for one year in
the air-tight bottles.

On account of the large number of variable factors involved (6 species
of seed, 5 kinds of containers, 13 storage points, and 3 temperature con-
ditions at each of these points), the general conclusions are by no means
fully supported by the results in every individual test. It is thought,
however, that the average results are a safe criterion of what may in
general be expected of these coniferous seed in storage.

Fresh seed, with the wings removed, of the species previously men-
tioned was obtained during the fall and winter of 1908-9 in the amounts
and from the sources indicated below:

Picea engelmanni, lo pounds, San Isabel National Forest, Colorado.

Pinus monticola, 55 pounds, Coeur d'Alene National Forest, Idaho.

Pinus contorta, 12 pounds, Deerlodge National Forest, Montana.

Pinus ponderosa, 70 pounds, Boise National Forest, Idaho.

Pinus strobus, 30 pounds, New York State.

Pseudotsuga taxifolia, 25 pounds, San Isabel National Forest, Colorado.

"When the seed was all brought together at Washington, D. C, it was
spread out thinly on a floor and fanned steadily for two days by means
of an electric fan. The object was to dry the seed coats thoroughly.

482 Journal of Agricultural Research voi. xxii.no. 9

Each lot of seed was then divided roughly into portions of about 600 to
800 seeds each, and these were distributed equally among the following
containers :

1. Ordinary manila paper coin envelopes.

2. Similar envelopes soaked in melted paraffin.

3. Cotton cloth bags.

4. Similar bags soaked in boiled linseed oil and dried.

5. Glass bottles which after filling were sealed air-tight with paraffin.
Seed of all six species stored in each of the five containers constituted

one test set of samples. For convenience in handling, shipping, and
storing, each test set was placed in a small wooden box lined with a wire
mesh to prevent the access of rodents.

POINTS OF STORAGE

It was one purpose of this study to determine whether seed deteriorated
in storage to a greater extent in one geographical region than in another.

AJjV-~

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Fig. I. — Map showing points at which coniferous seeds were stored to test effect of geographical locatioa

Thirteen points of storage, accordingly, as indicated below and on the
map (fig. i), were selected. These, it will be noted, are rather widely
scattered over the United States and afford a fair basis for arriving at
some conclusions concerning this particular aspect of seed storage.

POINT OP STOR.\Ge ' APPROXIMATE ALTITUD3

I. Ann Arbor, Mich 875 feet

a . Dundee, 111 700 feet

3. Fort Bayard, N. Mex 6, 500 feet

4. Halsey, Nebr 2, 700 feet

5. Ithaca, N. Y 800 feet

6. Lake Clear Junction, N. Y i, 600 feet

7. Lawrence, Kans 800 feet

8. New Haven, Conn 30 feet

9. Pikes Peak, Colo 9, 000 feet

10. Pocatello, Idaho 4, 500 feet

11. State College, Pa i, 150 feet

12. Warsaw, Ky 400 feet

13. Waukegan, 111 600 feet

Nov. 26, 1921 Storage of Coniferous Tree Seed 483

CONDITIONS OF STORAGE

Another point on which it was hoped this study would throw some
light was the eflfect of several conditions of temperature on seed in stor-
age. At each of the geographical points mentioned, accordingly, the
cooperators in the study were requested to store the seed where each of
the following conditions of temperature would prevail :

1. Ordinary indoor temperature, such as an office shelf where the
temperature would always be above the freezing point.

2. Fluctuating temperature, as in an outbuilding or unheated garret
where the temperature would follow rather closely the actual outdoor
variations. Proximity to a stable was to be avoided.

3. Fairly uniform low temperature, such as prevails in an unheated
basement or cellar.

To just what extent these conditions obtained and were entirely
comparable at all points of storage, it is not possible to say. It would
not be surprising if there were considerable differences, particularly in
the low-temperature conditions. It is believed, however, that they were
similar enough to warrant the drawing of general conclusions from the
tests.

PERIOD COVERED BY STUDY

The study was planned to cover a period of approximately five years.
The seed was sent to the 13 points of storage during March, 1909. In
January, 19 10, and again in January, 191 1, 19 12, and 19 14, three test
sets (one stored at each of the three temperature conditions) were for-
warded by express from each of the storage points to Washington,
D. C, for testing.

It is thus seen that tests were carried on after the seed had been in
storage for periods of approximately one, two, three, and five years.
There was no test of seed in storage for four years. From a practical
standpoint, at least, it seemed that tests covering a period of five years
would be sufficient. It is unhkely that seed in commercial quantities
at least will be stored for a longer time. As a matter of interest, how-
ever, a few of the seeds which had been stored in bottles were carried
over for another five years and tested during the year 19 19. This
phase of the study will be taken up in more detail later (p. 510).

SEED-TESTING OPERATION

The seed-testing operation was a simple but rather large undertaking.
There were carried on during each of the four years 195 tests for each
of the six species. Two hundred seeds were used in each test. Ordi-
nary greenhouse wooden flats about 14 by 18 by 4 inches in depth were
nearly filled with fresh sand, which was compacted and smoothed off;
the seed for each test was scattered uniformly over the surface, pressed
into the sand by means of a board, and then covered with yi inch to
% inch of sand. The flats were then set on greenhouse benches where
the sand was kept moist during the course of germination by sprinkling
it with an ordinary watering pot equipped with a fine rose or spraying
nozzle. The seeds were protected from mice by covering the flats with
frames made of fly screen. Ants were troublesome at first, but they
were successfully combatted by scattering napthalene flakes on the
benches. During the winter and spring months when artificial heat was
employed in the greenhouse, the temperature sought was about 70° F.

75308—22 3

484 Journal of Agricultural Research voi. xxn. No. 9

during the daytime and about 50° at night. There were, of course, some
variations in this, particularly as summer approached. On bright, sun-
shiny days in late spring or early summer, the temperature in the green-
house sometimes approached 100° during the middle of the day. By
that time, however, the germination tests were practically completed for
all except the slow-germinating eastern and western white pines. A
careful day by day record was kept of the germination. As the seeds
sprouted and developed a short radicle they were plucked out of the
sand and discarded. While this method of conducting germination tests
for all kinds of seed, particularly those which germinate very slowly like
the white pines, is not considered ideal, the results secured for the several
years are at least comparative.

CONCLUSIONS

In noting the conclusions, the reader should keep in mind that they
have reference to coniferous seed only, and that they are based upon
the results of one series of tests with only six species of coniferous seed
and may not accordingly be applicable to all coniferous seed, or even
to the same kinds of seed from other sources. It should also be remem-
bered that the seed used in this experiment ^vas thoroughly air-dried
before it was placed in air-tight storage.

(i) Storage of coniferous seed in the air-tight bottle is far superior in
every respect to storage in any other container. The average germina-
tion for the 5 -year period of seed stored in bottles over that stored in
the next best container was 22 per cent.

(2) Thoroughly air-dried coniferous seed stored in air-tight bottles
is little if at all affected by such differences in temperatures as exist
between a location where the temperature follows the natural fluctua-
tions, a location indoors where the temperature never falls below freez-
ing, and a location in an ordinary cellar or basement.

(3) Coniferous seed stored in air-tight bottles is little if at all affected
by the geographic location of the storage point.

(4) The quality of coniferous seed, by which is meant its value in
terms of both germinative energy and germinative ability, is much
superior in the case of seed stored in an air-tight bottle to that stored
in any other receptacle. This is manifest even at the end of one year
of storage.

(5) Following the air-tight bottle, the various containers, in the order
of their merit, fall into the following sequence: paper bag paraffined,
paper bag, cloth bag, and oiled cloth bag. It should be noted that an
ordinary paper bag closed at the top is superior to a cloth bag for seed
storage. The oiled cloth bag is practically worthless as a container.

(6) The use of any of the containers except the air-tight bottle results
in such rapid deterioration after one or t\vo years of storage under the
temperature conditions of this experiment as to render the seed, partic-
ularly of Engelmann spruce, Douglas fir, and white pine, of very little
worth.

(7) Storage at the indoor temperature is superior to that at the
fluctuating or low. Storage at the low temperature shows the poorest
results. This low temperature has reference not to a low uniform tem-
perature of freezing or less but to that of an ordinary cellar or basement.
The difference in germination percentage is not great under these three
conditions but is sufficient to make indoor storage preferable to the
other two conditions.

Nov. 26, 1921 Storage of Coniferous Tree Seed 485

(8) Some geographic locations are more favorable for seed storage
than others. Fort Bayard, Pikes Peak, Pocatello, and Lake Clear
Junction — all points of relatively high altitudes and, with the possible
exception of Lake Clear Junction, of low relative humidities — stand out
as exceptionally favorable localities. Four middle-western points,
Waukegan, Dundee, La\\Tence, and Warsaw, and one Atlantic sea-
board point. New Haven, stand out as unfavorable localities for seed
storage. Such points should apparently be avoided where ordinary
methods of storage are followed. No one of the geographic locations
shows marked superiority over another when the seeds are stored in
air-tight bottles.

(9). In respect to sustained vitality, the seeds employed in this study
range themselves in the following sequence, Avith the strongest first:
western yellow pine, lodgepole pine, western white pine, white pine,
Engelmann spruce, and Douglas fir.

WHAT THE STUDY SHOWS

The points brought out by the study can be shown better, it is thought,
by the accompanying tables and curves with a few comments than by
lengthy discourse.

EFFECT OF CONTAIN'ER

Table I, together with the curves (fig. 2), brings out what was very
evident during the progress of the study, the striking superiority of the
seeds stored in the air-tight bottles over those stored in any other con-
tainer. This is particularly true when the storage period extends beyond
one year and is more striking in the case of Engelmann spruce, Douglas
fir, and white pine than in that of lodgepole, western yellow, and western
white pines. The seeds of the former three species are apparently more
likely to deteriorate than those of the latter three and after two years
of storage are of little worth.

It seems safe to assume (barring any hypothesis of post-ripening of
the seed during storage) that the germination of the seed before it was
put in storage was at least equal to that of the seed stored in bottles at
the end of one year. Based on this assumption. Table I shows that the
average deterioration for all species has at the end of five years been for
seed stored in a paper bag 45 per cent; in a paper bag paraffined, 42.3
per cent; in a cloth bag, 47.8 per cent; in a cloth bag oiled, 51.4 per
cent; and in the air-tight bottle, 10.8 per cent. In this connection it
should be noted that Pinus ponderosa stored four years, Picea engelmanni
and Pseudotsuga taxifolia three years, and Pinus contorta two j^ears
show little if any decrease in the total germination of bottle-stored seed.
In fact, germination at the end of two and three years has in some cases
been greater than at the end of one year. The behavior of Pinus strobus
and Pinus monticola seed is a puzzle. It will be noted that the germina-
tion percentage of bottle-stored seed decreases through the second
and third years, but at the end of the fifth year (19 14) it equals or
betters that of the second year (191 1). The 1914 germination of these
two species with seed stored in the other four containers is practically
equal to the 1912 germination. Possibly the conditions for germination
in 1914 were somewhat superior to those in 1912, or there may have been
some physiological development to account for it.

486

Journal of Agricultural Research

Vol. XXII, No. 9

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Fig. 2. — Graphs showiug value of different kinds of containers for the storage of coniferous tree seeds

Nov. 26, 1921

Storage of Coniferous Tree Seed

487

Table I. — Germination percentages of seed stored in different containers and under
different temperature conditions

Seed tested for—

30
days.

33
days.

3i
days.

34
days.

95 1 80 93
days. days. days.

96
days.

Temperature and
container.

Picea engelmanni.

Pinus contorta.

1910

1911

1912

1914

Aver-
age for
all 4
years.

1910

1911

1912

1914

Aver-
age for
all 4
years.

Fluctuating tem-
perature;

Paper bag

Paper bag, par-
affined

Cloth bag

Cloth bag. oiled.
Bottle, air-tight.

47- 0

40.4
37-7
26. 7
67-1

38.8

43-2
27-5
20. 9
79-7

20.3

25.0
iS-o
II. 6

70. 0

10.3

11. I

6.7
8.3

54-4

29. I

29.9

21. 7
16. 9
07-8

66.0

74.0
55- 0
18.2
7S-0

55- I

62.3
47-3
13-5

73-2

40. 8

45-2
32-2
15.0
60. 0

34-6

44.0
36. 9
13-5
59-8

49.1

56.4
40.4
iS-o

67.0

Average for all
containers. . .

Average for all
containers
for all 4
years

43-8

42. 0

28.4

18.2

33-1

57-6

50-3

38-6

35-8

45-6

Indoor temperature:

Paper bag

Paper bag, par-
affined

Cloth bag

Cloth bag, oiled .
Bottle, air-tight.

Average for all
containers. . .

Average for all
containers
for all 4
years

52. I

55- I
51- 3
38.7
67.8

45- 7

51-5
39-5

31-8
74- 7

31- I

30. I
17-3
15-3
73-6

7-4

9. 2

7-9

4-5

61. 9

34. II 68. 8 1 bi. 2

36-5 77-2 57-4
29. 0 42. I i;i- 3
22. 6 16. 8 20. 8
69. si 72-1 75- 8

44-6

49-9
34-6
16.2

58- 4

36-9

45-9
21. 2
9-5
54-9

52-9

57-6
37-3
15-8
65-3

53. 0 48- 6

33- 5

18.2

38-3

55-4

53-3

40. 7

33- 7

45-8

I.,ow temperature;

Paper bag

Paper bag, par-
affined

Cloth bag

Cloth bag, oiled.
Bottle, air-tight.

30. 2 ; 19-2

40. 8 23-6
24-3 15- 9
16. I i 8. 3
67- 5 ] 77-2

10.5

"•3

6-3

■3

76.0

4-7

1.8

I. 2

57-8

IS- 7 ; 63. 9

2C. I : 70-7
12. I 49. 2
6. 5 ' 12. 7
(19. 6 : 72. 9

49.6

49-6
46.4
16.6

30- 7

34- 0
31-6
14-5
64. 2

29. 0

37-0
29. I
13-5
61.8

43-3

47-8
39-1
14-3
69. 0

Average for all
containers. . .

