741

“\ \ STUDY IN CEREAL RUSTS:

PHYSIOLOGICAL RACES

A THESIS SUBMITTED TO THE FACULTY
: OF
THE GRADUATE SCHOOL
| OF
THE UNIVERSITY OF MINNESOTA
BY.
se ELVIN CHARLES STAKMAN
IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

MINNEAPOLIS, MINN.
JUNE 1913

A STUDY IN CEREAL RUSTS:

PHYSIOLOGICAL RACES

A THESIS SUBMITTED TO THE FACULTY

OF

THE GRADUATE SCHOOL
OF

THE UNIVERSITY OF MINNESOTA

BY

ELVIN CHARLES STAKMAN

IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE. DEGREE.OF
DOCTOR OF: PHILOSOPHY

MINNEAPOLIS, MINN.
JUNE 1913

The University —
MAY 12 1h

ies Bie Ore CONT e Na Ss

Page
Bape Biologic POrms) 4 ..5.(85 <-. se see oy Ss ene cia ers es 7
Pee eal eke icici asl cite des Al aoa pees lowansiler sa-coue ayompeabee¥ials #bars 7
Biereriniemtals to. mtg td salar se aie eieere nee eye ee 10
Ieee Aa eer wie oe a aa ak igs msdn eee wyenedarensvabehe, «/ieteushehaiel siege 10
Experiments with Puccinia graminis hordet...........+----- 12
Gerleral statement iseie cc tenaroctehevs sein RTE arabs slots) a iejee =) 12
Inoculations on Ty€ ......-.ee see e ence rete eee e en eeees 12
neu ations: OW: Oats. <1. misters tenets «cys aim ener ohne meer of = 13
Inoculation after the use of anesthetics........-...++--- 13
Anocilations-on Wheat, cc... 02 eee oie soe we oe eo er erorele wr aieis 14
Summary of inoculations with Puccinia graminis hordei... 15
Experiments with Puccinia gramimis avende.......-.++++-+- 15
eeralistatenient «venient cs = ws berets oreo sieseueirs 145)
Imoculations OM Tye... 425. ce cj tere eee neem nere 15
Inoculations under ordinary conditions.............--- 15
Effect of high fertilization .......-.-2..s++eeeeeeeeeee 16
CCINGE AMESHNELICS: ais bad ss cle aw siname ete a Seni ajay aie ne 16
Pifoctrot deat Wj Ulye cmac dee ars eye ccdedteens «9 eet een ae, me 16
Refi IEIOMS OM WiHleat os ccc each ere + sete yes hee ae mew me « 17
Inoculations under ordinary conditions................ 17,
tactvot anesthetics: <..0cc sis vac sce wets «> ogc mau ales 17
EREGERO Tr ATIATIUIC Ge lee toe = eave won sco oe ok es Sees ernve ake carer nas 18
Effect: Of leaf injury 22.02.52. <5 62 cee ec ee ane 18
Summary of inoculations on wheat...........--+++-+-- 18
AMO cCHIAtIONSION DATICY. sc... eee ss o)s oe mina s)re's\e = Salysinas 19
Inoculations under ordinary conditions ................ 19
Inoculations after exposure to anesthetics.............. 19
Summary of inoculations made with Puccinia graminis
FREE Ee CRADLE RACV EE ary a No Pee REIS et to 19
Experiments with Puccinia graminis SOCUNES «a aisia ocala ay een 20
MeCilAtiONS OM WiHEAt. © acs. «cess seers nies Reid e ood eee ie yD 20
Ham culations st Orc OATS"... ni > scucuerale e siauasels late iotta (cus «ie rans ctn 20
Inoculations on’*barley .........2.s.-eee eee eee e ee eees 20
AGE TIAtIONS OMMeIMCOLI: |. .dcisieet esr Seenc'= cio eae aleteyctiate « lhacbs- Pal
Experiments with Puccinia graminis tritict......+.+++++.++- 21
Inoculations on barley ........-.2- sees eee eee eee e eens 21
Inoculations On TYE 10... eee cee eee eee ee te eee e tenes 21
Inoculation under ordinary conditions................. 21

d

6 CONTENTS

Inoculation after exposure to ether... 9 =). tee 21
Inoculations on Oats’ s. ./sc/./cele niamics oie atone ate oie teen ten 21
Inoculations under ordinary conditions................. 21
Inoculation after-expostire to ether... 2... 4-01. eee De,
Inoculations on oats and rye after the use of barley as a
“bridging form’? 1 .,4.20.2 5 sieeveetes ere ieee one ene ee 22
Summary of experiments with Puccinia graminis tritici.... 22
Effect of the aecidial stage on biologic forms................ 23
General statement... .3 224 24a Se06 cc coe Sn eae ZS
Experiments in 1012) 200 S50 82 Paka ie cb ne cer
Experiments in 1913). 2 acc). een Doe te eee 24
Adaptation of biologic: forms’ to new hosts. 2... 7. «2. seen 25
Summary of Part Dew). 2 PActey yee teeta ste ne eae 27
Part IJ. Rust-resistant varieties ‘of wheat... .-) =... 4+ 28
Historical... 2... e+ sie aa soadetelechonte ato userareeepeiclicele eae 28
Forms “which are resistant:.0.5 21,0 ieee eee ae 29
Field: observations <x iiakc.-lese te ae, seus 29
Experimental .. 2c. ss. sf eiPaieislel sletaepeiaets sie eeu a 30
Greenhouse trials, .2s.:.26 220s wate cies setae ee 30
Inoculations on resistant forms \.% eee hes ee ee 32
Metabolism of the host and rust resistance.......... event See ae
Effect of water content of soil: .2..h: 2/220 4:5 eee 34
Effect of fertilizers 21.) 4)\4: ete 52 eee eee eee 36
The nature of resistance}. {aso hee aie ae ape ee 40
Histological details of infection 4: 2 cio .4 4.522): 42
Infection’ of Minnesota:INio: s1O3. 0. 2: feat ee ; oe
Infection of Khaplt ... Jc 2: aioe = oie ee tote 44
The course of infection in other resistant forms........... 45
Minnesota No. 163 inoculated with Puccinia graminis avenae 46
Summary Of Part Taq. eine epee sere 0s oe 48
Bibliography $s curses Asim tee pettiness eee 50

Explanation’ of” Plates sr.c0)s 00 .ciocmae tee cine ae ee 59

A STUDY IN CEREAL RUSTS

PHYSIOLOGICAL RACES

PART I. BIOLOGIC FORMS

HISTORICAL

Cereal rusts were known as destructive plant pests by the ancients.
Observations were made on the effect of weather and location on their
prevalence. The comparative susceptibility of the various cereals
received some attention. Theophrastus speaks of the varying suscepti-
bility of different cereals, as does Pliny, who says that barley is less
likely to rust than are other grains. In modern times many observa-
tions have been made, but many of these were only incidental.

Dietel (1887) calls attention to a certain amount of morphological
and physiological variation in rust fungi, but does not definitely estab-
lish the fact that there are distinct biologic forms. Previous to his
time Puccinia graminis Pers. was considered a single species found
on cereals and various grasses. However, in 1894 Eriksson (1894-1)
showed that, although the morphology of the fungus on the different
cereal hosts varied but slightly, there was a distinct specialization in
parasitism. He therefore divided the different species of rusts into
subdivisions which he termed “formae speciales.” Puccinia graminis,
the only species extensively used in the present investigation, he di-
vided into five formae speciales, two of which he mentioned as being
sharply demarcated and the remainder as being probably also distinct.

All of these forms of Puccinia graminis were capable of produc-
ing aecidia on various species of Berberis. It therefore occurred to
Eriksson that perhaps his forme speciales would be equalized when
grown upon the alternate host. This, however, he found not to be the
case. He concluded that the forms were physiologically distinct even
when grown upon Berberis. In fact he was led to believe that they
became more firmly fixed after a period on the barberry.

This discovery stimulated much research concerning the physiolog-
ical relationships of the Uredineae. In the United States Hitchcock
and Carleton (1894) made observations at about the same time that

fh

8 A STUDY IN (CBREALRUSES,

Ieriksson was making his in Sweden. Their conclusions pointed to
the fact that there was not much danger of rust from one cereal infect-
ing another. The same phenomenon was observed in rusts on a wide
variety of hosts. Magnus (1894 and 1895), Rostrup (1894), Klebahn
(1896), Dietel (1899), Ward (1901), Bandi (1903), and others firmly
established the fact that this specialization of parasitism was quite
common in the various rusts. To the formae speciales of Eriksson
various names were applied. Schroeter (see Magnus 1894, p. 360)
called them Schwester-Arten, and Rostrup (1894, p. 40) called them
biologische Arten, while Hitchcock and Carleton (1894) referred to
them as physiological races.

Neger (1902) found evidences of a similar condition among the
Erysiphaceze. Marchal (1902) conducted a large number of cross-inoce-
ulations with Erysiphe graminis and concluded that there was a fairly
large number of “races specialisées.”’ These did not differ essentially
in any morphological character. He showed (1903) and Salmon
(1903-2) later showed that the ascospores behaved in the same way
with regard to this specialization of parasitism as did the conidia.
Reed (1905) has shown that there may be more than one physiological
race upon a single genus of host plant.

Magnus was one of the first to try to explain the phenomenon of
specialization of parasitism. He distinguishes (1894, p. 366) be-
tween ‘‘Gewohnheitsrassen” or adaptive races and biologie forms.
The former name he applies to such forms as merely show difference
in infection power, while those which are fixed he calls biologic forms.
Long association with one host plant, he says, may bring the develop-
inent from Gewohnheitsrassen to biologic forms. This he showed
(1895) to be true not only of rust fungi but of others as well. Dietel
(1899) expressed a somewhat similar opinion, his idea being that
formerly a given species attacked a variety of hosts, but that it be-
came more and more specialized to form first Gewohnheitsrassen and
then biologic forms. Eriksson (1902, p. 657) says that rust forms adapt
themselves. Where a certain host is present in large numbers, and
climatic conditions are favorable, changes take place in favor of the
new host. These changes are expressed not only in the vitality of the
iungus but also in a higher degree of systematic firmness. The new
rust form, he says, becomes separated from its sister forms of parallel
origin and becomes “‘scharf fixiert.’’ His conclusion is, “Das Phanomen
der Spezialisierung steht nicht langer da als der Exponent eines dem
‘Schmarotzer innewohnenden, launenhaften und unerklarlichen Triebes,
seue Formen zu produzieren. Dieser Trieb wird durch die umge-
benden Verhaltnisse—die vegetative Unterlage und das Klima,—
unter denen der Parasit lebt, in eine bestimmte Richtung geieitet.”
This, Eriksson (1902, pp. 606 and 654) thinks, accounts for the fact

BIOLOGIC FORMS 2

that the specialization has taken a different course in Sweden from
that it has followed in the United States. The most widely grown
crops would naturally be the ones on which the particular biologic
forms adapted to them would attain their highest development. There-
fore, the fact that a rust shows particular relationships in one country
does not by any means preclude the possibility of a quite different set
of relationships in another country.

The idea that the fungus changes its habits as a result of environ-
ment is substantiated by many observations. Perhaps an extreme case
of such a tendency is found in the life history of Puccinia graminis in
Australia. McAlpine (1906, p. 21) states that the teleutospores seem
unable to infect the barberry in Australia, and, according to his obser-
vations, the fungus is quite rapidly being reduced to a reproduction
by uredospores only. This 1s accounted for by the absence of the bar:
berry.

Eriksson (1896, p. 339) shows that closely related host forms
are somewhat similar in their relation to rust. He says, however, that
the taxonomic relationship of host plants does not entirely determitie
the specialization of the rust form. Ward (1901) in his work on the
rust (Puccinia dispersa) of the bromes states that the closeness of re-
iationship of hosts is the determining factor in the ability of the rust
to pass successfully from one host plant to another. Freeman (1902)
also concluded that the farther removed a species of Bromus was tax-
onomically from the plant serving as a host for the rust the less prob-
ability there was of infection. Ward showed further (1903) that some
forms of bromes might act as bridging species in enabling the rust to
pass indirectly from one group of bromes to another, although direct
transfer was impossible. Salmon (1904) showed that the same thing
was true of Erysiphe graminis D. C. Freeman and Johnson (1911}
have found that barley can act as a bridging form enabling Puccinia
graminis to increase its range of infection power. Salmon (1904 and
1°05) showed that the range of infection possibility of Erysiphe
graminis forms may also be increased under certain cultural conditions.
By injuring leaves and subjecting plants to heat and anesthetics he
was able to infect normally immune forms.

The conception of a biologic form, then, is that it represents a
tendency toward adaptation. This tendency may be due to various
causes, the evidence being that it depends largely on the availability
of host species. Hitchcock and Carleton (1894), Carleton (1899), and
Freeman and Johnson (1911) investigated quite thoroughly the mat-
ter of biologic forms of Puccinia graminis in the United States. Free-
man and Johnson (1911, p. 27) give the following as the biologic
forms of this rust in the United States:

P. graminis tritici (stem rust of wheat) on wheat and barley.

10 A ‘STUDY IN‘CEREAL RUST S

P. graminis hordet (stem rust of barley) on barley, wheat, and

P. graminis secalis (stem rust of rye) on rye and barley.

P. graminis avenae (stem rust of oats) on oats.

If these forms are merely adaptations it ought to be possible to
change their parasitic tendencies by restricting or changing their en-
vironment. It ought to be possible to break down the biologic forms
under conditions abnormal for host or parasite. Various methods
have been tried in attempting to break down the specialization of para-
sitism of different fungi. Salmon’s work along this line on the Ery-
siphaceae has already been mentioned. Ray (1903) states that by
subjecting maize to ether vapor and then inoculating it with spores of
Ustilago zeae, the resulting infection was much more virulent than
that on plants not so treated.

Comparatively little work of this nature has been done with Puc-
cuua graminis. The fact that physiological races of rusts behave dif-
ferently under different conditions has been known for some time.
Much of the information was, however, gathered from incidental
observations. In breeding wheats for the purpose of obtaining rust-
resistant forms it would be very helpful to be able to correlate certain
characters with rust resistance. For this reason this phase of the
question, from both practical and scientific points of view, is of much
importance. The same is true of physiological races. It is important
to know if they can be broken down by means of a high degree of soil-
fertilization, if they become generalized by growing on the alternate
host, and if they adapt themselves readily to new hosts. The present
investigation was therefore undertaken with the object of determining
the possibility of developing and breaking down physiological races
and of obtaining definite information concerning the factors influencing
varying resistance in immune or semi-immune varieties of wheat.

EXPERIMENTAL.

METHODS

The rusts used in making inoculations were obtained originally
from their respective hosts in the fields at University Farm, St. Paul,
Minnesota. They were then artificially transferred to plants growing
in the greenhouse. Transfers were made to new plants about once
every three weeks until the rust had been confined to its own host for
at least twelve successive transfer generations. In nearly all the exper-
iments with biologic forms the rust had been confined to its own host
for at least twenty generations, thus giving assurance that it was the
particular biologic form desired.