Average for all
containers
for all 4
years

35.8 28.8

2C. 9

13.6

' 53-9

i
1
24.8

47-9

35- 0

34- I

42- 7

Average for all 3
temperatures:

Paper bag

Paper bag, par-
affined

Cloth bag

Cloth bag, oiled
Bottle, air-tight

43- I

45-4
37-8
27. a
67-5

34-6

39-4

27. 6
20.3
77-2

20. 6

22. I
12.9
9.1

73- 2

e.8
8.3

5-S
4- 7
58.0

26. 3 ! 66. 2

28. 8 74-0
21. 0 48. 8
15-3 IS- 6
69.0 1 73-3

55- 3

56. 4
48-3
17.0
75-4

38- 7

43-0
32-8
15-2
60. 9

33-5

42-3
25- 7
12. 2
58.8

48-4

53-9
38-9

15- S

66.9

Average for all
containers
and tem-
I)eratures

44.2

39-8

27.6

16.7

55- 6

50- 5

38.1

34-5

488

Journal of Agricultural Research

Vol. xxir. No. 9

Table I. — -Germination percentages of seed stored in different containers and under
different temperature conditions — Continued

Seed tested for—

days.

34
days.

,34

days.

103 117

days. 1 days.

117

days.

113

days.

Temperature and
container.

Pseudotsuga taxifolia.

Pinus ponder osa.

1910

1911

1913

1914

Aver-
age for
all 4
years.

1910

1911

1912

19 '4

Aver-
age for
all 4
years.

Fluctuating tem-
perature:

Paper bag

Paper bag, par-
affined

Cloth bag

Cloth bag, oiled.
Bottle, air-tight.

22.7

25-7

14.3

3-9

44-3

14-7

■ 30.3
ii-S
6.6

Si-S

6.9

6.6
6-3
1.8

4S-S

0. 5

1.4
. I
. I

30- 4

II. 2

13-5
S.o
31

43.9

76.0

75-6
75-2
71.6

78.3

63- 9

61. I
63- 7
60.6
72.2

55- S

38.1

. 34-8

43.7

74-4

43-3

30.2
42.4
32.6
72.8

63.3
61.3

39.0
53-4
74- 4

Average for all
containers...

Average for all
containers
for all 4

33. 3

20- 9

13-4

6-3

IS- 8

7S-3

64- 3

s3-3

4S.7

61.6

Indoor temperature:

Paper bag

Paper bag, par-
affined

Cloth bag

Cloth bag, oiled.
Bottle, air-tight.

28.7

34-5
34.7
II. 3
49.1

14-3

17-7

12-3

3-3
48.6

4-3

6.0

3- 7

•9

45-8

•4

•9

•S

•7

33.6

13. 0

14.8
10.3
4.1

43- 0

7S-a

77-1
77.3
74-4
76. I

67.7

67-8
65.8
61.8
72.1

62.6

63-6
62.6
S6.4
7S-3

SI- 5

S8.i
44.6
38-3
76.6

64-3

66.7
62.6
S7-7
75- 0

Average for all
containers.. .

Average for all
containers
for all 4
years

39.7

19- 3

13. I

6.2

16.8

76.0

67.0

64. t

53-8

65.3

Low temperature:

Paper bag

Paper bag, par-
affined

Cloth bag

Cloth bag, oiled .
Bottle, air-tight .

13- S

19. 6
8-S
6-5

49.8

7-7

9.8
6.0
6.9

50.8

•7

2.6
•3

47-7

1-3

1.4

1.6

■ I

33-9

5-8

8.4

4. I

3-4

45-6

73.1

71.8
73-9
68.3

78. S

48.1

S3- 4
49.2
SO-S
67-3

42.6

30- S

38.9
39-4
7S-9

32- S

32.6
29-9
37.6
75.0

48.8

S2-I
48.0
49.0

74-2

Average for all

containers

Average for all
containers
for all 4
years

19. 6

16.2

10.3

7-7

13- S

72.9

S3- 7

49- S

41-5

54- 4

Average for all 3
temperatures:

Paper bag

Paper bag, par-
affined

Cloth bag

Cloth bag, oiled .
Bottle, air-tight.

21. 6

36.6
iS.S
7-3

47- 7

"■3

IS- 9
9.9
S-6

so. 3

S-I

3-4

•9

46- 3

•7

I. 3

• 7

•3

3I-0

9-7

13. 3

7-5

3-S

43-8

74-4

74-8
75-4
71-4
77-6

S9-9 S3- 6

60.8 S7-4
S9- 6 S2- I
S7- 6 48- 2

70. S 75- 2

43-1

47.0
39-0
36-2
74-8

S7-8

60. 0
56.5
47-9
74- S

Average for all
containers
and tem-
peratures....

2.vS

i8.8

11.9

6.8

74-7

6r-7 S7-3

48.0

Nov. 26, 1921

Storage of Coniferous Tree Seed

489

Table I.— Germination percentages of seed stored in different containers and under
different temperature conditions — Continued

Seed tested for—

days.

126

days.

129 126
days. days.

days.

126
days.

129 126
days. days.

Temperature and
container.

Pinus strobus.

Pinus monticola.

1910

1911

1912

1914

Aver-
age for
all 4
years.

1910

1911

1912

1 Aver-
years.

Fluctuating tem-
perature:

Paper bag

Paper bag, par-
affined

Cloth bag

Cloth bag, oiled .
Bottle, air-tight .

40. 6

44.0
30-5
24-5
56.0

27-3

29-9

18.4

7-9

46. S

8.8

9.9

8.3

5-0

30-7

9.8

8.9

8.6

2. 2

49.2

21.7

23.2
16. s
9-9
45- 6

36.0

36. 7
30-3
18.7
41. 1

24.7

27. 0
II- 3
10.4
27-7

6.2

5-7
30
4-7
7-8

6.0

7.S
4-4
3-0
25-9

18.2

19.2
12.3
9 2
25.6

Average for all
containers...

Average for all
containers
for all 4
years

39- I

26. 0

12- S

IS- 7

23-3

32.6

20. 2

s-s

9-4

16. 9

Indoor temperature:

Paper bag

Paper bag, par-
affined

Cloth bag

Cloth bag, oiled.
Bottle, air-tight.

46.6

49-9
3SO
23. 2
58. 5

28.7
32.9

13-4

II. 3

SO. 6

9.6

12.8
S.8
5- I

33.6

4-4

4.0

3-2

1-7

SO- 7

22.3

24.9
14.4
10.3
48.4

36-2

39-2
32-1
22. 0
39-7

26.3
24.2

is- 1

13-0

24. s

7-0

7-3
3-8
3-6
9-2

6.2

9-9

4-3

3-6

33-9

18.9

2a 2
13.8
10.6
26.3

Average for all
containers.. .

Average for all
containers
for all 4
years

42.6

27- 4

13-4

12.3

24.1

33-8

20.6

6.2

II. 6

18. 1

Low temperature:

Paper bag

Paper bag, par-
affined

Cloth bag

Cloth bag, oiled .
Bottle, air-tight.

31-7

37-8
27-3
16. 2
58.9

23-3

27.9

12. 2

6.8

48-9

4.9
9.0

I. 2

• 7
27. 0

S-o

4.6

2. 2

46. 2

16.2

19-8

10.7

6.1

4S-3

33.6

34-7
26.7
19-3
44.8

17-9

22. 2
10.7
6.2
22.3

6.0
6.2

3-7
.9

7-7

3-3

S-4

2. 2

1-3

28.8

IS- J

17. I
10,8
6.9
26. 0

Average for all
containers.. .

Average for all
containers
for all 4
years

34-4

23.8

8.6

II. 8

19. 6

31-8

16. 0

4.9

8.2

IS-'

Average for all 3
temperatures:

Paper bag

Paper bag, par-
affined

Cloth bag

Cloth bag, oiled
Bottle, air-tight

39-6

43-9
30-9

21-3

37.8

26.5

30.2
14. 7
8.6

48.7

7. 8 1 6. 4

10. 6 ' 5-8
S- I 4-7
3.6^ 1.6

30. 4 ' 48. 7

20. I

22.6
13- 9
8.8
46.4

35-3

36.9
29-7
20. 0
41.9

23. 0

24-5
12.4
9-9
25.0

6.4

6.4
3-5
3-1
8.2

5-2

7-6

3-6

2.6

29-5

17- S

18.9
12.3
8.9

26.2

Average for all
containers
and tem-
peratures . . .

38.7

25- 7

II. 5 13-4

32.7

1
18.9 S-5

9-7

490

Journal of Agricultural Research

Vol. XXII. No. 9

Table I. — Germination percentages of seed stored in different containers and under
different temperature conditions — Continued

Temperature and containers.

Fluctuating temperature:

Paper bag

Paper bag, parafifined

Cloth bag

Cloth bag, oiled

Bottle, air-tight

Average for all containers

Indoor temperature:

Paper bag

Paper bag, paraffined

Cloth bag

Cloth bag, oiled

Bottle, air-tight

Average for all containers

Low temperature:

Paper bag

Paper bag, parafifined

Cloth bag

Cloth bag, oiled

Bottle, air-tight

Average for all containers

Average for all 3 temperatures:

Paper bag

Paper bag, parafifined

Cloth bag

Cloth bag, oiled

Bottle, air-tight

Average for all species.

51-3
55. 5
43- 7
31- I
60.6

40.8
45.9

23. 2
62. I

46. 7

39. 7
37-3

37- 5
40. 6
3C. o
20. o
58.5

37-3

23- I
25. I
19.9
14.5
SI. I

26.

56-5
28,3
21-3
16.3
49-3

28.3

27.6
31. I

18.3
13-4

48.

12-3
14.3
II. I
9. I
50. 6

16. o
18.7
13-3

9.6

50. 2

Average

for all 4

years

for all

species.

31.6
33.9
26. 3
18.0

54.6

32. 9

34. I
36.8
'7-9
30. I
54. 7

34.7

27. 6
20. 8

28.4

Possibly the germination test was not continued long enough to deter-
mine the comparative germinative ability of the seed. WTien the test of
bottle-stored seed was continued in 191 2 for 270 days, an average germi-
nation of 58 per cent for Pinus monticola and 76 per cent for Pinus strobus
resulted. This is in contrast to germination percentages of 8.2 and 30.4
for the 129-day period shown by Table I. Again, in 1914 when the test
was continued for 155 days, the average germination percentages were
42 and 58.5, as contrasted with 26.2 and 48.7 for the 126-day period,
also shown in Table I. When the germination figures are curved, how-
ever, they indicate that had the tests been continued longer during the
years 1910 and 191 1, there would have been a considerable increase in
the germination percentage shown by the tests of those years. It is
believed, accordingly, that the figures in the table are a very fair cri-
terion of the comparative value or condition of the seed for the years in
question. This does not explain the reason for the sudden jump in ger-
mination of bottle-stored seed in 19 14. Frankly, the writer is not able
to assign any definite reason. The delayed germination in nurseries of
these two seeds, particularly Pinus monticola, always is a source of trouble,
and it is not surprising that in these tests they have not followed a
course similar to the other seed. There is much to be learned about
these two species.

In addition to pointing out the very evident superiority of air-tight
storage. Table I shows that the other containers should in respect to their
merit be placed in the following sequence : paper bag paraffined, paper bag,

Nov. 26. 192 1 Storage of Coniferous Tree Seed 491

cloth bag, cloth bag oiled. This is somewhat interesting. Small lots of
seed of a few pounds are perhaps stored more often in a cloth bag than
in any other container. This study indicates that on the average an
ordinary heavy manila type of paper bag would, if tied at the top, be
superior to the cloth bag. If the paper bag can be treated with a coat
of paraffin, it will be still better. In fact the germination percentage of
Pinus contorta in the paraffined paper bag was slightly greater than, and
of P. ponderosa nearly equal to, that of the sealed bottle after one year's
storage. The oiled cloth bag is decidedly inferior. This is much less
pronounced with P. ponderosa than with the other species. Whether
the general inferiority of this container is due to penetration of the seed
coat by the oil and consequent injury of the embryo, to the prevention
of access of water to the embryo when the seed was sown, or to some
other cause is unknown. Regardless of the reason, the truth is evident
that such a container should be avoided in storing any of these seeds.

The superiority of the bottled seeds over those in other containers is in
all probability due to the almost complete suspension of physiological
activity by the seed thus stored. (This was not verified by any experi-
ruents undertaken in connection with this study.) The two conditions
essential for such activity are warmth and moisture. The seed in the
bottles was of course exposed to high enough temperature to induce
respiration, but the necessary amount of moisture for any great degree
of activity was not present. Such moisture as was present in the seed
or bottled air could not be increased by additions from without. On
the other hand, the seed in the other containers was intermittently sub-
jected to both temperature and atmospheric moisture conditions sufficient
at times to induce rather active respiration. Such respiration can be
carried on only by using up food material stored in the seed itself, with
the consequent gradual weakening of its germinative ability. The seed
stored in the paraffined paper bag and the plain paper bag were, it is
believed, less subject to changes of atmospheric moisture than that
stored in the ordinary cloth bag. The rate of deterioration was in
consequence less rapid.

It is quite generally held by forest tree seed investigators that the
true criterion of the quality of seed is not alone its germinative ability
or viability (germination percentage) but rather this germinative ability
in conjunction with germinative power or energy. Rapidity of germina-
tion (the germination percentage at the end of a certain period of time)
is the measure of this germinative energy. This period is measured from
the date of sowing the seed through the time that germination is pro-
ceeding steadily and rapidly and at the end of which it starts to fall off
rather abruptly. If the progress of germination is plotted and curved,
the point at which the curve begins to fall off rather abruptly and flatten
out will represent the number of days which should be selected for that
species. Now, the quality of the seed will be determined by the mean
of the germination at the end of this period and the final germination
percentage.