The seeds of the host plants were planted in rich loam soil in four-

BIOLOGIC FORMS 11

inch clay pots. Only ten plants were left in each pot and the first leaf
of each was inoculated when six or seven days old. The plants were
trimmed whenever necessary so as to leave only the one inoculated leaf
on each plant. Fresh, viable uredospores were used for inoculations
except where otherwise specified. The spores were put on the leaves
with a flat inoculating needle which had been previously moistened
in order that the spores might better adhere to the leaf surface. The
pots were then placed in shallow pans filled with water, or on wet
sand, and covered with bell jars for forty-eight hours. In nearly all
cases a fine film of moisture covered the leaves during a considerable
part of the time that they were under the jars. This, together with a
moderate temperature, made the conditions for infection ideal. After
the removal of the bell jars the plants were kept on greenhouse benches
in such locations as to reduce to the minimum the danger of accidental
infection.

The grains used were the following, the numbers, except where
specified, being Grain Investigation numbers of the United States
Department of Agriculture:

Fife wheat, Minnesota No. 163

Velvet Blue Stem wheat, Minnesota No. 169

Minnesota No. 188 wheat, a cross between White Fife and Ladoga
Fife

Manchuria barley, Minnesota No. 105

Early Gothland oats, Minnesota No. 295

Swedish rye, Minnesota No. 2 ‘

Kubanka 1516—Nos. 8 and 9 pedigreed—Dickinson, N. Dak., 1910

Kubanka 2094

Arnautka 288

Arnautka 1431

Iumillo 1736 (1736-II-3 selected at Amarillo, Tex., 1910)

Einkorn (Triticum monococcum) 2433—Nos. 4, 6, 7, and 8 pedi-
greed—Dickinson, N. Dak., 1910

Emmer (Triticum dicoccum) 1522

Khapli (an Indian Emmer)

Of these the Kubankas, Arnautka, and Iumillo are varieties of
Triticum durum. The durums generally have the reputation of being
-more resistant to rusts than are the ordinary wheats (Carleton 1905, p.
9). Einkorn, also, is quite resistant while the emmers vary greatly,
one used in this work, G. I. No. 1522, not being very resistant. MKhapli
is an emmer obtained from India by E. C. Johnson, formerly Cereal
Pathologist in the office of Grain Investigations, United States Depart-
ment of Agriculture. It has no Grain Investigation number. It is the
most resistant of all the forms used. |

12 A STUDY IN CERBAL RUSTS

EXPERIMENTS WITH PUCCINIA GRAMINIS HORDEI

GENERAL STATEMENT

Freeman and Johnson (1911, p. 20) state that the barley stem
rust is more versatile than any of the other biologic forms of the cereal
rusts, and, further, that the range of infection of a given form is in-
creased after having been transferred to barley. The results of the
author, in general, agree with those of Freeman and Johnson, although
the percentages of infection are in some cases quite different.

INOCULATIONS ON RYE

In the first series 20 leaves were inoculated, after which the pots
were kept under bell jars for 48 hours. In 8 days very distinct signs
of infection began to appear and on the tenth day distinct pustules
were quite numerous. Eventually 16 leaves out of 18 showed distinct
pustules. Two of the leaves had been killed. Very evidently not all
of the mycelial wefts developed pustules, since pustules were often
intermingled with typical, yellowish rust flecks and green islands. The
pustules were in all cases small, most of them less than a millimeter in
size, but they were fairly numerous. There were some fairly large
areas in which no pustules developed, but which contained a large
number of green islands.

In the second series 40 leaves were inoculated, 10 of which eventu-
ally produced pustules. Of the remaining 30 leaves 29 were very
strongly flecked in such a manner as to show conclusively that infec-
tion had taken place although no pustules had been formed.

The results of both series indicate that rye is easily infected by
the stem rust from barley. Out of 58 leaves, 26 were infected, indi-
cating greater ease of infection than in case of the strain used by
Freeman and Johnson (1911, p. 19) in their experiments. The 29
leaves which are indicated as having been strongly flecked would at
first sight have been counted as having pustules. However, close
examination showed that there were no ruptured pustules.

Although a fairly high percentage of successful infections can be
obtained, rye is by no means a congenial host for Puccinia graminis
from barley. This is shown by the fact that the pustules are always
small. Long areas of the leaf may be killed and in this area there may
be many unruptured pustules. Often an area extending across the
entire leaf and one centimeter or more in length may be completely
killed. (See Plate I, A.) This may not contain a single pustule, but
close examination reveals the fact that there are many green islands,
some of which have a yellowish tinge in the center. These latter look
very much like unruptured pustules. Histological examination re-
veals the fact that the mycelium has spread, the host cells have died,
and wefts of mycelium have formed, either directly under, or a slight

BIOLOGIC FORMS 13

distance below, the epiderm. These wefts may send up a few hyphae
which resemble those normally producing spores. In some cases a
few small, abortive spores are formed, while in other cases, none are
produced.

This phenomenon very closely parallels that occurring in some of
the resistant forms of wheat. Attention will be called to this more in
detail later on. It will be sufficient here to emphasize the fact that
rye, when inoculated with Puccinia graminis spores from rye, does not
show any of these dead areas (see Plate I, B), the leaves remaining
green and producing pustules vigorously. When the spores, however,
are taken from bariey, infection takes place, but there is not such a per-
fect relationship set up between host and parasite as to enable both to
live and thrive for a long time.

INOCULATIONS ON OATS

The first attempt to infect oats with stem rust from barley failed
absolutely. For this reason the greatest precautions were taken to
furnish optimum conditions for germination of spores and develop-
ment of the mycelium. Of 104 leaves inoculated at various times, not
one produced pustules. Of this number 8 were slightly flecked, but
showed no tendency to form pustules. In fact, the flecks were ex-
tremely small and very few occurred on each leaf. In some cases
there was but a single fleck. These might quite easily have escaped
detection on some of the leaves had they not been invariably situated
in the inoculated area. Their position in this area indicates quite
strongly that they were true rust flecks. In these areas the tissues of
the leaves seemed to have been killed outright. However, the mycelium
must have been very restricted in its development since the diameter
of a spot never exceeded one millimeter.

Inoculation after the use of anesthetics—

In the first trial two pots of oats were exposed to the fumes of
ether for 15 minutes. Immediately after the exposure they were
inoculated with fresh spores of Puccimia graminis from barley and
then placed under a bell jar in a shallow pan of water. Two pots
were exposed to chloroform fumes for an equal length of time and
inoculated. At the same time 25 leaves were inoculated under normal
conditions, as a check.

Of those leaves exposed to ether, 4 out of a total of 20 developed
slight flecks but no pustules; neither were there any unruptured pus-
tules, which are commonly found when infection is not normal. Of
those exposed to chloroform 2 out of 19 developed pustules in 12
days. These pustules were very small, but were unquestionably pus-
tules of stem rust. A few of the leaves were so indistinctly flecked
that it was doubtful if they were true rust flecks.

’

14 A STUDY IN CEREAL RUS TS

In the second trial plants were again exposed to ether and chloro-
form, but the time was increased from 15 to 20 minutes. An attempt
was made to insure conditions for successful infection. It was ob-
served that a film of moisture formed on the leaves at night and the
temperature was moderate, insuring successful infection if that was
ordinarily possible.