By final genninative percentage is not meant here the absolute final
but rather that at the end of a reasonable period. Accepting this hypoth-
esis, Table II was prepared, and the curves in figure i were drawn from
it. They represent the quality of each species of seed stored in each
container at the end of one, two, three, and five years. The periods

492

Journal of Agricultural Research

Vol. XXII, No. 4

selected for the measure of germinative energy and final germination
were as follows:

Engelmann spruce . .

Dou^^las fir

Lodgepole pine

Western yellow pine.

White pine

Western white pine..

Period of

«emunati%-e

final

ger-

energy.

mination .

105

115

The germinative energy periods were selected through the use of the
germination curves as the guide. Other investigators may not agree
with these periods, and the writer will admit that other lots of seeds
might indicate periods of different length. The period of final germi-
nation is the same as that shown in Table I for the year 1910. It was
necessary for comparative results to select a period not longer than
that over which the tests were conducted in any one of the four years.
The tests were discontinued sooner in 19 10 than in any other year, and
this led to the selection of the test period during that year.

Table II. — Quality of seed after storage for i, 2, j, and 5 years

Species.

Picea engelmanni .

Pinus ponderosa.

Pin us contorta.

Paper bag

Paper bag paraffined .

Cloth bag

Cloth bag oiled

Bottle air-tight

Paper bag

Paper bag paraffined .

Cloth bag

Cloth bag oiled

Bottle air-tight

Paper bag

Paper bag paraffined .

Cloth bag

Cloth bag oiled

Bottle air-tight

Paper bag

Paper bag paraffined .

Cloth bag

Cloth bag oiled

j Bottle air-tight

Pinus strobus , Paper bag

Paper bag paraffined .

I Cloth bag

j Cloth bag oiled

I Bottle air-tight

Pinus monticola Paper bag

Paper bag paraffined .

Cloth bag

i Cloth bag oiled

• Bottle air-tight

Pseudotsuga iaxifolia.

Container.

Quality expressed in percentage after stor-
age for —

I year.

42. O
44.2
35-6
26. o
66.6
52.2
53- 8
.54-9
36-4
64.8

59- o
66.4

44-7
15. o
70.4
20.8
26.0
15.0
6.8

46.3
29.9

32-9
24. o
17.0
49.6

] years.

19-3

33.8
38.6
26.8

19-3
76.6
39-8
41. o
39-6

39-7
58.0

.54-3
55-2
47-4
17.8

73-9
II. 2
14. 6
8.8
5-2
47. 2

13-2

13.6

7.8

5-8

26.6

17.8

18.2

9.4

7.8

16.6

3 years.

11. 7

12. 4

5
55

27'

39
38,
26,
46,

33-^
37-6
27. 7
13-3
56.3
2.8

3-3
2. 2
.6
32.4
3-0
4.2

14.8
3-2

2.4

1.6

1-4
2.9

S years

5-4

6 6

4 4

3-6

35- o

33-4

35.8

21- 5

20. I

47-5

36. I

33.8

ig. 3

9-9

55. 3

•5

•9

•5

. 3

27. 6

2-5

3. 2

22. 6

3- r
4-4
3. 2

I- 5
17.3

Nov. a6, t9it Storage of Coniferous Tree Seed 493

These curves are interesting. They emphasize more than ever the
superiority of air-tight storage over any of the other methods. It will
be recalled that, judged by final germination percentage only (Table I),
the quality of lodgepole and western yellow pine seed stored in paraffined
paper bags was at the end of one year practically equal to that of seed
stored in the bottles. The curves adequately dispel this idea of equality.
The bottle-stored seed of western yellow pine and lodgepole pine excels
that in paraffined paper bags by 1 1 and 4 per cent, respectively. Further-
more, at the end of five years the bottle-stored seed of all species except
western yellow pine is practically equal or superior to that stored for only
one year in cloth bags, and the bottle-stored seed of western yellow pine
is superior to that stored for two years in any of the other containers.
Douglas fir, Engelmann spruce, and lodgepole pine seed stored in bottles,
western yellow pine in oiled cloth bags, lodgepole pine in cloth and oiled
cloth bags, and western white pine in paper and paraffined paper bags
show some appreciation in quality at the end of the second year over that
at the end of the first ; there is in general a marked and fairly uniform de-
terioration of seed for a 3-year period, after which it is less rapid ; the pre-
viously expressed relative merits of the various containers is confirmed —
that is, in the order of their merit they should be ranged in the sequence
of air-tight bottle, paper bag paraffined, paper bag, cloth bag, and oiled
cloth bag. The oiled cloth bag is so inferior that it should receive no
consideration at all for seed storage purposes.

EFFECT OF TEMPERATURE

Of the three conditions of temperature under which the seed was
stored. Table I clearly indicates that the highest average germination
percentages were secured with that stored at the indoor temperature,
followed in order by the fluctuating and low temperatures. The differ-
ences in the average germination percentage for the indoor and fluctuating
temperatures is only 1.8 per cent, but the superiority of indoor tempera-
ture conditions over those in an ordinary basement or cellar is indicated
by an average excess germination of 6 per cent. This general superiority,
it will be noted from the table, is consistent with all of the species in-
volved. Here again, however, the superiority of air-tight storage is evi-
denced by the fact that the seed stored in bottles at the low temperature
shows no inferiority but in fact a slight superiority (0.3 and 0.4 per cent)
over that stored under indoor and fluctuating temperature conditions.

Leaving the general averages in Table I and analyzing the results in
Tables IV to IX, it will be noted that the superiority of seed stored at
the indoor over that stored at the fluctuating and low temperatures seems
to vary with the species about as follows: Engelmann spruce, Douglas
fir, western yellow pine, western white pine, eastern white pine, and
lodgepole pine. It will be noted that the two species, Engelmann spruce
and Douglas fir, most susceptible to deterioration in storage were the
most favorably affected by storage at the indoor temperature. The
better results from storing at the indoor temperature was most marked
at Halsey, New Haven, Lawrence, and Ann Arbor. Poor germination
resulting from storage at low temperature was most pronounced at New
Haven, Pikes Peak, Pocatello, Waukegan, Halsey, and State College.

494

Journal of Agricultural Research

Vol. XXII, No. 9

Table III. — Germination percentages of seed at the several storage points

Location.

Ann Arbor

Dundee

Fort Bayard

Halsey

Ithaca

Lake Clear Junction

Lawrence

New Haven

Pikes Peak

Pocatello

State College

Warsaw

Waukegan

Picea engelmanni.

I9I0

1911

191 2

1914

53-7

44-9

28.3

13-2

25-7

18.5

IS- 9

12-9 1

6s-7

66.5

56.8

30- s !

45-6

44- 7

22- S

II- 4

43-5

41.9

24.9

14-4

SI- 3

56.7

3S-9

16.3

22. I

18.4

12.4

3-4

42.7

29- 3

19. I

6.1 !

59-6

53-8

S4-2

43- 0

49-5

61.6

47- S

2S-0

46.4

32-5

19. 6

14. I

30- 0

15-6

II. 6

12.3 !

40.5

33-4

20. 9

14.0 1

Aver-
age.

Pseudotsuga laxifolia.

35- o
18.2
54-9
31- I
31-2
40. I
14. I
24-3
52-7
45-9
28- 2
17.4
27. 2

191 1

191 2

16.7

10. 2

13.8

9.9

34-4

18.8

15-9

8.9

17-3

II- 7

31- I

15- I

11.9

9-7

II. 0

9-3

28.0

21. 6

25-6

14-7

12-5

9-3

II. 0

8-3

IS- 5

10.4

8.;
9'
13- ■

Location.

Pinus contorta. Pinus ponderosa.

1910

1911

1912

1914

MV: 1 --

191 1

1912

-- ^agr

Ann Arbor

56.0

36.8

44-2
36.9
46-5
39-9
39-7
40- S
30.8
38.3
50. 2
41.8
32- I
24-3
33-3

22. 9
28.2
SI- 9
32-4
30.1
40. 6
22.3
29. 0
S5-6
44- I
32-5
24-3
33-3

40. 0
42.8

55-6

77.6
75-2
78.1
73.8

59-3
69.9
75-1
72-5
67- 4
66.9

58.4
45-8
49-2
61. I
52- 7
52-3
69.8

68. e

Dundee

52
69
61
64
70
44
38
56
64
66
35
68

8
8
6
8

I
8

6
6

Fort Bayard

Halsey

63. 2 j 60. 7
so- 9 i 57- I
S3-S S3-0
52-9 SO-S
S8-S 1 43-8
56. 1 44. 8
66. 1 cs: T

72- 7 73- 9

Ithaca

44-1 I 7S-S
46. I 1 7S- 8
38.9 68.8
42. I . 74- 4

57-5 ' ■ 7S-7
51.0 1 75-8
40. 9 ! 71. 8
34- 7 76- I

63. 5 67. 8

65. 2 , 69. 5

Lake Clear Junction.
Lawrence

New Haven

28. 2 46. 8

Pikes Peak

57- 4 59-7
64. 9 66. 6

Pocatello

63.8
48.7
so. 3
48. s

54-4

SO- 2

39-9

SS-8

State College

49. I 60. 0

Waukegan ... .

47-3 65-4

Location.

Pinus strobiis.

Pinus monticola.

1910

1911

191 2

1914

Aver-
age.

I9I0

1911

1912

1914

Aver-
age.

Ann Arbor

36-4
29-3
51-8
40-3
39-1
48-9
20. 7
35- 2
49-9
54-3
36.9
23-4
35-6

26.3
21.8
44-9
24.9
26. 9
36.0
10.0
15- 5
35-0
40-5
23-5
9-9
19-5

10.5
8.2

29-3
10. I
8.7
13-5
7-9
6.8
19.9
19. 0
9-4
5-6
7.6

10. 2
10.9
16. 7
10. I
10. 2

14. 2
II. 0

20. 9
17.6
35-7
21.4

21. 2
28.2
12.4
t6. A

32.8
30.8
42.6

33-9
31.0
31- I
23-1
35-3
38.2
35-9
33-9
27-6
29-3

II. 0
19.9

52. I
37-9
28.7
19.8

8-5
11. 6
10. I
14.0
14.0

5-3
Ij- 7

6.4
5-6
6-5
6-1
5-1
5-8
4-6
1.6
7-1
10. 3
6.9
2. I
4-6

11.9
9-3

27-9
S-9

7.0
12. 7
7-3
5-4
16. 0
9.1
4-3
4-7
4-3

15. 5

Dundee

16.4

Fort Bayard

Halsey

32.3
21. 0

Ithaca

18.0

Lake Clear Junction.

Lawrence

New Haven

17-4
10.9

Pikes Peak

30- 6 1 33.9
20.0 t 33.5
9.4 I 19.8

17.9

State College

Warsaw

14-8
10. 0

Waukegan

9.8

18. 1

12-9

Location.

Ann Arbor

Dundee

Fort Bayard

Halsey

Ithaca

Lake Clear Junction

Lawrence

New Haven

Pikes Peak

Pocatello

State College

Warsaw

Waukegan

Average of all species.

46. 1
38.2
55-8
44-8
45- I
49.6
34-5
43- 7
53-3

36.8

26.3
39- o

34-6

28.0
21.6
38.0
24.8
25-8

30. 2
18.4
19-0

34-9

33-0

24- o

14-5

Average

ofaU

species

for all 4

years.

21-4
IS- 6
35-5
20.8

21-5

2S-8
II. 8
13-8
35-3
28.3
19.0
13- I
19 I

32.0
27.0
46. 1
33-2
32.9
37-3

22- 5

35- 7
40- 1
39- c
29-3
21. 7
29-9

Nov. 26, 1921

Storage of Coniferous Tree Seed

495

Average for each
container at all 3
temperatures for
all 4 years.

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496

Journal of Agricultural Research

Vol. XXII, No. 9

&i J

•5r°

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Storage of Coniferous Tree Seed

497

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498

Journal of Agricultural Research

Vol. XXII, No. 9

Average for each con-
tainer at all 3 tem-
peratures for all 4
years.

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Journal of Aarictiltural Research

Vol. XXII, No. 9

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Storage of Coniferous Tree Seed

503

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5o8

Journal of Agricultural Research

Vol. XXII. No. 9

Various experiments with tree seed go to prove that storage at a
uniformly low temperature (o° to 32° F.) is preferable to that at higher
temperatures. It is easy to understand that at such low temperatures
there would be little if any physiological activity of the seed and con-
sequently little wasting of stored-up food and energy. Had the seed
in this study been stored at such low temperatures it is very probable
that it would have excelled in germination that stored at the other two
temperatures. In this study, however, the seed was stored in basemient
or cellar where the summer temperature was doubtless fairly high.
Moreover, as is usually the case in such locations, the atmospheric
humidity was also doubtless high. The conditions, in fact, during
summer were such as to be more conducive to physiological activity
than under the fluctuating or indoor conditions of storage. Hence, it
is reasonabe to suppose that there was more of such activity with the
consequent deterioration of the seed. The lesson to be learned from this
particular phase of the experiment is to avoid ordinary basements and
cellars for storing seed in unsealed containers, and that in northern
temperate climates storage indoors where the temperature never goes
below freezing is preferable to storage where the temperature follows
the natural variations.

EFFECT OF GEOGRAPHICAL LOCATION

Ordinarily, the seed dealer will perhaps have to store any seed which
he possesses at his own establishment regardless of its location. It v^nll
be a matter of interest, however, to learn that some geographic locations
seem much more suited for the purpose than others. For seed in con-
tainers other than the sealed bottle, there was a very consistent superiority
shown in the germination of that stored in some locations and a very
consistent inferiority of that stored in others. Table III brings this out.
Note, for instance, the consistently high relative germination of seed
stored at Fort Bayard, Pikes Peak, Pocatello, and Lake Clear Junction;
on the other hand, note the relatively low germination of seed stored at
Dundee, Lawrence, New Haven, Warsaw, and Waukegan. These are
averages for all containers. Knowing, now, that seed in air-tight bottles
is not so much affected by adverse conditions as that in other containers,
it is believed, accordingly, that it should not be considered in determin-
ing the effect of the geographic location upon stored seed. The effect
is better shown by the behavior of seed stored in the cloth bags. Using
the averages shown in Tables IV to IX, it is found that the towns range
themselves in the following order:

Town.