Of the 20 leaves exposed to ether, 11 became very distinctly
‘flecked, but there were no evidences whatever of successful pustule
formation. In the chloroform series one leaf out of 20 produced a
small pustule and 8 became clearly flecked. There were 20 check
leaves, and, of these, 4 became flecked. The flecking was, however,
rather indistinct.

Out of a total of 193 inoculations only 3 resulted in pustule-for-
mation, and these only after exposure to chloroform. Of the remain-
der, 31 were flecked, but this flecking was not always sharp on the
control plants. In the other cases, however, it was distinct. The spots
were always less than one millimeter in diameter, and usually much
smaller. It appeared that this small area of the leaf had been killed,
thus preventing the further spread of the mycelium and precluding
the possibility of pustule formation. In no case was there any indica-
tion whatever that large areas of the leaf were involved, as was the
case when rye was inoculated with the rust from barley. In this con-
nection it may be mentioned that Freeman and Johnson (1911, p. 19)
were able to get 7 successful infections out of a total of 35, but the
pustules were always very small. It may be that the difference is due
to the use of various strains of the rust. The fact that there are strains
of the same biologic form seems to be quite definitely indicated. In
any case this seems to furnish an example of rather extreme incom-
patibility between host and parasite. In some cases it would seem that
the infection threads of the fungus are checked almost immediately ;
or that they may gain a temporary foothold, only to kill the cells upon
which they depend for nourishment and then develop but little further.

INOCULATIONS ON WHEAT

There is‘no question but that the stem rust from barley, in this
country, usually passes to wheat with practically the same degree of
readiness with which it passes to barley. In the’ trials made with this
form this appeared to be the case whether susceptible or resistant forms
of wheat were used. Inoculations on Arnautka, Khapli, and emmer
show that the barley rust is quite as capable as is the wheat rust of
attacking these varieties.

Pritchard (1911-1, pp. 181, 182) cites evidence tending to show
that the forms on wheat and barley in North Dakota are distinct. This
is not the case, however, with the strains used in the experiments in
Minnesota.

BIOLOGIC FORMS 15

SUMMARY OF INOCULATIONS WITH PUCCINIA GRAMINIS HORDEI

Wheat and barley are not included since in nearly every case 100
per cent of infection results. The denominator in each case represents
the total number of leaves inoculated, and the numerator, the number
of leaves which developed pustules.

Barley rust to:

Oats; 3-43 8 slightly flecked
Oats after exposure to ether qty; 15 flecked

Oats after exposure to chloroform see 8 flecked

Rye 34; 29 very strongly flecked

It will thus be seen that barley rust -does not find either rye or oats
a congenial host. It was transferred to rye much more easily in these
experiments than in those reported by other investigators in this coun-
try. Without exposing the host plants to anesthetics, however, no suc-
cessful infection of oats was obtained. By the use of ether and chloro-
form the possibility of infection was somewhat increased, as evidenced
by the formation of pustules on a small percentage of leaves and the
increased percentage of flecked leaves. In addition to this, the flecks
were much more distinct than those on the check plants.

EXPERIMENTS WITH PUCCINIA GRAMINIS AVENAE

GENERAL STATEMENT

This rust in this country is supposed to infect no cereal except
oats, although it is capable of infecting a number of grasses. Carleton
(1899, p. 60) was unable to transfer it successfully to wheat, barley,
or rye. Eriksson (1902, p. 601) mentions it as being found on oats
and 18 species of grasses in Sweden. Freeman and Johnson (1911,
p. 22), however, find that it can be transferred to barley also, and they
report that Derr succeeded in obtaining direct transfers from oats to
wheat and rye.

INOCULATIONS ON RYE

Since Derr, according to Freeman and Johnson (1911, p. 22),
obtained but one successful infection by inoculating rye with stem
rust of oats, a large number of trials were made with this form, espe-
cially since the same authors (l.c. p. 23) assert that under favorable
conditions these can undoubtedly be made.

Inoculations under ordinary conditions—

The results of inoculations made under average conditions show
clearly that it is possible to transfer the rust from oats to rye. Al-
theugh the percentage of entirely successful infections was small, the

16 A STUDY GAIN: CERBALYRUSES

appearance of inoculated plants would lead one to believe that the rust
transfers with greater ease than it really does. Out of a total of 55
leaves inoculated only 3 produced pustules. These pustules, although
small, were very distinct. Of the remaining leaves, 13 were clearly
flecked. It is the appearance of these so-called flecked leaves which is
often misleading. When looking at them from a distance they some-
times appear to be quite badly rusted. Closer examination, however,
shows that there are no pustules. Sometimes the flecks are so distinct
as to suggest the appearance of unruptured pustules.

The effect of high fertilzation—

A rich loam was well mixed with rich barnyard manure. Rye
was planted in two pots and the leaves inoculated in the usual manner.
Evidences of infection appeared at the usual time. It was found thai
the severity of infection was much greater than that on plants grown
in unfertilized soil. Of a total of 18 leaves 9 developed pustules and
the other 9 were distinctly flecked. The areas of infection on these
plants were much larger than on those grown in ordinary soil. The
leaf tissues were very clearly killed, sometimes in areas a centimeter
long, and in these areas small pustules appeared. The appearance
was very characteristic of semi-normal infection. The mycelium spread
fairly well, but the host cells were killed and only small pustules were
aeveloped.

The etfect of anesthetics—

Plants were inoculated after exposure to ether, chloroform, and
nitrous oxide for periods ranging in different experiments from 5 to
15 minutes. The difference between the check plants and those ex-
posed to anesthetics for 10 minutes was fairly distinct. This was not
apparent so much in the number of pustules or flecks, but rather in the
sharpness of the flecks. The following were the results:

After exposure to ether ae 26 flecked

After exposure to chloroform ee 13 flecked

After exposure to nitrous oxide oe 6 flecked

It will thus be observed that in point of numbers the difference
between the success of these infections and those on the check plants
was not great. However, a real difference in severity of infection,
although not remarkable, did exist. On the other hand the plants in
highly fertilized soil developed a larger number of pustules and also a
more typical infection.

Effect of leaf injury—

A number of leaves were injured, just previous to inoculation, by
being punctured in many places with a sterilized needle point. Spores
were then placed on this injured area in large numbers. The epidermis

BIOLOGIC FORMS 17

was stripped from others and the inoculations then made. The results
were not different from those obtained on check plants. Only one
pustule developed on one leaf out of a total of 28. Six of the remain- -
ing leaves were flecked, but the flecks remained small. In no case did
the virulence of infection equal that on plants grown in highly fertilized
soil.

The stem rust of oats, then, can be transferred directly to rye.
This was accomplished most easily when the plants were grown in
heavily manured soil. Exposing them to various anesthetics before
inoculation seems to increase the virulence of infection slightly, while
leaf injury had no apparent effect. In the various trials, under differ-
ent conditions, 27 out of 236 leaves developed pustules and 73 were
flecked. Under conditions which might exist in the field 12 out of 73
leaves produced pustules and 22 were flecked.

INOCULATIONS ON WHEAT

The stem rust of oats can be transferred to wheat only with great
difficulty. Carleton (1899, p. 60) did not succeed in obtaining infec-
tion in his experiments. Freeman and Johnson (1911, p. 22) made
100 inoculations but none was successful. They report, however, that
Derr was able to make transfers.

Inoculations under ordinary conditions—

The fact that the oat rust is transferred with great difficulty to
wheat became apparent very soon. Out of 108 inoculations only one
was entirely successful, although 5 leaves became slightly flecked.
Very evidently the infection threads, after having grown among the
tissues of the leaf, must have died, since only very minute flecks de-
veloped, and these in only a few cases.