Fort Bayard

Pikes Peak

Pocatello

Lake Clear Junction

Ithaca

Halsey

Ann Arbor

State College
Waukegan. ..
New Haven .
Dundee ....
Lawrence . . .
Warsaw. . . .

Average
gennina-
tion per-
centage for
all 4 years
of seed
stored in
cloth bags.

23.0
20. 6

18.3

16. o

13.2

9-7

Nov. 26, 1921

Storage of Coniferous Tree Seed

509

Fort Bayard stands out as an exceptionally favorable storage point,
and Pikes Peak, Pocatello, and Lake Clear Junction follow it rather
closely. On the other hand, New Haven, Dundee, Lawrence, Warsaw,
and Waukegan seem especially unsuited as locations for seed storage in
ordinary containers. There was not enough information collected in con-
nection with this study to show the reason why some points give so much
more favorable results than others. It seems probable that it is due to
some climatic factor or factors which in turn have their effect upon the
physiological activities of the seed. Fort Bayard, Pikes Peak, Pocatello,
and Lake Clear Junction are in high altitudes, and the first three places
at least experience conditions of low relative atmospheric humidity.
Waukegan, Dundee, Lawrence, and Warsaw are middle western loca-
tions of moderate altitude which experience severe temperature fluctua-
tions and higher relative humidity than the first three locations men-
tioned. During the summer when such changes would most affect seed
in storage. New Haven experiences higher relative hum.idities than the
middle western locations and severe climatic fluctuations are somewhat
less marked there. Regardless of the reason, the study indicates that
middle western points and perhaps Atlantic coast points should if possible
be avoided as locations for storing coniferous tree seed in ordinary
containers, especially for periods of several years. This conclusion for
Atlantic coast points may not be entirely warranted as it is based on
results at only one station.

There is, however, comfort for those wishing to store seed in such
locations when the germination of bottle-stored seed is examined. Based
on the average germination percentage of such seed for all species and all
years, the towns line up in the following sequence :

Towa.

Dundee

Fort Bayard
Waukegan . .
Pikes Peak.

Ithaca

Pocatello . . .
Halsey

Average
germina-
tion per-
centage for
all 4 years
of seed
stored in
air-tight
bottles.

Town.

Lawrence

Lake Clear Junction

Ann Arbor

New Haven

State College

Warsaw

Average
germina-
tion per-
centage for
all 4 years
of seed
stored in
air-tight
bottles.

This tabulation indicates, and it can be corroborated by inspection of
Tables IV to IX, that the bottle-stored seed in this study was not affected
by climatic conditions at the points of storage. Two of the points,
Dundee and Waukegan, which the study indicates were very unfavorable
storage points when ordinary methods of storage are followed, appear in
the case of bottle-stored seed to be among the most favorable locations.
Here again the lesson is obviously to use air-tight methods of seed storage.
The geographic location of the storage point will then be of little or no
consequence.

5IO Journal of Agricultural Research voi.xxii. N09

RESULTS OE STORAGE AT END OF TEN YEARS

Reference was made (p. 483) to the fact that some of the bottle-stored
seed was carried over for another 5 years and then tested again. The
seed so carried over was that of each of the six species which had previous-
ly been stored at the indoor temperature at Dundee, 111. P'ollowing the
test during the winter of 1914, the bottles were resealed air-tight and then
stored until January, 19 19, on a shelf in the Forest Service office building
at Washington, D. C. The 1914 test of these seed showed a germina-
tion percentage of 71.5 for Engelmann spruce, 43 for Douglas fir, 61.5
for lodgepole pine, 82 for western yellow pine, 74 for western white pine,
and 56.5 for white pine.

The test in 19 19 was carried on for 167 days under the same conditions
as in previous years. Engelmann spruce, Douglas fir, and white pine
germinated not at all, while lodgepole pine germinated to the extent of
9 per cent, western yellow pine 22 per cent, and western white pine 6.5
per cent. Although the test was continued for 167 days, the lodgepole
pine had completed its germination in 90 days, western yellow pine in
75 days, and western white pine in 130 days. Due to the fact that
these seeds were exposed to the air at the end of 5 years, although the
bottles were afterwards resealed, this particular part of the experiment
does not truly indicate whether seed can be successfully stored for 10
years without great deterioration. It does give an idea of the relative
sustained vitality of the species concerned. It has been rather apparent
all through the study that as respects general sustained vitality, the
species will rank in the following sequence, the more vital species being
placed first: Western yellow pine, lodgepole pine, western white pine,
white pine, Engelmann spruce, and Douglas fir. If the seeds were se-
cured from different sources than those of this study, the sequence
might be altered.

SUvSCKPTlBILlTY OF THE DIFFERENT Vx\RIETIES OF
SWEET POTATOES TO DECAY BY RHIZOPUS NIGRI-
CANS AND RHIZOPUS TRITICI

By L. L. Harter. Pathologist, and J. I,. Weimer, Pathologist, Office of Cotton, Truck,
and Forage Crop Disease Investigations, Bureau of Plant Industry, 'United States
Departinent of Agriculture

INTRODUCTION

Although Rhizopus yiigricans Ehrb. has generally been regarded as
the cause of the softrot of the sweet potato (Ipomoea batatas Lam.),
Harter, Weimer, and Lauritzen^ have shown by recent experiments that
a similar decay may be caused by the following additional species of the
genus: tritici Saito, nodosus Namysl, reflexns Bainier, delemar (Boid),
Wehmer and Hanzawa, oryzae, Went and Pr. Geerligs, maydis Bruderl.,
arrhizus Fischer, and artocarpi Racib. Their investigations showed that
in order to obtain infection with the dififerent species it was necessary to
incubate the potatoes at a temperature suited to the growth of the par-
ticular species of Rhizopus with which they were inoculated. They group
the different species into high, intermediate, and low temperature forms,
R. nigricans Ehrb. and R. tritici, the two species concerned in the
present investigations, belonging to the low and intermediate forms,
respectively.

The parasitism of the dififerent species of Rhizopus was determined by
them for the Yellow Jersey variety of sweet potatoes only. Although
softrot occurs on all the varieties, it is the general belief that some of
them are more resistant to decay than others. The Jersey type of sweet
potatoes, for example, is recognized as a poor keeper, while some of the
varieties grown in the South are thought to keep well in storage. The
present investigations were conducted in order to throw more light on
the relative susceptibility of a number of the commercial varieties to
infection and decay by R. nigricans and R. tritici.

R. nigricans was employed for the major portion of the inoculations
because it is the species commonly found under storage-house conditions
and seems to be responsible for most of the loss. R. tritici although not
so common as R. nigricans is a very parasitic species, especially under
artificial conditions. A comparison of these two species gives some idea
of the results that may be expected from them, one requiring relatively
low and the other one an intermediate temperature, or at least a tem-
perature considerably above that recommended for the storage of sweet
potatoes.

METHODS OF EXPERIMENTATION

The susceptibility to infection and decay by R. nigricans was deter-
mined for the following varieties of sweet potatoes: Big Stem Jersey,
Little Stem Jersey, Southern Queen, Porto Rico, Dooley, Georgia, Pierson,
Dahomey, Triumph, Gold Skin, Haiti, Nancy Hall, Early CaroHna, Yellow

1 Harter. L. L., Weimer, J. L., and Lauritzen, J. I. the decay of sweet potatoes (ipomoea
batatas) produced by different species of rhizopus. In Phytopathology, v. ii, no. 7, p. 279-284.
Literature cited, p. 2S4. 1921.

Journal of Agricultural Research. Vol. XXII, No. 9

Washington, D. C. Nov. 26, igar

aas Key No. 0-258

(511';

512

Journal of Agricultural Research

Vol. XXII, No. 9

Belmont, and Red Brazil. The parasitism of R. tritici was determined
for all the above-named varieties except the last four.

The mechanical operations involved in carrying out these experiments
are identical with those previously employed ' and will not be discussed
here. The potatoes inoculated with R. nigricans and R. tritici were
incubated at temperatures of from 20° to 22° and 30° C, respectively,
these temperatures having been found in previous experiments to be
favorable for the growth of these two organisms. Records of the per-
centage of infection and of the progress of decay were made at the end
of 48 hours after inoculation and every day thereafter to the close of the
experiment. The experiments were allowed to run for from four to six
days.

EXPERIMENTAL DATA

The results of the inoculations of the different varieties of sweet pota-
toes with R. nigricans are shown in the following table. The figures in
columns i and 2 were obtained by taking an average of the results of the
several experiments. The data presented in the second column are based
on an estimation of the percentage of the total amount of decay when
the experiments were terminated.

Table I. — Percentage of sweet potatoes infected and the estimated percentage of decay at

the end of the experimeii t

Variety,

Porto Rico

Big Stem Jersey. ..

Triumph

Pierson

Gold Skin

Little Stem Jersey

Georgia

Early Carolina. .. .

Percent-

Per-

age of

cent-

decay

age

at the

end of

infec-

the

tion.

experi-

ment.

7=^

100

73 1

100

Variety.

Nancy Hall

Florida

Red Brazil

Haiti

Dahomey

Southern Queen
Yellow Belmont
Dooley.

Per-
cent-
age
of
infec-

Percent-
age of
decay
at the
end of
the

tion.

experi-
ment.

100

97
87

53
23

100
100

93
98

The results show that a large percentage of the potatoes became infected
by the method employed. There were cases, especially among the more
resistant varieties, where only a very small amount of the tissue about
the well decayed. In view of this fact it was frequently difficult to decide
whether infection had actually taken place or whether the small amount
of decay was due to an enzym in the inoculum at the time the inocula-
tions were made. The writers finally decided to regard as infected all
potatoes which had been softened for i cm. or more beyond the margin
of the well. It is not unlikely that the percentage of infection of some
of the varieties, especially those which resisted further decay, was actu-
ally placed too high by this method.

' H.^RTER, L. L., Weimer, J. L,., and Adams, J. M. R. sweet-pot.ito storage-rots. In Jour. Agr.
Research, v. 15, no. 6, p. 337-368, pi. 21-27. 1918. Literature cited, p. 366-368.
Barter, I,, h., Weimer. L., and Lauritzen ,J. I. op. cit.

Nov. 26, J92I Susceptibility oj Sweet Potatoes to Rhizopus 513

Although it was easy to estimate the relative amount of decay between
very susceptible and very resistant varieties, it was more difiBcult to
determine the percentage of total decay at the end of a given time. The
percentage of the entire potato that would be decayed at the end of a
certain number of days depended to a considerable extent on the size
and shape of the potatoes. Naturally a larger percentage of a small
sweet potato would be decayed in a given time than of a large one; also
a short to nearly spherical potato would be completely decayed sooner
than a long, cylindrical one. As to shape, the potatoes differed greatly.
The potatoes of some varieties were short and chunky, while others were
long. In view of these facts the writers wish to emphasize the danger of
putting too much reliance on small differences. The results, however,
show some wide differences which the authors believe to be a fair estimate
of the relative susceptibility of the different varieties.

With respect to their susceptibility to decay by R. nigricans, the dif-
ferent varieties of sweet potatoes can be divided roughly into three
groups as follows: i, very susceptible; 2, quite resistant; and 3, inter-
mediate. To the first belong Gold Skin, lyittle Stem Jersey, Georgia,
Early Carolina, Red Brazil, Haiti, Yellow Belmont, and Dooley; to the
third group belong Porto Rico, Big Stem Jersey, Triumph, Pierson,
Florida, and Dahomey; and to the second, Nancy Hall and Southern
Queen. As might be expected, a considerable variation exists between
the different varieties of a single group. Furthermore, there is no sharp
line of separation between the more resistant and the more susceptible
varieties of two contiguous groups, the differences frequently being no
greater than that which exists between varieties of the same group.
The Gold Skin is by far the most susceptible variety studied, decay be-
ing completed considerably in advance of that of any of the other vari-
eties. The Little Stem Jersey is likewise very susceptible and with the
iGold Skin stands out conspicuously as regards the rapidity with which
t decays. The Big Stem Jersey, a variety grown extensively in
the northern range of the sweet-potato belt, decays fairly rapidly and
completely, and must be ranked high as a susceptible variety in the
intermediate group. Nancy Hall and Southern Queen are the only
representatives of the resistant group. Although the table shows a high
percentage of infection, the amount of decay was always small, and
within the limits of these experiments they must be regarded as much
more resistant than any of the other varieties. The Florida is the only
other variety that approximates these two in resisting decay by R.
nigricans.

Although the object of these experiments was primarily to determine
the susceptibility of the different commercial varieties of sweet potatoes
to decay by R. nigricans, one single set of inoculations was made with
R. tritici, using the same varieties with the exception of the four already
mentioned. R. tritici was found to be parasitic on all the varieties.
One outstanding fact as a result of these experiments seems to be that
the Nancy Hall and Southern Queen, varieties which are especially re-
sistant to R. nigricans, are rather susceptible to decay by R. tritici. A
further comparison, therefore, was made of the relative parasitism of
these two species on Nancy Hall and Southern Queen, using the Little
Stem Jersey, a very susceptible variety, as a control.

After inoculation of the three varieties in the usual way with R.
tritici and R. nigricans they were divided into two lots. One lot was in-

514

Journal of Agricultural Research

Vol. XXII, No. 9

cubated at 30° C, a temperature favorable for R. tritici, and the other
lot at 20° to 22°, a temperature favorable for the growth of R. nigricans.
None of the potatoes inoculated with R. nigricans and incubated at 30"^
became infected, showing that this temperature is unfavorable to in-
fection by this fungus. On the other hand, all those inoculated with
R. tritici became infected and were completely decayed in three or four
days. A direct comparison of these two organisms at 30° was therefore
impossible. The percentage of infection as well as the total amount of
decay of all three of these varieties by both organisms when incubated
at a temperature of 20° to 22° for seven days is shown in Table II.