Effect of anesthetics—

Plants were inoculated after exposure to ether and chloroform
for periods of from 1 to 15 minutes. The best results were obtained
after exposure to ether for 5 minutes. In this case 3 leaves out of 50
developed pustules while all the other leaves were flecked. These
pustules and flecks appeared 10 days after inoculation, this being
slightly longer than a normal incubation period. None of the check
plants showed any distinct signs of successful infection, while the
flecks on the plants which had been exposed to ether were very con-
spicuous, forming a sharp contrast with the check plants. These
flecks were scattered all along the line of inoculation and had the
appearance of young, unruptured pustules.

This series offered the best evidence that anesthetics may have
some influence in rendering a plant more susceptible to a rust than it
would otherwise be. In subsequent trials, exposing the plants for a

18 A STUDY IN CEREAL RUSTS

greater length of time, no pustules were developed on any of the 40
leaves inoculated. Nine of these leaves were, however, quite strongly
flecked. The use of chloroform did not result in the formation of
pustules, although there was a distinct advantage over the check plants
both in number and definiteness of flecks. In all, 40 plants were inocu-
lated and of these 19 became distinctly flecked. After the use of ether,
then, 3 out of 90 leaves were successfully infected, and 56 were sharply
flecked, and these, added to the totals after the use of chloroform, give
3 pustules and 75 infected leaves which developed no pustules out of
130 trials.

Effect of manure—

Wheat was planted in two pots containing a rich loam very heavily
fertilized with rich barnyard manure. Of the 20 leaves inoculated, 6
developed pustules and the rest were strongly flecked. In this case,
however, there was a possibility that the pustules developed as a result
of accidental infection. The flecks were apparently due to the arti-
ficial inoculation, since such flecks have never been observed after
direct inoculation with spores from either wheat or barley, or after
the inoculation of wheat with wheat or barley rust. It is therefore
quite certain that the pustles, which were normal, were the result of
accidental infection, while the flecks, which were exactly like those
developed on a semi-immune form, were the result of artificial inocula-
tion.

Effect of leaf injury—

In one experiment, 16 leaves were pricked full of holes in an
area of one centimeter or more. They were then inoculated, and 4
became flecked, but no pustules developed. In another experiment the
epiderm was stripped from 29 leaves immediately before inoculation.
Although 10 became flecked, the flecks were extremely minute and no
pustules were developed. Histological examination showed that the
spores had sent out germ tubes in large numbers. These tubes grew
among the host cells, but true infection did not take place. Sections
of these plants were made and examined. It was clearly evident that
jeaf injury did not increase the chances for infection. The hyphae did
not develop better than did those in normally inoculated plants.

Summary of inoculations on wheat—

Out of a total number of 283 leaves inoculated under varying
conditions only 4 developed pustules and 113 became flecked, showing
that, although the rust of oats can sometimes develop on wheat, it can
seldom attain to pustule formation. The severity of infection, always
very slight, can be increased somewhat by exposing plants to be in-
oculated to anesthetics. The experiment with high fertilization of

BIOLOGIC FORMS 19

soil seems to indicate that here, as well as in the case of rye, inoculated
with oat rust, the mycelium is enabled to develop more extensively
and possibly produce more spores than when ordinary soil is used.

INOCULATIONS ON BARLEY
The results of this series of inoculations were very similiar to
those obtained by other investigators. The behavior of the rust was
typical of its usual behavior on an uncongenial host plant. The flecks
were small in nearly all cases and were especially sharp when plants
had been exposed to anesthetics.

Inoculations made under ordinary conditions—

These were not especially successful. No pustules were developed
on 50 inoculated leaves but 10 of the leaves became flecked.

Inoculations after exposure to anesthetics—

Ether and chloroform were used. The period of exposure varied
at different times from 5 to 15 minutes. Plants exposed only 5 minutes
did not appear different from the check plants. After exposure for
15 minutes the flecking was more distinct than on the check plants.
Only one pustule developed in the entire number of trials, this being
after exposure to ether. There were 47 leaves inoculated after expo-
sure to ether, and of these one developed a pustule and 15 were fleck-
ed. The flecks in this case had every appearance of unruptured pus-
tules. No pustules were developed after exposure to chloroform,
but 7 leaves out of 50 became flecked. Of 147 leaves inoculated 32
were flecked, but only one showed a pustule.

SUMMARY OF INOCULATIONS MADE WITH PUCCINIA GRAMINIS AVENAE

Oat rust to:

Wheat tae 5 flecked

Wheat after exposure to ether a0: 56 flecked

Wheat after exposure to chloroform oo ; 19 flecked

Wheat after leaf injury es 14 flecked

Wheat plants grown in manured soil ae:

Barley 3 10 flecked

Barley after exposure to ether ,',; 15 flecked

Barley after exposure to chloroform 5 i 7 flecked

Rye eS: 13 flecked

Rye after exposure to ether a 26 flecked

Rye after exposure to chloroform 22e% 13 flecked

Rye after exposure to nitrous oxide shu 6 flecked

Rye after leaf injury sh 14 flecked

Rye grown in highly fertilized soil =", ; 9 flecked

20 A STUDY IN CEREAL RUSTS

The rust can be transferred successfully from oats to any one of
the other cereals. It infects rye much more easily than wheat or bar-
iey. Anesthetics help to break down the barriers as does a high degree
of soil fertilization. It should be mentioned that this fact seems due,
not to any new ability the rust fungus has of attacking an uncongenial
host, but to an increased capacity for development. Here again evi-
dence of the possible nature of resistance is offered by the inoculated
plants. The flecks, whether large or small, consist in many cases of
dead tissue of the host plant. Histological examination shows that the
fungus gains entrance but cannot develop to any extent in these areas
which are killed by the fungus itself.

EXPERIMENTS WITH PUCCINIA GRAMINIS SECALIS

These experiments were not very extensive and were made with
the idea of determining rather the character than the number of suc-
cessful infections.

INOCULATIONS ON WHEAT

In all 70 leaves were inoculated. None of them produced pustules,
but 18 became flecked. The flecks were distinct but in no case were
they large. There seemed to be much less dead tissue than is the case
in many of the other forms, as for instance rye inoculated with barley
rust. In fact the flecks were hardly noticeable unless one looked very
carefully for them. Apparently they were due to the death of host-
plant cells in the inoculated area.

INOCULATIONS ON OATS

In addition to a small number of control inoculations, some were
made after exposure to ether and others after exposure to chloroform.
The anesthetics apparently made no particular difference in the suc-
cess of the attempts, although there were more flecked leaves after ex-
posure to chloroform. Nothing out of the ordinary appeared so only
the summaries are given:

After chloroform, 5 min. aus 5 slightly flecked
After ether, 5 min, .°,; 2 slightly flecked
Under ordinary conditions =°,; 1 slightly flecked

INOCULATIONS ON BARLEY

After exposure to ether for 5 minutes, 16 out of 21 inoculated
leaves became very distinctly flecked. There were many of these
flecks all along the line of inoculation. They were very suggestive of
the flecking in other forms in which the mycelium was known to have
spread to some extent. However, no pustules were developed. Judg-
ing from the experience with other forms, if these plants had been
highly fertilized they might have developed pustules. Of the check

BIOLOGIC FORMS 21

plants, only 4 out of 40 became flecked. No pustules were developed.

INOCULATIONS ON EINKORN

Einkorn was inoculated with rye rust both after exposure to
ether for 15 minutes and without having been so exposed. The length
of time in ether fumes was 15 minutes. No pustules developed on
any of the leaves, 20 being used in each series.

EXPERIMENTS WITH PUCCINIA GRAMINIS TRITICI
INOCULATIONS ON BARLEY

It has long been a well-established fact that stem rust from wheat
can easily attack barley. The infection in trials made by the writer
was found to be normal and practically as virulent as if spores had
been taken directly from barley. In the one series tried especially for
the purpose 30 out of 30 leaves became characteristically infected.
There is no evidence whatever of uncongeniality between host and
parasite.

INOCULATIONS ON RYE
Inoculation under ordinary conditions—

In the control experiments 6 out of 30 leaves developed pustules
and 20 were strongly flecked. In fact, they were so strongly flecked
that flecking hardly expresses properly the appearance developed.
Long areas on the leaf were killed outright. In these dead areas very
small green islands were often found, some of which contained un-
ruptured pustules. All the pustules were very minute, and some had
ruptured the epiderm. It was another very good example of semi-
compatibility between host and fungus. The host leaves did not suffer
very great injury; the fungus was enabled to spread to a certain ex-
tent but succeeded in producing only small pustules.

Inoculation after exposure to ether for five minutes—

The results here were very striking. Fifty leaves were inoculated,
6 of which produced pustules, while every leaf was infected with the
mycelium of the rust fungus. The character of the infection was much
the same as was that on the check plants. The areas were perhaps
more extended. On some leaves there were dead areas 3 centimeters
long, while ordinarily they were not so long on the check plants.

INOCULATIONS ON OATS
Inoculations under ordinary conditions—

Direct inoculation of oats by spores from wheat has not met with
success on the part of either Carleton (1899, p. 54), who, however.
reports a doubtful case, or Freeman and Johnson (1911, p. 18), who
cite Derr as authority for the statement that this direct transfer can
be made.

22 A STUDY IN CEREAL RUSTS

Attempts to transfer directly from wheat were unsuccessful. Not
a large number were made, but of the 46 attempts no leaves produced
pustules and only 2 of them developed flecks. The flecks were very
small and not very distinct. In this connection it may be mentioned
that when oat leaves were inoculated with aecidiospores derived from
wheat teleutospores one leaf out of 56 developed pustules. This ap-
pears to indicate that direct transfer can be made. The success of the
inoculation was probably not due to the fact that the rust had passed
through the barberry stage. This seems especially true since the
other cereals behaved in the same way toward the aecidiospores as
they did toward the corresponding uredospores.

Inoculation after exposure to ether—

Only 30 leaves were inoculated after having been in ether fumes
for from 3 to 5 minutes. No pustules were developed, but 10 of the
leaves were flecked. There was a perceptible difference between the
check plants and those of this series, although it cannot be said that
the results were very striking.

INOCULATIONS ON OATS AND RYE AFTER THE USE OF BARLEY AS A
BRIDGING FORM

The work done on bridging species has already been mentioned.
Since it is almost impossible to infect oats directly with wheat aecidio-
spores or uredospores, attempt was made to transfer aecidiospores de-
veloped from wheat teleutospores first to barley and then to infect
oats with the resulting uredospores. The attempt was made with
fourth- and fifth-generation uredospores from barley, these uredo-
spores having been derived from wheat-rust aecidiospores. Successful
infection took place in one out of 39 attempts, 4 leaves being distinctly
flecked.

The rye plants are possibly slightly more severely attacked if the
rust is transferred first to barley, 16 out of 19 of the plants inoculated
becoming infected.

SUMMARY OF EXPERIMENTS WITH PUCCINIA GRAMINIS TRITICI
Wheat rust to:

Barley 39

Rye 3°53 20 flecked

Rye after exposure to ether =5,; 43 flecked

Rye after barberry and 4 generations on barley +

Oats, uredospores vsi 2 flecked

Oats, aecidiospores sy

Oats after exposure to ether .°,; 10 flecked

Oats after barberry and 4 generations on barley fe 4 flecked

——EEE——

BIOLOGIC FORMS 23

EFFECT OF THE AECIDIAL STAGE ON BrioLocic Forms
GENERAL STATEMENT

Eriksson (1894, pp. 292-309) was one of the first to suggest the
possibility of breaking down biologic forms by means of the aecidial
generation. He came to the conclusion, however, that this could not
be done, that, as far as the cereal rusts are concerned, they behave in
exactly the same manner when transferred through barberry as they
do in the uredinial stage. Salmon (1903, p. 159) and Marchal (1903,
p. 280) showed that with biologic forms of the Erysiphaceae the rela-
tions were exactly the same whether ascospores or conidia were used.
There is not an exact parallelism between the two since the cereal
rusts are heteroecious. However, the cases are somewhat similar since
a sexual fusion intervenes in both. Arthur (1910, pp. 227-228) con-
cludes that although there is distinct specialization of parasitism in
Puccima poculiformis (Jacq.) Wettst. (Puccinia graminis Pers.) on
various grasses, this specialization breaks down in the aecidial stage.
The barberry would, then, serve as a bridging form between the vari-
ous grasses. Jaczewski (1910, pp. 356-357), on the other hand, does
not find this to be the case with biologic forms of Puccinia graminis
on cereals and grasses in Russia. His experiments support Eriksson's
claim that the barberry does not change the physiological specialization
of the various strains when they produce aecidia.

EXPERIMENTS IN 1912

In connection with the experiments described by the writer it
should be mentioned that the aecidia used in one set of experiments
were developed in the field but there was very slight chance for acci-
dental infection. Wheat straw very badly affected with rust in the
teleutospore stage was tied around barberry bushes. There was no
other rusted straw of any kind within considerable distance, so that
there was little chance of accidental infection. These aecidiospores
which were developed from wheat rust were transferred to wheat,
barley, oats, rye, and einkorn. The following were the results:

RESULTS OF INOCULATIONS OF CEREALS WITH AECIDIOSPORES OF PUCCINIA GRAMINIS
TRITICI DEVELOPED IN THE FIELD

Grain

Wheat

Barley

Oats

Rye

Einkorn

*Somewhat doubtful.

24 A STUDY IN CEREAL, RUSTS

It will be noticed that the percentages of infection are practically
the same as are those developed from uredospore inoculation. This
is true also of the character of the infection.

On the rye plants, for instance, there was the same characteristic
spotting and the same small, abortive pustules. The pustules on rye
were all very small and there was no observable increase in virulence.
The same thing is true of einkorn. On oats but one rather doubtful
pustule developed, indicating that the aecidial stage in no way broke
down the barriers in this case.

EXPERIMENTS IN 1913

In the fall of 1912 the barberry bushes in the rust plat were sur-
rounded with badly rusted wheat straw, the rust being in the teleuto-
spore stage. In the spring of 1913 the aecidiospores were used in
inoculating the four common cereals and einkorn. The resuits were
surprising since it was supposed that the aecidia had been developed
from the wheat-rust teleutospore sporidia. The results of the various
trials are given below:

RESULTS OF INOCULATIONS OF CEREALS WITH AECIDIOSPORES FROM FIELD BARBERRIES

; TRIALS
Grain a. Total
I Il
Wheat u8e 2 is)
0 25 95
0 0 0
Oats 40 30 T00
32 16 6 6
Barley 40 27 oT
40 " 55
Rye 40 BE) BO
oot 12 2 ie
Einkorn eo 2 7

It will readily be seen that there would naturally be much doubt
as to the origin of these aecidia from wheat-rust teleutospore sporidia.
These aecidiospores were being used in spraying wheats in the rust
nursery for the purpose of developing a rust epidemic. The results
were very discouraging. There was a great deal of Agropyron repens,
badly affected with the teleutospore stage of Puccinia graminis, near
the barberry bushes. Experiments were therefore started to determine
whether or not the aecidia on barberries might not have been devel-
oped from this source rather than from the wheat-rust material. This
view seemed all the more reasonable when it was observed that the
Agropyron repens was very severely affected with the uredospore
stage of rust, while the wheat, although it had been thoroughly and
persistently sprayed with water containing the aecidiospores, had only
a few scattered pustules of rust.