Table II. — Percenlaije of infection and estimated percentage of decay of three -varieties of
sweet potatoes by R. nigricans and R. tritici after seven days at 20° to 22° C.

Variety,

Organism.

Percentage
of infection.

Percentage
of decay.

Southern Oueen

R. nigricans

R. tritici

R. nigricans

100

100
100

Nancy Hall

85
40

Little Stem Jersey

R. tritici

R. nigricans

R. tritici

100

An examination of this table shows that at this temperature the Little
Stem Jersey variety was decayed much more readily than either Nancy
Hall or Southern Queen, by both organisms. However, decay was much
more rapid by R. tritici than by R. nigricans, being completed by the
former in three to four days and not quite completed by the latter in
seven days. The potatoes of the Nancy Hall and Southern Queen varie-
ties were 100 and 40 per cent and 85 and 15 per cent decayed by R. tritici
and R. nigricans, respectively, at the end of seven days. A comparison of
the three varieties shows that the estimated amount of decay caused by
R. nigricans at the end of the experiment was about 15, 40, and 80 per
cent for Southern Queen, Nancy Hall, and Little Stem Jersey, respec-
tively. The total amount of decay caused by R. tritici, on the other hand,
was 85, 100, and 100 per cent, respectively. The results therefore show
that the Little Stem Jersey is quite susceptible to decay by both species,
while Nancy Hall and Southern Queen are very resistant to decay by
R. nigricans only. The percentage of infection of the Nancy Hall potatoes
was 95 and 80 by R. tritici and R. nigricans, respectively, while the
Southern Queen potatoes were 100 and 70 per cent infected by the same
organisms. On the other hand, Little vStem Jersey potatoes were 100 per
cent infected by both species.

These results indicate what occurs at temperatures of 20° and 30° C.
under the conditions existing in these particular experiments. A tem-
perature of 30° proved to be too high for R. nigricans and 20° to 22°, while
not optimum for either organism, is probably more favorable for decay
by R. tritici than by R. nigricans. On the other hand, if a much lower
temperature had been employed it would have been more favorable to
R. nigricans but less favorable to R. tritici. The results, however, do
show a considerable difference in the three varieties with respect to their
relative susceptibility to decay by the two species of Rhizopus at the
temperatures tried.

Nov. 26, 1921 Susceptibility of Sweet Potatoes to Rhizopus 515

SUMMARY

(i) R. nigricans is parasitic on the following varieties of sweet potatoes :
Porto Rico, Big Stem Jersey, Triumph, Pierson, Gold Skin, Little Stem
Jersey, Georgia, Early Carolina, Nancy Hall, Florida, Red Brazil, Haiti,
Dahomey, Southern Queen, Yellow Belmont, and Dooley. R. tritici was
found to be parasitic on all the above-named varieties except Early Caro-
lina, Florida, Red Brazil, and Yellow Belmont, on which it was not tried.

(2) With respect to their susceptibility to decay by these two species of
Rhizopus, the varieties can be separated roughly into three groups- — first,
those that are very susceptible ; second, those that are very resistant ; and,
third, those that are intermediate between the first and second. To the
first group belong the Gold Skin, Little Stem Jersey, Georgia, Early Caro-
hna. Red Brazil, Haiti, Yellow Belmont, and Dooley; to the second,
Southern Queen and Nancy Hall ; and to the third, Porto Rico, Big Stem
Jersey, Triumph, Pierson, Florida, and Dahomey.

(3) Nancy Hall and Southern Queen, the two most resistant varieties,
were more susceptible to decay by R. tritici than by R. nigricans at a
temperature of from 20° to 22° C. Neither of these varieties, however,
deca5'-ed as readily as the Little Stem Jersey under similar conditions and
used as a control.

(4) The results of these experiments show that all the varieties tried
(16) are more or less susceptible to decay by R. nigricans but that there
are some varietal differences. Likewise, the results show that those
varieties which decayed most readily under the conditions of these
experiments are the ones which have been observed to decay most readily
under commercial storage-house conditions.

75308—22 5

Vol. XXII DECEMBER 3, 1921 No. 10

JOURNAL OF

AGRICULTURAL
RESEARCH

CONTENTS AND INDEX
OF VOLUME XXII

PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE

WITH THE COOPERATION OF THE ASSOCIATION OF

LAND-GRANT COLLEGES

WASHINGTON, D. C.

GOVERNMENT PRINTING OFFICE

1923

EDITORIAL COMMITTEE OF THE

UNITED STATES DEPARTMENT OF AGRICULTURE AND

THE ASSOCIATION OF LAND-GRANT COLLEGES

FOR THE DEPARTMBNT

K. F. KELLERMAN, Chairman

Phytiologist and Associate Chkf, Bureau
of Plant Industry

B. W. ALLEN

CUrf. Office of Bxptriment Stations

C. L. MARLATT

Entomologist and Associat* Chief, Bureau
of Entomolocy

FOR THB ASSOCIATION

J. G. LIPMAN

Dean, Stale College of Agriculture, mtd
Director, New Jersey Agricultural Experi-
ment Station, Rutgers College

W. A. RILEY

Entomologist and Chief, Ditision of Enkh
mology and Economic Zoology, Agricnt-
tural Experiment Station of the Untsersity
of Minnesota

R. L. WATTS

Dean. School of Agriculture, and Dirtttof,
Agricultural Experiment Station, The
Pennsylvania State College

All correspondence regarding articles from the Department of Agriculture should be
addressed to K. F. Kellerman, Journal of Agricultural Research, Washington, D. C.

All correspondence regarding articles from State Experiment Stations should be
addressed to J. G. Lipman, New Jersey Agricultural Experiment Station, New
Brtmswick, N, J.

ERRATA AND AUTHORS' EMENDATIONS

Page 54, line i. should read "Fj or Fi" instead of "Fa or F."

Page s8. Table V, under last column the first number should read 33 instead of 23.

Page 68, line 24, should read "with a nutrient solution" rather than "with an nutrient solution."

Page 69, lines 9 and 11, should read "O. A. Pratt" instead of "A. C. Pratt."

Page 76, line 3, should read "Plate 11, A" instead of "Plate 11, B."

Page 80 and following, in Plates 10 and 11, fis^res A and C of Plate 10 should be interchanged with figures
A and C of Plate 11.

Page 174, line i, should read "causal" not "casual."

Pages 175 and 177, and Plates 2a and 24, running head should read, "Transmissible Mosaic Disease of
Chinese Cabbage " instead of " Transmissible Mosaic Disease of Cabbage."

Page 178, colored plate is by J. Marion Shull.

Page 242, line 8, should read "Humphrey, as reported by Jones (5)," not "Humphrey (4) states . . . ."

Page 282, line 32, should read "at each treatment" instead of "to each treatment."

Page 283, line 17, should read "nitric acid," not ''nitri acid."

INDEX

Page
ABSORPTION OF COPPER FROM THE

SOIL BY POTATO PLANTS 281-387

Acer —

nibrum, host of Xylotrechus colonus 195-198

saccharum, organic acids 221-229

Achillea ptarmica, susceptibility to Puc-

cinia triticlna 152-172

Acid —

amino, in G€orgia velvet beans 15

citric, in grapefruit 263-279

diainiao, in Georgia velvet bean 15

hydrocyanic, in Sudan grass 125-138

:iialic, in Pyrui coronaria 221-229

phosphoric, effect on hydrocyanic acid in

Sudan grass 135

succinic, in Pyrus coronaria 22 1-229

sulphuric, effect on hydrocyanic acid in

Sudan grass. 133-135

Acid producers in corn meal 181-188

Acid-sugar ratio in grapefruit 263-279

Acids—
effect on hydrocyanic acid in Sudangrass. 133-135
organic, of Pyrus coronaria , Rhus glabra, and

Acer sacckarutn 221-229

Aconitum—
Fischeri, resistance to teliosporesof Pticcinia

triticiiia 155-172

Lycoctonutn, susceptibility to Puccinia

triticiiia 152-172

Napellus, susceptibility to Puccinia triti-

cina 152-172

Actaea spicaia, resistance to teliospores of

Puccinia tritichia 155-172

Actinotnyces sp. in com meal 187-188

AECIAL STAGE OF THE ORANGE
LEAFRUST OF WHEAT, PUCCINIA

TRITICINA ERIKS 151-172

Aecidiuin —

Anchiisae, pathogenicity on wheat 152

Berberidis, in life cycle of Puccinia grami-

nis 151-172

Ranunculacearum 7 Thalictri Havi. Syn.

Aeculiiim Thalictri Jiavi.
Thalictri flavi, rust on Agropyron repens. . . 164
Aegopodiuin Podagraria, susceptibility to

Puccinia triticina 152-172

A gropyron —
caninum, resistance to aeciospores of Puc-
cinia triticina 163-172

cristatum, resistance to aeciospores of Puc-
cinia triticina 163-172

desertorutn, resistance to aeciospores of

Puccin'a triticina 163-172

inerme, resistance to aeciospores of Puccinia
triticina 163-172

Page

^4 gropyron — Coniinued.
repens, resistance to aeciospores of Puccinia

triticina 163-172

Rickardsonii, host of Puccinia Agropyri. . . . 165
Smithii, resistance to aeciospores of Puc-
cinia A gropyri 165

t&nerutn, resistance to aeciospores of Puc-
cinia triticini 163-172

Agrostemma Githago, susceptibility to Puc-
cinia triticina 152-172

Agrostis —

borealis, host of Puccinia triticina 164-172

orthogonia. Syn. Porosagrotis orthogonia.

Akis muricota. Syn. Embaphton muricatum .

Albumen, egg, effect on growing chicks 146

Albumin in Georgia velvet bean 15

Alfalfa, effect of soil temperature on develop-
ment of nodules 17-31

Alkaline solution, efl'ect on hydrocyanic acid
in Sudan grass 135-136

Alternaria sp. on pitted grapefruit 277

Atnbrosia artemisiifolia, glucosid 4

Amino acid. See Acid, amino.

Ammodrainus savannaruni bim avtilatu s ,
enemy of Porosagrotis orthogonia 313

A mygdalus persica—
effect of temperature on damage from rots 452-465
host of Lecaniu m corni 191

Aiichusa —
arvensis, host of aecial stage of Puccinia

rubigo-vera 151-173

ofvcinaUs, host of aecial stage of Puccinia
rubigo-vera 151-172

Ancylostoma—

caninum, nonhemolytic effect 383

duodenak, hemotoxins 382-432

Anemone —
canadensis, resistance to teliospores of

Puccinia triticina 155-173

cylindrica, resistance to teliospores Puccinia

triticina 155-172

japonica, resistance to Puccinia triticina. . 155-173
ranunculoides, susceptibility to Puccinia
triticina 152-173

A noploccphala —

perfoliata, hemotoxins not found 383

pHcata, hemotoxips not found 382

Anthocyanin in maize 2-31

Anthoxanthum odoratum, host of Puccinia
triticina 164-172

Anticoagulins in hookworms 418-420

Aphids, carriers of mosaic of Chinese cab-
bage 173-178

Aphis, sugar-beet root. See Pemphigus betae.

Apple blotch. See Phyllosticia solitaria.

5i8

Journal of Agricultural Research

Vol. xxir

Page
Apple, crab. See Pyrus coronaria.
Aqiiilegia —
alpina, resistance lo teliospores of Puccmia

trilicina iSS"'?*

canadensis, resistance to teliosi>ores of

Puccinia trilicina 155-173

chrysantha, resistance to teliosjwres of

Puccinia trilicina 155-172

glandulosa, resistance to teliospores of

Puccinia trilicina 155-172

olympica, resistance to teliospores of

Puccinia trilicina 157-172

Skinneri, resistance to teliospores of

Puccinia trilicina 155-1 72

xnilgaris, resistance to teliospores of

Pucciniii trilicina 155-172

Army cutworm. See Cliorizagrotis auxiliaris.
Arrhenatiierum clalis, resistance lo aeciospores

of Puccinia trilicina 163-172

Ascaris —

conocepkala, cause of anemia 385-432

luinbricoides, hemotoxins 385-432

Ash, bone, effect on growing chicks 145-149

Asb. See Fraxinus sp.

ASH CONTENT OF THE AWN.
RACHIS. PALEA, AND KERNEL
OF BARLEY DURING GROWTH

AND MATURATION 433-449

A spergillus —

candidus in corn meal 187-188

Jlavus in corn meal i8i-i88

futnigalus in corn meal 185-188

niger in corn meal 181-188

ockraceous in com meal 187-188

re pens in com meal 181-188

tamari in corn meal 185-188

terreus in com meal 185-188

Awn of barley, ash content 433-449

Bacillus —

coli communis, effect on aspartic acid 224

nigcr in corn meal 186-188

phytophlhorus, cause of balckleg potato

tuber-rot 81-92

radicola —

effect of sulphur 102-110

used in tests of effect of soil temperature

on nodule development 20-31

tetanus, blood toxin 381

Bacteria —

in cheese 93, 100

in corn meal 181-188

Bacterium aerogenes in com meal 183-188

Barbaraea vulgaris, susceptibility to Puccinia

trilicina 152-172

Barley, ash content 433-449

Bartlett, H. H., and Sando, Charles E.:
NOTES ON THE ORGAJ^IC ACIDS
OF PYRUS CORONARIA, RHUS
GLABRA, AND ACER SACCHA-

RUM 221-229

OCCURRENCE OF QUERCETIN
IN EMERSON'S BROWN-HUSKED

TYPE OF MAIZE 1-31

Bfiin, velv-jt. See Stizolobium deeringianum.
Beetle, pine. See Dendroct&nus inonticolae.

Page

Beetles, cerambycid, host-selection princi-
ple 189-220

Beets, sugar, dryrot canker 47-52

Bcrberis vulgaris —
failure to become infected from basidio-

spores of Puccinia rubigo-vera 152 -i 72

susceinibility to Puccinia trilicina 152-172

BIOLOGICAL ANALYSIS OF THE
SEED OF THE GEORGIA VELVET
BEAN, STIZOLOBIUM DEERING-
IANUM 5-is

BIOLOGY OF EMBAPHION MURICA-

TUM 333-334

Black locust. See Robinia pseudacacia.

Black mold rot, caused by Rhi2o pus nigri-
cans. 451-465

BLACKLEG POTATO TUBER-ROT
UNDER IRRIGATION 81-92

Blight, chestnut. See Endothea parasitica.

Blotch-
apple. See Phyllosticla solitaria.
plum. See Phyllosticla congesta.

Blue mold rot. See Penicillium cxpansum.

Bone ash, effect on growing chicks 145-149

Bonnetia compla, parasite of Porosagrotis
orthogonia 313

Boshnakian, Sarkis, and Leighty. Clyde E. :
GENETIC BEHAVIOR OF THE
SPELT FORM IN CROSSES BE-
TWEEN TRITICUM SPELTA AND
TRITICUM SATIVUM 335-364

Boving, Adam G., and Wade, J. S.: BI-
OLOGY OF EMBAPHION MURICA-
TUM 323-334

Brassica —

japonica, mosaic disease 173-178

pekincnsis, mosaic disease 173-178

rapa, mosaic disease 173-17S

Bromus —

ciliatus, host of Puccinia Agropyri 165

Porter i, host of Puccinia allernans 165

Brooks, Charles, and Cooley, J. S.:
TEMPERATURE RELATIONS OF

STONE FRUIT FUNGI 451-465

TRANSPORTATION ROTS OF STONE
FRUITS AS INFLUENCED BY OR-
CHARD SPRAYING 467-477

Brown-huskcd maize, quercetin 1-31

Brovm rot. See Sclcrolinia cinerea.

Bustomum phlebnlomum, heinptoxins 415-432

Cabbage, Chinese. See Brassica pekinensis.

Calcium-
carbonate, effect on-
growing chicks 139-149

plant growth 102-110

toxicity of copper sulphate 281

lactate, effect on growing chicks 145-149

sulphate, effect on plant growth ■ 102-110

Callidium —

antennatum, host selection 194-220

janthinum, host selection 203-220

Camassia esculenta, resistance to teliospores
of Puccinia trilicina 155-172

Campanula rolundifolta. susceptibility to
Piiccinia trilicina 152-17;

Oct. i-Dec. 3, 1921

Index

519

Page

Canker of sugar beets 47-52

Calvin, J. W., et al.: NUTRIENT RE-
QUIREMENTS OF GROWING
CHICKS: NUTRITIVE DEFICIEN-
CIES OF CORN 139-149

Camallanus ainrricanus, oxyhemoglobin in
body fluid 392-432

Carbonate —
calcium, effect on —

growing chicks 139-149

plant growth 102-110

toxicity of copper sulphate 281-287

sodium, efiect on hydrocyanic acid in Sudan
grass 135-136

Casein, effect on growing chicks 139-149

Caslanca dentala, host of —

Hyperplalys maciilaius 217-219

Xylotrechus cuhnus 195-198

Cattle hookworm. See Bustomum phkboto-
muni.

Cattle, maintenance requirement 115-121

Celtis occidentalis, host of —

Cyllene picius 198-203

Molorchus bimaculatus 212-213

Centaurea Cyanus, susceptibility to Puccinia
triticina 152-172

Cerambycid beetles, host -selection principle. 189-220

Cfrcis canadensis, imrrmnity to Molnrchus
bimaculatus 212-213

Cestode hemolysins 420-432

Cheese, bacteria 93-100

Cherries, effect of —
orchard spraying on transportation rots. . 467-477
temperature on damage from rots 441-465

Chestnut. See Castanea dentata.

Chestnut blight. See Endothea parasitica.

Chinese cabbage See Brassica pekinensis.

Chlorid, sodium, effect on growing chicks. . 145-149

Chorizagrotis auxiliaris, comparative des-
tructiveness of Porosagrotis orthogonia 289

Cimicifuga racemosa, resistance to teliospores
of Puccinia triticina 155-172

Citelhis richardsoni, enemy of Porosagrotis
orthogonia 313

Citric acid. See Acid, citric.

Citromyces sp. in corn meal 187-188

Cladosporium sp. on pitted grapefmit 277

Clematis —
Douglasii, resistance to teliospores of Puc-
cinia triticina 155-172

fiatninula, probable aecial host of Puccinia

triticina 153-172

Fremontii, resistance to teliospores of Puc-
cinia triticina 155-172

heraclaefolia, resistance to teliosirares Puc-
cinia triticina 155-172

ligustici folia, resistance to teliospores of

Puccinia triticina 155-172

orientnlis, resistance to teliospores of Puc-
cinia triticina 155-172

paniculata, resistance to teliospores of Puc-
cinia triticina 157-172

recta, resistance to teliospores of Puccinia
triticina 155-172

Page

Clematis — Continued.
virginiana, resistance to teliospores of Puc-
cinia triticina 155-171

■citalba, probable aecial host of Puccinia
triticina 153-171

Clonorchis sinensis, hemotoxins 380-432

Colletotrichum sp. on pitted grapefruit 277

COMPARATIVE VIGOR OF Fi WHEAT
CROSSES AND THEIR PARENTS.... 53-63

COMPARISON OF THE PECTINASE
PRODUCED BY DIFFERENT SPEC
SPECIES OF RmZOPUS, A 371-377

Coniferous tree seed, storage 479-510

Cook. F. C: ABSORPTION OF COPPER
FROM THE soil, BY POTATO
PLANTS 281-287

Cooley, J. S., and Brooks, Charles: TEM-
PERATURE RELATIONS OF STONE
FRUIT FUNGI 451-465

Copper, absorption from soil by imtato plants
281-287

Copper sulphate, toxicity to potato plants. . 281-287

Com, nutritive defeciencies 139-149

Com meal, flora 179-188

Cornus florida, host of —

Hyperplalys maculatus 217-219

Molorchus bimaculatus 212-213

Coronaria floscuculi, susceptibility to Puc-
cinia triticina 152-172

Corticium vagum, cause of dryrot canker of
sugar beets 47-52

Crab apple. See Pyrus coronaria.

Craighead, F. C: HOPKINS HOST-
SELECTION PRINCIPLE AS RE-
LATED TO CERTAIN CERMBYCID
BEETLES 189-220

Cutworm —
army. Chorizagrotis auxiliaris.
pale western. See Porosagrotis orthogonia

289-322

Cyanidin, glucosid, in maize 2-31

Cyllene —

crinicoriiis, host selection 203

picius, host selection 193-220

Delphinium —
ajacis, resistance to teliospores of Puccinia

triticina 155-172

"Belladonna," resistance to teliospores of

Puccinia triticina 155-172

consolida, resistance to teliospores of Puc-
cinia triticina 155-172

geyeri, resistance to teliospores of Puccinia
triticina 155-172

Dendroctonus monticolae, parasite on Pinus
contorta 189-220

Dextrose in grapefruit 263-279

Diamino acid. See Acid, diamine.

Digestion, liberation of hydrocyanic acid. ... 127

Diphyllobothrium latum, hemotoxins 381-432

Dipotassium phosphate, effect on growing
chicks 145-149

Dipylidium caninum, hemotoxins 382-432

Dogwood. See Cornus florida.

DRYROT CANKER OF SUGAR BEETS,
A 47-5«

520

Journal of AgrictUtural Research

Vol. XXII

Page
Echium vulgare, susceptibility to Puccinia

triticina 152-172

Edson , H . A . , and Shapovalov , M . : B LACK-
LEG POTATO TUBER-ROT UNDER

IRRIGATION 81-92

EFFECT OF SOIL TEMPERATURE UP-
ON THE DEVELOPMENT OF NODU-
LES ON THE ROOTS OF CERTAIN

LEGUMES 17-31

Egg albumen, effect on growing chicks 146

Eleodes contiisum, resemblance to Embapkion

iuuricalum 323

Elymu.1 —
ausiralis, resistance to aeciospores Puccmia

triticina 163-172

canadensis, resistance to aeciospores of Puc-
cinia triticina 163-1 72

glauctis, resistance to aeciospores of Puc-
cinia triticina 163-172

triticoides, resistance to aeciospores of Puc-
cinia triticina 163-172

virginicus, resistance to aeciospores of Puc-
cinia triticina 163-172

Embapkion —
concavum, resemblance to Embapkion muri-

catutn 314

muricatum —

biology 323-334

control 333

description 326-332

distribution 324-325

enemies 332-333

Endotkea parasitica, food of Neoclytus capraea . 193
Euxoa ochragaster, connection with Porosagro-

tis orthogonia 290

Fasciola hepatica, hemotoxins 380-432

FERTILITY IN SHROPSHIRE SHEEP. 231-234
Festuca Thicrberi, host of Puccinia Cockerel-
liana 16s

Fisher, D. F.. and Brooks, Charles: TRANS-
PORTATION ROTS OF STONE
FRUITS AS INFLUENCED BY OR-
CHARD SPRAYING 467-477

Flavone in maize 3-31

FLORA OF CORN MEAL 179-188

Fraxinus sp., host of —

Neoclytus capraea 210-211

Xylotrechus colonus 195-198

Fruit fungi, temperature relations 451-465

Fruits, stone, influence of orchard spraying

on rots 467-477

Fungi, of stone fruits, temperature relations. 451-465
FURTHER STUDIES ON RELATION
OF SULPHATES TO PLANT GROWTH

AND COMPOSITION loi-iio

Fusarium —

eumartii, effect of temperature 66-80

hyperocysporum, effect of humidity 71-80

oxysporum —

cause of rot of Irish potatoes 65-80

confused with causal organism of black-

l^e potato tuber-rot 81-92

fadicicola —
confused with causal organism of black-
leg potato tuber-rot 81-92

effect of temperature 66-80

Page

Fusarium — Continued.

trichothecioides, effect of temperature 66-So

sp.—

in corn meal 187-188

on pitted grapefruit 277

trichothecioides, isolated from potato black-
leg tubers 82

lubirivorum, effect of temperature 66-80

vasinfectum var. pisi, host selection 191-220

Gardner, Max W.. and Kendrick. James B.t

TURNIP MOSAIC 123-124

SOYBEAN MOSAIC 111-114

GENETIC BEHAVIOR OF THE SPELT
FORM IN CROSSES BETWEEN TRI-
TICUM SPELTA AND TRITICUM
SATIVUM 335-364

Geographical location, effect on coniferous
tree seed in storage 508-510

Georgia velvet bean. See Stizolobium deering-
ianum.

Glechov'.a hederacea, susceptibility to Puccinia
triticina i5J-i7»

Globulins in Georgia velvet bean 15

Glucosid of quercetin in maize 2-31

Gluten, effect on growing chicks 144

Goss, R. W.: TEMPERATURE AND
HUMIDITY STUDIES OF SOME FU-
SARIA ROTS OF THE IRISH POTATO 65-80

Gracilaria stigmatella, change in nidification. . , 191

Grape —
See Vitis vinifera.
wild. See Vilis sp.

Grapefruit, ripening and storage ;63-279

Graphidium strigosuni, heruoto.xins 3(57-432

Grass, Sudan, hydrocyanic acid 1^5-138

Griffee, Fred: COMPARATIVE VIGOR
OF Fi WHEAT CROSSES AND THEIR
PARENTS 53-63

Ground squirrel. See Citellus richardsoni.

Growth of plants, effect of sulphur loi -no

Kackberry. See Celtis occidcntalis.

Haemoncltus contortus, hemotoxins 387-431

Halbersleben, D. L., et al.: NUTRIENT
REQUIREMENTS OF GROWING
CHICKS: NUTRITIVE DEFICIEN-
CIES OF CORN 139-149

Harlan, Harry V., and Pope, Merritt N.:
ASH CONTENT OF THE AWN.
RACHIS, PALEA, AND KERNEL OF
BARLEY DURING GROWTH AND

MATURATION 433-449

Harter, L. L., and Weimer, J. L.:
A COMPARISON OF THE PECTINASE
PRODUCED BY DIFFERENT SPE-
CIES OF RHIZOPUS 371-377

SUSCEPTIBILITY OF THE DIFFER-
ENT VARIETIES OF SWEET POTA-
TOES TO DECAY BY RHIZOPUS
NIGRICANS AND RHIZOPUS TRI-
TICI S"-SiS

Hawkins, Lon A.: A PHYSIOLOGICAL
STUDY OF GRAPEFRUIT RIPENING
AND STORAGE 263-279

HEMOTOXINS FROM PARASITIC
WORMS 379-43»

Oct. i-Dec. 3, 1921

Index

521

PilgC

He polka sp., resistance to leliospores of Puc-

cinia triticina 155-172

Heterodera schachti, host selection 192

Histidin in Georgia velvet bean 15

Hogan, A. G., et al.: INFLUENCE OF
THE PLANE OF NUTRITION ON
THE MAINTENANCE REQUIRE-

ilENT OF CATTLE 115-121

Hoowkorm —
of cattle. See Bustomum phlebotomum.
of dog. See Ancylosloma cannium.
of man. See Necator americanus.
of man, Old World. See Ancylostinna
duodenale.
HOPKINS HOST-SELECTION PRINCI-
PLE AS RELATED TO CERTAIN

CERAMBYCIDi: BEETLES 189-220

Hordeum —
caespitosuin, resistance to aeciospores of

Puccinia triticina 163-172

gussoneanum, resistance toXaeciospores of

Puccinia triticina 163-172

jubatum —

host oilPuccinia Agropyri i6s

resistance to aeciospores oilPuccinia triti-
cina 163-172

murinuiii, resistance to aeciospores of Puc-
cinia triticina 163-172

pusilluut, resistance to aeciospores of Puc-
cinia triticina 163-1 72

vuhare, resistance to' 'aeciospores of Puc-
cinia triticina 163-172

Homed lark. See Otocoris alpestris leuco-
laeina.