BIOLOGIC FORMS 25

Barberry bushes which had been in the cellar all winter were set
out in the field and covered with a heavy muslin cage. Badly rusted
straw of wheat was tied around one bush and that of Agropyron repens
around another. None of the check plants developed any aecidia while
those surrounded with straw were very badly affected. The Agropyron
repens material produced mature aecidia 10 days earlier than the wheat
material and the aecidia were also developed in greater abundance.

RESULTS OF INOCULATIONS WITH AECIDIOSPORES AND UREDOSPORES FROM WHEAT
AND AGROPYRON REPENS

Wheat
aecidia

Wheat
uredospores

A. repens
aecidia

A. repens
uredospores

Grain

6 30
Wheat 27 30

0 0
Oats 29 40

12 30
Barley 30 30

Rye

The field barberries were very probably infected with the rust from
Agropyron repens, as will be readily observed by referring to the two
tables. Further, the various biologic forms do not show any apparent
change as a result of having been transferred to barberry, thus con-
firming the results obtained in previous experiments.

These inoculations, although not extensive, show quite clearly
that Puccinia graminis tritici and Puccinia gramims from Agropyron
repens do not seem to develop any greater range of infection possi-
bility for cereals after having lived for a time on the alternate host—
the barberry. The incubation period, even on wheat, barley, and ein-
korn, was a little longer in these experiments than that of uredo-devel-
oped mycelium.

ADAPTATION OF Brotocic Forms To NEw Hosts

Magnus (1894, p. 362) was one of the first to suggest that a
particular biologic form might, by constant association with one host,
change its physiological capabilities to such an extent as to make a
new race out of it. This view was also expressed by Dietel (1899, pp.
81 and 113) who gave it as his opinion that a given rust formerly at-
tacked a number of plants but by long association with one form be-
came narrowed to this form more closely, possibly retaining also a
somewhat weakened capability of attacking other forms. These
authors distinguish between adaptation races (Gewohnheitsrassen) and
true biologic forms, the tendency being, under favorable conditions,

26 A STUDY IN CEREAL RUSTS

for the former to develop into the latter. Eriksson (1902, p. 657) also
expresses this view in a somewhat modified form. Ward (1902-1)
shows that adaptation of Puccinia dispersa takes place. Klebahn (1904,
pp. 152-167) cites numerous experiments to show that this may be
the case. Miss Gibson (1904, pp. 184-191) grew a number of suc-
cessive generations of rust on resistant host varieties, but, in that time,
found little adaptational tendency. Massee (1904, p. 17) explains the
resistance and susceptibility of various plants to parasitic fungi on the
ground of the presence or absence of chemotactic substances in the
host. He contends that saprophytes can be educated to become para-
sites. This would also apply in large measure to biologic forms. Sal-
mon (1905, p. 183) grew Erysiphe graminis from wheat on Hordeum
sylvaticum for five generations and found no diminution in the power
to infect the original host. Freeman and Johnson (1911, p. 28) con-
clude that “the host plants exercise a strong influence, not only on
the physiological and biological relationships, but in some cases even
on the morphology of the host.”

An attempt was made to test this matter. The object was to de-
termine the change, if any, in the physiology of the rust as evidenced
by its power of infection and in its morphology as evidenced by changes
in spore dimensions. For this purpose Puccinia graminis tritici was
grown on Minnesota No. 163, a susceptible wheat, and on einkorn 2433.
The einkorn, in the first few trials, was apparently one of the most
resistant of the Triticums to the wheat stem rust. The rust was trans-
ferred to einkorn and grown on this host through successive genera-
tions for 19 months, transfers being made, on an average, once every 3
weeks. After one year on einkorn the rust seemed to be much more
virulent than the original wheat rust had been. Einkorn plants were
inoculated with this einkorn rust and with uredospores from wheat.
In both cases there was 100 per cent of infection, but the virulence of
the infection was quite different. On the leaves inoculated with rust
from einkorn, leaf areas one to two centimeters long were affected,
being well covered with pustules shedding spores in great abundance.
Individual pustules were from one to three millimeters long and nearly
as broad, giving every indication of a severe rust attack. On the plants
inoculated with wheat rust, on the other hand, the pustules were al-
ways smaller, although fairly numerous. Few of them were as much
as one millimeter long and many of them did not rupture at all.

Some wheat plants were inoculated with rust from einkorn and
others with the rust taken directly from wheat. Here again there wasa
considerable difference in virulence of infection. The pustules developed
from einkorn-rust inoculations were fairly large and numerous. Some
of them were two millimeters long, although the average length was
less than this. The infection on wheat inoculated with wheat rust was

BIOLOGIC FORMS 27

much more severe. Areas of the leaf two centimeters in length were
often almost covered with pustules, some of which attained a length of
seven millimeters.

A number of other trials were made, the last after the rust had
been confined to einkorn for 17 months. In one experiment the re-
sults were just the opposite of what were expected. This is explained
by the fact that lack of greenhouse space necessitated keeping the ein-
korn plants inoculated with einkorn rust in a draught of cold air.
There were only a few wheat uredospores available in inoculating the
wheat, so, in this experiment, einkorn-by-einkorn inoculations were not
so successful as einkorn-by-wheat. In subsequent trials, however, un-
der uniform conditions, the results first described were substantiated.

The conclusion, then, is justified that by confining Puccima gram-
imis tritici to einkorn for successive generations throughout a year or
more the rust adapts itself somewhat to its new host and loses, at least
to a slight degree, its power to infect the original host. It would no
doubt require a very long period of time to fix this character in the
plant to such a degree as to make it a new biologic form. But there is
very evidently such an adaptational tendency (see Plates II and III).
It must be noted, however, that this new character is not so firmly
fixed that it cannot be overbalanced by environmental factors. The ex-
perimental production of new forms is apparently possible, but a long
period of time is required.

The change in the fungus manifests itself not merely in the para-
sitic tendency toward the host but in the morphology as well. Wheat
and einkorn were inoculated with spores from the same plant. The
uredospores after growing for a year on wheat averaged 37.85x 22.76
while those grown on einkorn for a year measured 33.58x 21.79 y,
When einkorn was inoculated with Puccimia graminis tritici aecidio-
spores, the resulting uredospores were more nearly identical with the
wheat-rust spores in length. The width, however, remained practically
the same. The average size of these spores was 35.92 x 21.69 yu.

SUMMARY OF PART I

1. Direct transfers of Puccinia gramims have been made from
oats to both wheat and rye. The rusts from oats and barley used in
these experiments could be transferred to rye more easily than those
used by Carleton or those used by Freeman and Johnson. The barley
rust, however, did not prove as versatile as the strain used by Freeman
and Johnson.

2. The use of anesthetics has some effect in rendering an immune
plant slightly more susceptible to the rust; leaf injury apparently had
no effect.

3. High fertilization, by increasing the virulence of the attack

28 A STUDY IN CEREAL: RUSTS

on semi-immune forms, may have some influence in breaking down
biologic forms.