Host-selection principle 189-220

Hucker, G. J.: MICROSCOPIC STUDY OF

BACTERIA IN CHEESE 93-100

Humidity, effect on Fusaria rots of Irish po-
tatoes 65-80

HYDROCYANIC ACID IN SUDAN

GRASS 125-138

Hydrogen-ion concentration, effect on devel-
opment of nodules ou legiunes 30

Hydrophyllum appendiculatuni, resistance to

teliospores of Puccinia triticina 155-172

Hydroxid, sodimn, effect on hydrocyanic

acid in Sudan grass 135-136

Hylolrupes ligneus, host selection 207-210

Hyperplatys —

aspersus, host selection 217-219

maculatus, host selection 2 1 7-219

Hystrix Hyslrix, resistance to aeciospores of

Puccinia triticina 163-172

hnpatiens sp., resistance to teliospores of Puc-
cinia triticina 155-172

INFLUENCE OF THE PERIOD OF
TRANSPLANTING WESTERN
WHITE PINE SEEDLINGS UPON
THEIR BEHAVIOR IN NURSERY

AND PLANTATION 33-46

INFLUENCE OF THE PLANE OF NU-
TRITION ON THE MAINTENANCE

REQUIREMENT OF CATTLE 115-121

Ipomoea batata:, susceptibility of different

varieties to Rhizopus spp 511-515

Irish potato. See Solatium tuberosum.

Page
Iron sulphate, effect on growing chicks. . . . 145-149
.Tackson, H. S., and Mains, E. B.: AECIAL
STAGE OF THE ORANGE LEAF-
RUST OF WHEAT, PUCCINIA TRI-
TICINA ERIKS I5I-I7J

Jones, Fred Rcuel, and Tisdalc, W. B.: EF-
FECT OF SOIL TEMPERATURE
UPON THE DEVELOPMENT OF
NODULES ON THE ROOTS OF CER-
TAIN LEGUMES 17-31

Jones, L. R., and Walker, J. C: RELA-
TION OF SOIL TEMPERATURE AND
OTHER FACTORS TO ONION SMUT

INFECTION 235-263

Kafir, hydrocyanic acid 137

Kendrick, James B., and Gardner, Max W.:

SOYBEAN MOSAIC 111-114

TURNIP MOSAIC 123-124

Kernel of barley, ash content 433-449

Knautia arvensis, susceptibility to Puccinia

triticina 152-172

Lactate.'calcium, effect on growing chicks. 145-149
Lark; horned. See Otocoris alpestris leuco-

laema.
Lnunaca';asplenifolia, suspected accial host of

Puccinia triticina 153-172

Leafiusl, orange.£ See Puccinia triticina.
"Leak," confused with blackeg potato

tuber-rot 81-92

Lecanium —

corni, host selection 191

robiniarum, host selection 191-220

LeFevre, Edwin, and Thom, Charles:

FLORA OF CORN MEAL 179-188

Legumes, effect of soil temperature on devel-
opment of nodules 17-31

Lcighty, Clyde E., and Boshnakian, Sarkis:
GENETIC BEHAVIOR OF THE
SPELT FORM IN CROSSES BE-
TWEEN TRITICUM SPELTA AND

TRITICUM SATIVUM 335-364

Ligustrum vulgare, susceptibility to Puccinia

triticina 152-172

Liopus alpha, host selection 194-220

Liriodendron tulipifera —

host of Hyperplatys maculatus 217-219

immunity to Neoclytus erythrocephalus . . . 213-315
Lithospermum purPureo-coeruleum, suscepti-
bility to Puccinia triticina 152-172

Locust. See Robinia pseudacacia.
Lodgepole pine. See Pinus contorta.
Lythrum Salicaria, susceptibility to Puccinia

triticina 152-172

Macracanthorhynchus hirudinaceus, hemo-

toxins 387-432

Magnesimn sulphate, effect on growing

chicks 145-149

Mains, E. B., and Jackson, H. S.: AECIAL
STAGE OF THE ORANGE LEAF-
RUST OF WHEAT, PUCCINIA TRI-
TICINA ERIKS 151-172

Maize —

nutritive deficiencies 139-149

quercetin in brown-husked 1-31

Malic acid. See Acid, malic.
Maple, red. See Acer rubrum.

522

Journal of Agricultural Research

Vol. XXII

Pase

Maple, sugar. See Acer saccharuni.

Meal, corn, flora 179-188

Melandryum album, susceptibility to Puc-
cinia triticina 152-172

Mertensia virginica, resistance to teliospores of
Puccmia triticina 155-172

Mesquite. See Prosopis juliflora.

Metarrkizium anisopkae, parasite of Emba-
phion muricatum 332

MICROSCOPIC STUDY OF BACTERIA
IN CHEESE 93-100

Miller, Harry G.: FURTHER STUDIES
ON RELATION" OF SULPHATES TO
PLANT GROWTH AND COMPOSI-
TION lOI-IIO

Mold, blue. See Penicillium expansuvi.

Mold rot, black, caused by Rhizoptts nigri-
cans 451-465

Molds in com meal i8i-i88

Molorchus bimaculatus, host selection 212-213

Monilia. See Sclerotinia cinerea.

Moths rubra, host of Cyllene pictus 198-203

Mosaic —

of Chinese cabbage 173-178

of soybean 111-114

of turnip 123-124

Mulberry. See Morus rubra.

Mulliceps midticeps, hemotoxins 382-432

Mumford, F. B., Hogan, A. G., and Salmon,
W. D.: INFLUENCE OF THE PLANE
OF NUTRITION ON THE MAIN-
TENANCE REQUIREMENT OF
CATTLE 115-121

Mussehl, F. E., Calvin, J. W., Halbersleben,
D. L., and Sandstedt, R. M.: NUTRIENT
REQUIREMENTS OF GROWING
CHICKS: NUTRITIVE DEFICIENCIES
OF CORN 139-149

Mustard. See Brassica japonica.

Myosotis —
alpestris, failure to become infected from

basidiospores of Puccinia rubigo-vera . . . 152-172
arvensis, failure to become infected from

basidiospores of Puccinia rubigo-vera. . . 152-172
palustris, resistance to teliospores of Puc-
cinia triticina 155-172

scorpoides, resistance to teliospores of Puc-
cinia triticina 155-172

sp., susceptibility to Puccinia triticina. . . 152-172

Myzus persicae, carriers of mosaic of Chinese
cabbage 173-178

Nasturtium sp., susceptibility to Puccinia
triticina 152-172

Necator americanus, hemotoxins 383-432

Necosmopora vasinfecta, host selection 191-220

Neoclytus —

capraea, host selection 193-220

erythrocephalus , host selection 213-215

Nitrate, sodium, effect on plant growth. . . . 102-110

Nitrates, effect on development 011 nodules
on legumes 18

Nitrogen, effect of soil temperature on assim-
ilation by legumes 17-31

Nodules, effect of soil temperature on devel-
opment 17-31

Page

Nonnea rosea, failure to become infected from
basidiospores of Puccinia rubigo-vera 152-17*

NOTES ON ORGANIC ACIDS OF PY-
RUS CORONARIA, RHUS GLABRA,
AND ACER SACCHARUM 221-220

Notholcus lanatus, resistance to aeciospores of

Puccinia triticina 163-172

NUTRIENT REQUIREMENTS OF
GROWING CHICKS: NUTRITIVE
DEFICIENCIES OF CORN 139-149

Oak, white. See Quercus alba.

OCCURRENCE OF QUERCETIN IN
EMERSON'S BROWN-HUSKED
TYPE OF MAIZE 1-31

(Ocneria) Porthetria dispar, host selection 191

Oneideres cingulata, host selection 194-220

Onion smut. See Urocystis cepulae.

Orange leaf rust. See Puccinia triticina.

Orange, osage. See Toxylon pomiferunu

Ornithogalum umbellatum, resistance to telio-
spores of Puccinia triticina 155-172

Osage orange. See Toxylon pomiferum.

Otocoris alpestris Icucolaema, enemy oi Poroses
gratis orthogonia 313

Palea of barley, ash content 433-449

PALE WESTERN CUTWORM (POROS-
AGROTIS ORTHOGONIS MORR.) . . . 289-322

Paloverde. See Parkhisonia microphylla.

Parker, J. R., Strand, A. L., and Seamans,
H. L.: PALE WESTERN CUTWORM
(POROSAGROTIS ORTHOGONIA
MORR.) 289-322

Parkinsonia microphylla, host of Cyllene crmi-
cornis 203

Pastinaca sativa, susceptibility to Puccinia
triticina iS2-i7«

Peach. See Amygdalus persica.

Pectinase produced by species of Rhizopus. 371-377

Peleteria robusta, parasite of Porosogrolis or-
thogonia 3 13

Pemphigus betae, carrier of cortichim. vagii m.. . 50-52

Penicillin m —

expansu m in com meal 185-188

lutcu m in com meal 185-188

oxalicu m in corn meal 185-188

purpurogenum 185-188

sp. on pitted grapefruit 277

Penta-acetyl-quercetin in maize 3-31

Perilitus eleodis, parasite of Embaphion muri-
catum 332

Phacelia —
Purshii, resistance to teliospores of Puccinia

triticina 155-172

tanaceti folia, resistance to teliospores of Puc-
cinia triticina 155-172

Phillyrea sp., susceptibility to Puccinia triti-
cina 152-172

Phosphate, dipotassiiim, effect on growing
chicks 145-149

Phosphoric acid. See Acid, phosphoric.

Phratora vitellinoe, inherited changes in habits 191

Phyllosticta —

congesta, cause of plum blotch 365-370

solitaria, cause of apple blotch 365

Oct. i-Dec. 3, 1931

Index

523

Page
PHYSIOLOGICAL STUDY OF GRAPE-
FRUIT RIPENING AND STORAGE,

A 263-279

Picea engelmanni, storage of seed 481-510

Pigmentation of maize 1-31

Pine—
lodgepole. See Pinus contorta.
western white. See Pinus tiionticola.
yellow. Pinus pandetosa.
Pine beetle. See Dendroctonus inonticolac.
Pinus —
contorta—

host of Dendroctonus tnonticolae 189-220

storage of seed 481-510

monticola —
influence of period of transplanting on

growth of seedlings 33-46

storage of seed 481-510

ponderosa —
threatened by Dendroctonus Tnonticolae. 189-220

storage of seed 481-510

sirobus, storage of seed 481-510

PLUM BLOTCH, A DISEASE OF THE
JAPANESE PLUM, CAUSED BY
PHYLLOSTICTA CONGESTA HEALD

AND WOLF 365-370

Plum, Japanese. See Prunus triflora.

Poa nemoralis var. firmula, host of Puccinia

triiicina 164-172

Poplar, yellow. See Liriodendron tulipifera.
Porosagrotis —
delorata. Syn. Porosagrotis orthogonia.
orthogonia —

control 307-313

distribution 291

economic importance 305-306

history 289-290

life history 293-305, 314-320

natural enemies 313-314

Porthetria dispar, host selection 191-220

Pope, Merritt N., and Harlan, Harry V.:
ASH CONTENT OF THE AWN, RA-
CHIS, PALEA. AND KERNEL OF
BARLEY DURING GROWTH AND

MATURATION 433-449

Potato-
Irish. See Solanum tuberosum.
sweet. See Ipomoea batatas.

Potato tuber-rot, blackleg 81-92

Prosopis juUfiora, host of Cyllene crinicornis. . 203
Prunella vulgaris, susceptibility to Puccinia

triticina 152-172

Prunes, effect of —
orchard spraying on transportation rots. . 471-477

temperature on damage from rots 452-465

Prunus triflora, host of Phullosticta congesta . 365-370

Pseudotsuga taxifolia, storage of seed 481-510

Puccinia —
Agropyri —

ascia on Ranunculaceous hosts 154

rust on Thalictnim dasycarpum 165

alternans^

aecia on Ranunculaceous hosts 154

rust on Bromi's Porleri 165

borealis, rust on A grostis borealis 164

Page
Pwccmja- Continued.
bromina, pathogenicity to Symphytum

officinale 154-172

Clematidis, aecia 151-172

Cockerelliana, rust on Festuca Thurberi 165

dispersa, brown rust of wheat 152-172

glumarum, on wheat 151-172

graniinis, on wheat 151-172

obliterata, rust on Thalictrum alpinum 165

perplexans, aecia on Ranunculaceous hosts. . 154
Persisiens, aecia on Ranunculaceous hosts. . 154

rubigo-vera, on rye 151-172

straminis. Syn. Puccinia rubigo-vera.

triticina, aecial stage 151-172

Pulmonaria —

inontana, host of Puccinia bromina 154-172

officinalis, failure to become infected from

basidiosporesof Puccinia rubigo-vera 152-172

Pyrus coronaria, organic acids 221-229

Pythium debaryanum, confused with causal

organism of blackleg potato tuber-rot 81-92

Quamasia hyacinthina. Syn. Camassia esculenia.

Quercetin, in brown-husked maize 1-3 1

Quercus alba, host of Neoclytus capraea 210-211

Rachis of barley, ash content 433-449

Ragweed. See Ambrosia artemisiifolia.

Ram, relation of age to fertility 232-233

Ranunculus—

susceptibility to Puccinia triticina 152-172

aconitifolius, resistance to teliospores of

Puccinia triticina 155-172

acris —
failure to become infected from basidios-
poresof Puccinia rubigo-vera 152-172

resistance to teliospores of Puccinia tri-
ticina 155-172

asiaticus, susceptibility to Puccinia triti-
cina 152-172

auricomus, susceptibility to Puccinia triti-
cina 152-172

bulbosus, failure to become infected from

basidiosporesof Puccinia rubigo-vera. . . . 152-172
Ficaria, susceptibility to Puccinia triti-
cina 152-172

flammula, susceptibility to Puccinia triti-
cina 152-172

lanuginosus, susceptibility to Puccinia

triticina 152-172

repens, susceptibility to Puccinia triticina. 152-172
Read, J. W., and Sure, Bamett.: BIOLOG-
ICAL ANALYSIS OF THE SEED OF
THE GEORGIA VELVET BEAN,
STIZOLOBIUM DEERINGIANUM .... 5-15
Redbud. See Cercis canadensis.
Red maple. See Acer rubrum.
RELATION OF SOIL TEMPERATURE
AND OTHER FACTORS TO ONION

SMUT INFECTION 235-262

Rhanius —
cathartica, failure to become infected from

basidiospores of Puccinia rubigo-vera .... 152-172
Frangula, failure to become infected from
basidiosporesof Puccinia rubigo-vera. . . . 152-172
Rhizoctonia solani, similarity to Corticium
vagum 51

524

Journal oj Agricultural Research

Page
Rhizopus —
arrhizus —

pathogenicity on sweet potato S'l

production of pectinase 372-377

artocarpi —

production on pectinase 372-377

pathogenicity on sweet potato 511

chinensis, production of pectinase 372-377

deUmar —

pathogenicity on sweet potato 511

production of pectinase 372-377

maydis —

pathogenicit y on sweet potato 511

production of pectinase 372-377

microspoTus, production of pectinase 372-377

nigricans —

in com meal i8s-iS8

pathogenicity on different vurieties of

sweet potatoes sn-sis

production of pectinase 372-377

temperature relations 451-465

nodosiis—

pathogenicity on sweet potato 511

production of pectinase 372-377

oryzae —

pathogenicity on sweet potato 511

production of pectinase 372-377

reftexus —

pathogenicity on sweet potato 511

production of pectinase 372-377

spp., production of pectinase 371-377

irHici —

pathogenicity on sweet potato 511

production of pectinase 371-377

Rhus glabra, organic acids 221-229

Richards. B. L.: A DRYROT CANKER

OF SUGAR BEETS 17-52

Ribes Grossularia, susceptibility to Puccinia

triticina 152-172

Roberts, Elmer: FERTILITY IN SHROP-
SHIRE SHEEP 231-234

Roberts. John VV.: PLUM BLOTCH, A
DISEASE OF THE JAPANESE PLUM,
CAUSED BY PHYLLOSTICTA CON-

GESTA HEALD AND WOLF 365-37°

Robinia pscudacacia, host of —

Lecanium corni 191-220

'Xyloirechus colonus 195-198

Rogers, E. C: INFLUENCE OF THE
PERIOD OF TRANSPLANTING
WESTERN WHITE PINE SEED-
LINGS UPON THEIR BEHAVIOR IN

NURSERY AND PLANTATION 33-46

Rot-
black mold, caused by Khizoptis nigricans 451-465
blue mold. See Penicillrum expansum.

brown, caused hy ScUrotinia cinerea 451-465

Rots, transportation, influence of orchard

spraying 4*7-477

Roughage requirements of chicks 146

Rum^z acelosa, susceptibility to Puccinia

triiicina 152-172

Salmon. W. D., et al.: INFLUENCE OF
THE PLANE OF NUTRITION ON
THE MAINTENANCE REQL^RE-
MENT OF CATTLE 115-1M

Page
Sando. Charles E., and Barllett, H. H.:
NOTES ON TNE ORGANIC ACIDS OF
PYRUS CORONARIA. RHUS GLA-
BRA. AND ACER SACCHARUM.. 221-229
OCCURRENCE OF QUERCETIN IN
liMERSON'S BROWN- HUSKED

TYPE OF MAIZE i-ji

Sandstedt, R. M.. et al.: NUTRIENT RE-
QUIREMENTS OF GROWING
CHICKS: NUTRITIVE DEFICIEN-
CIES OF CORN 139-149

Schistosoma japonicum, hemotoxins 387-432

Schultz, E. S.: A TRANSMISSIBLE
MOSAIC DISEASE OF CHINESE
CABBAGE, MUSTARD, AND TUR-
NIP 173-17S

Schwartz. Benjamin: HEMOTOXINS

FROM PARASITIC WORMS 379^432

Sclerotinia cinerea, temperature relations. . . 451-465
Seamans. H. L., et al.: PALE WESTERN
CUTWORM (POROSAGROTIS OR-

THOGONIA MORR.) 289-322

Secale cerealc, susceptibility to aeciospores of

Puccinia triticina 163-172

Seed of coniferous trees, storage 479-510

Sequoia sempenirens, host of HyhtruPes lig-

neus 209-210

Sermn, efifect on hookworm hemolysin 41 2-413

Shapovalov, M., and Edson, H. A.: BLACK-
LEG POTATO TUBER-ROT UNDER

II^TUOATION 8i-9»

Sheep, Shropshire, fertility 231-234

Shropshire sheep, fertility 231-234

Sitanion Hystrix, susceptibility to aeciospores

of Puccinia triticina 163-172

Smut—
of oats. See Ustilago avenae.
onion. See Urocystis cepulae.
stinking, of wheat. See TiUelia tritici.
Sodium —
carbonate, efitect on hydrocyanic acid in

Sudan grass 135-136

chlorid, effect on growing chicks 145-149

hydroxid, effect on hydrocyanic acid in

Sudan grass 135-136

nitrate, effect on plant growth 102-110

sulphate, effect on plant growth 102-1 10

Soil temperature, effect on —

nodules 17-31

onion smut 235-262

Solanum tuberosum —

absorption of copper from soil 281-287

Fusaria rots 65-80

Sorghums, hydrocyanic acid 125-138

Soybean meal, effect on growing chicks 145

SOYBEAN MOvSAIC 111-114

Soybeans, effect of soil temperature on

nodule development 23

Sparrow, western grasshopper. See Ammo-
dranius s^vannarum bimaculatus.

Spelt fonn, behavior in wheat crosses 335-364

Spraying, influence on transportation rots of

stone fruits 467-477

Squirrel, ground. Citellus richardsoni.

Sterility in wheat crosses 58-61

Oct. i-Dec. 3, 1921

Index

525

Page

145-14Q
139-149
102-110

lOI-IIO

145-149

Stomach worm of sheep. Sec Hacmcnckits

contortus.
Stone fruit fungi, temperature relations. . . . 4^1-465
Stone fruits, influence of orchard spraying on

rots ,

407-477

STORAGE OF CONIFEROUS TREE

SEED

„, , . 479-.S10

Strand, A. L., et al.: PALE WESTERN
CUTWORM (POROSAGROTIS OR-

THOGONIA MORR.) 289-322

Stinking smut of wheat. See Tilktia tritici.
Succinic acid. See Acid, succinic.
Sudan grass. See Grass, Sudan.
Sugar-beet root aphis. See Pemphigus hUae.

Sugar beets, dry-rot canker 4.-,,

Sugar maple. See Acer sacckarum.

Sugars in grapefruit 26^-70

Sulphate-

calcium, effect on plant growth 102-110

copper, toxicity to potato plants 281-287

iron, effect on growing chicks

magnesimu, effect on growing chicks

sodium, effect on plant growth

Sulphates, relation to plant growth

Sulphur, effect on growing chicks

Sulphuric acid. See Acid, sulphuric.
Sumac. See Rkus glabra.
Sure, Bamett, and Read, J. W.: BIOLOGI-
CAL ANALYSIS OF THE SEED OF
THE GEORGIA VELVET BEAN,
STIZOLOBIUM DEERINGIANUM
SUSCEPTIBILITY OF THE DIFFER-
ENT VARIETIES OF SWEET POTA-
TOES TO DECAY BY RHIZOPUS NI-
CRICANS AND RHIZOPUS TRITICI. 511
Swanson, C. O.: HYDROCYANIC ACID

IN SUDAN GRASS

Sweet potatoes. See Ipomoea batatas.
Symphytum —
asperrimum, failure to become infected from

basidiospores of Puccinia rubigo-vera 152-172

officinale —

host of Puccinia bromina

susceptibUity to Puccinia trilieina..
Taenia —

saginata, hemotoxins iSo- ,-,-,

,. , , 300^433

sclruin, hemotoxms ,go .-^

Tapeworm. Sec Diphyllobothriunt laiunt.

Taraxacum officinalis, failure to become in-
fected from basidiospores of Puccinia rubigo-
vera

TEMPERATURE AND HUmidity^^ '^'
STUDIES OF SOME FUSARIA ROTS

OF THE IRISH POTATO 65-80

Temperature —

effect on hookworm hemolysin 413-414

soil, effect on—

nodules j^,^ ^

onionsmut 235-262

TEMPERATURE RELATIONS OP

STONE FRUIT FUNGI 4S1-465

Thalictrum —
angustifolium, susceptibility to teliospores
of Puccinia triticina j^5_j ^ j

125-138

154-172
152-172

157-173

157-172

• 157-172
of

• 155-172
. . . 165

240
24a

_,, , Page

1 n-dltctrum — Continued.

aquilegifolium, susceptibility to teliospores

of Puccinia triticina 155-172

dasycarPuvi, susceptibility to teliospores of

Puccinia triticina

Dclavayi, susceptibility to teliospores of"

Puccinia triticina

dioicum. resistance to teliospores of Puccinia

triticina

Fendleri. host of Puccinia CockeTelliana .... 165

foetidum. host of Puccinia triticina 164-173

viinus —

resistance to teliospores of Puccinia trit-
icina

,. ... 155-173

adtanttfolxum. susceptibility to teliospores

of Puccinia triticina 157-172

polycarPum, susceptibility to teliopores of

Puccinia triticina

polygamum. resistance to teliospore;

Puccinia triticina

sparsiflorum. host of Puccinia alfernans
Thorn, Charles, and LeFevre, Edwm-

FLORA OF CORN MEAL jjg-.ss

Thysanosoma adinioides. hemotoxins 421-432

Tilletia tritici, effect of —

soil moisture

soil temperature

Tillotson,C. R.: STORAGE OF CONIFER-

^Uf^^^ESEED ,„.3.„

Tisdale. W. B., and Jones, Fred Rcuel-
EFFECT OF SOIL TEMPERATURE
UPON THE DEVELOPMENT OF
NODULES ON THE ROOTS OF CER-
TAIN LEGUMES

Toxylon Pomiferum, host of Cyllene pictus. ^98-203

TRANSMISSIBLE MOSAIC DISEASE OF

CHINESE CABBAGE. MUSTARD

AND TURNIP.A 173-178

TRANSPORTATION ROTS OP STONE
FRUITS AS INFLUENCED BY OR-
CHARD SPRAYING 467_4„

Trichostrongylus retortaeformis, hemotoxins . 387-432
Trichuris —

depressiuscula, hemotoxins

triciura, hemotoxins

Triticum —
aegilops, susceptibility to aeciospores of Puc-
cinia triticina ,63-172

aesttium, host of Puccinia triticina 151-172

capilatum. behavior of the spelt form in

crosses , ^

^ , 335-364

com Pactum —

behavior ot the spelt form in crosses .

vigor of Fi of crosses

dicoccum—

behavior of spelt form in crosses ^^^

vigor of Fi of crosses " , ^^^^

dicoccoides, behavior of spelt form in crosses 361
durum —

behavior of spelt form in crosses

vigor of F 1 of crosses

polonicum, effect of crossing 335-364

satixum. behavior of spelt form in crosses

with T. spella

17-31

420
384-432

• 335-364
, ... 54-63

335
54-63

335-346

526

Journal of Agricultural Research

Vol. XXII

Page
Trilintm— Continued,
spelt a, behavior of spelt form in c rosses with

T. sativum 33S~364

turgidum, behavior of spelt form in crosses. 335
vulgare —

behavior of spelt form in crosses 335-36^

vigor of Ki of crosses. S4-63

Syu. Tritkum aestivum.
TroUius europeiis, resistance to teliospores of ^

Puccinia trilicina 1 5S~i72

Tuber-rot, blackleg 81-9

Tulip tree. See Liriodendron tulipifera.

Turnip. See Brassica rapa. " ^

TURNIP MOSAIC 123-12

Tussilago Farfara, susceptibility to Puccinia

triticina 152-1 7a

Urocystis cepulae, effect of soil temperature 235-26*
Urlica dioica —
failure to become infected from basidiospores

of Puccinia ritbigo-vera 152-172

susceptibility to Puccinia triticina 152-172

Ustilago —

avenae, effect of soil temperature 242

levis, effect of soil temperature 242

Valeriana dioica, susceptibility to Puccinia

triticina 152-172

Velvet bean. See Stizolobium deeringianum.
Vitis—

vinifera, succinic acid 224

sp., host of Cyllene pictus 198-203

Page
Wade, J. S., and Boving, Adam G.: BIOL-
OI.OGY OF EMBAPHION MURI-

CATUM 323-334

Walker, J. C! , and Jones, L. R. : RELATION
OF SOIL TEMPERATURE AND OTH-
ER FACTORS TO ONION SMUT IN-
FECTION 235-262

Water, hot, effect on hydrocyanic acid in Su-
dan grass 131-133

Weather, effect on hydrocyanic acid in Sudan

grass 136

Weimer, J. L., and Harter, L. L.:
A COMPARISON OF THE PECTINASE
PRODUCED BY DIFFERENT SPE-
CIES OF RHIZOPUS 371-377

SUSCEPTIBILITY OF THE DIFFER-
ENT VARIETIES OF SWEET POTA-
TOES TO DECAY BY RHIZOPUS
NIGRICANS AND RHIZOPUS TRIT-

ICI s"-sts

Western white pine. See Pinus monticola.

Wheat crosses, vigor of Fi seedlings 53-63

Wheat, orange leafrust 151-172

Whipworm. See Trichuris trichiura.

White oak. See Quercus alba.

White pine, western. See Pinus monticola.

Worms, parasitic, homotoxins 379-43»

Xylotrcchus colonus, host selection 195-220

Yellow pine. See Pinus ponderosa.
Yellow poplar. See Liriodendron tulipifera.

New York Botanical Garden Librar

3 5185 00263

3756

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