4. There appears to be a physiological and even a slight mor-
phological change in the rust fungus when grown continuously on a
semi-immune host. The physiological change manifests itself as an
adaptation to the new host, which, however, is very gradual.

5. There are indications that biologic forms of cereal rusts, at
least Puccinia graminis tritici, do not lose their specialization tenden-
cies when grown on barberry.

6. The degree of incompatibility of host and parasite varies
greatly. In semi-compatible forms, fairly large leaf areas are some-
times killed, indicating a killing of host cells by fungus and consequent
death of the mycelium itself. In this respect they resemble very closely
some of the rust resistant forms of wheat. The biologic forms of rusts,
therefore, with susceptible and immune varieties of host plants, throw
light on the question of the nature of resistance to Puccinmia graminis.

PART II. RUST-RESISTANT VARIETIES OF WHEA®

HISTORICAL

It has long been a matter of common observation that some wheats
are more resistant than others to the attacks of Puccimia gramims and
other rusts. Among the earlier observers Henslow (1841, p. 3), La
Cour (1863, p. 326) and Little (1883, p. 634) note that some wheats
are less injured by rust than are others. Bolley (1889, p. 16) observes
that those varieties least susceptible to rust are “hardy, stiff-strawed
wheats, having smooth, fibrous leaves.’’ Anderson (1890, p. 84) says
that hard, flinty wheats are more rust-resistant than others. He thinks
it may be due to the large amount of silica in the hard wheats. Cobb
(1892) advanced his “mechanical theory” to explain resistance. It
was due, according to his idea, to morphological characters of the
host, namely, thick cuticle, waxy coating, and small stomata. Hitch-
cock and Carleton (1893, p. 12) also correlated resistance with morpho-
logical characters, asserting that resistant forms had stiff, upright
leaves with a thick epidermis. Eriksson (1895, p. 199) and many
others since have shown, however, that a wheat resistant to one spe-
cies of rust is not necessarily resistant to another species, thus indicat-
ing a rather delicate relationship as the basis of resistance.

Eriksson and Henning (1896, pp. 332-365) were unable to sub-
stantiate Cobb’s mechanical theory, since morphological characters

RUST-RESISTANT VARIETIES OF WHEAT 29

could not always be correlated with resistance. They suggest that re-
sistance is of a complex chemico-physiological nature and is inherent
and fairly constant within the plant. Ward (1902) decided in connec-
tion with Puccinia dispersa on the bromes that resistance was indepen-
dent of any recognizable morphological character and suggested that
the problem was much the same as that dealing with the factors gov-
erning fertility and sterility of stigmas to pollen. Biffen (1907, p. 128)
concludes that resistance to Puccinia glumarum is independent of any
discernible morphological character. He reasserts this principle in a
later work (1912, pp. 421-429). Bolley (1908), although not positive,
inclines to the view that disease-resistance is physiological rather than
morphological in its nature. Cook and Taubenhaus (1912) show that
various vegetable acids are toxic to parasitic fungi and that the amount
of some of these acids present depends on the stage of ripening of the
fruit. Jones, Giddings, and Lutman (1912, p. 83) conclude that the
resistance of potato tubers and leaves to Phytophthora infestans is due
to something either largely or wholly within the tissues. The consen-
sus of opinion among the more recent investigators seems to be that
there is a very delicate balance maintained between host and parasite.
This balance is dependent; to a certain extent, on the environmental
conditions under which host plants are grown. This, however, as well
as other phases of the problem, will be discussed more fully under the
various sections into which the question naturally subdivides itself.

FORMS WHICH ARE RESISTANT

Attention has frequently been called by various observers to the
fact that freedom from disease does not necessarily indicate resistance.
Varieties which mature early may escape the disease, and various other
factors, mainly ecological, may influence the degree of resistance.
Careful experiments, however, have shown that some varieties of
Triticum durum are really resistant. The resistance of durum wheats
varies with locality according to Bolley’s observations (1906, p. 662).
Consequently to determine absolute resistance the plants should be
grown under controlled conditions. Further, they should be subjected
to conditions favorable for infection.

FIELD OBSERVATIONS

Field observations were made on various forms which were grown
in a rust plat. An epidemic was induced by spraying frequently with
water containing a large number of spores of Puccinia graminis tritici.
Frequent observations were made on the amount of rust. Final notes
were taken at ripening time. The percentage indicates in each case
the estimated percentage of resistance, assuming absolutely immune
forms to have a resistance of 100 per cent.

30 A STUDY IN CEREAL RUSTS

ESTIMATED PERCENTAGES OF RUST ON VARIETIES GROWN IN 1911 anp 1912

1911 1912
Per Per
Variety Cent Variety Cent
Einkorn 2433
_ Dickinson 1910 Ped. No. 4.. 92 Minnesota No. 169........... 15
BNO hoe riches ci ee 90 Lamillo 1736,.../eache ae 98
INGSEA tee mira herein 93 Minnesota No! 18S5-4-ceneenee 30
INOS eden aoe cae ee a Einkorn -2433° ¢...504 00s See 96
TIninatllag 1736) 445, Sits. s atte fee 95 Khapli! 00.) 2a ie cee eee 95
Kubanka 1516 No. 8.......... 15 Minnesota No. 169 ........... 35
Keubankas 1I5160No0. 95-2255: 24 10 Arnautka. :Z288: {).cic~ soe eee 97
Minnesota Noy 163s-e serene 10 Arnautka 1431... «4:46 67

It will be noticed that in 1912 the resistance was slightly greater
than in 1911. This may be accounted for by the fact that in 1911 the
grains were sown late, giving the rust ample opportunity to develop
fully.

EXPERIMENTAL

GREENHOUSE TRIALS

The varieties tested for resistance in the greenhouse were: Minne-
sota No. 163, Minnesota No. 169, Kubanka 1516, Nos. 8 and 9, Ku-
banka 2094, Iumillo 1736, einkorn 2433, Nos. 4, 6, 7, and 8, emmer
1522, Arnautka 288, and Khapli. Of these, Minnesota No. 163, Min-
nesota No. 169, Kubanka 1516 did not prove resistant. The behavior
of einkorn has already been discussed under adaptation of biologic
forms. Very careful observations were made on the others to deter-
mine as accurately as possible their comparative resistance. The inocu-
lations were made with fresh, viable spores and the plants put under
bell jars 48 hours after inoculation.

The incubation period varies with temperature conditions, both
high and low temperatures lengthening the period very perceptibly.
No experiments with this particular object in view were made, but
numerous observations brought the fact out very clearly. Under the
same conditions on Minnesota No. 163 the incubation period is shorter
than on any of the resistant forms. With an average temperature of
about 65 degrees Fahrenheit and a variation of from 40 to 75 degrees
and a relative humidity of about 55 per cent, pustules appear on Minne-
sota No. 163 in 7 or 8 days. In case of Iumillo the period is usually
about 2 days longer, although considerable variation was found. Em-
mer has an incubation period of about 11 days, Arnautka, 12 days, and
Khapli, 14 days. Arranged in order of their susceptibility these vari-
eties are as follows: .

RUST-RESISTANT VARIETIES OF WHEAT 31

Variety Incubation Period
MRPCIRIES OLA ONG.) LOGiraicieisvateleratareisi= aes iere: afer Wisvorahe araiaielors /cielaiei eicvele's 7 days
Merete POE Aiea ee os siers Taine e aiafates civie sGis ti eyo a a dele o/acjere’a sian 9 days
ETT A) Gel oR ee ee ee reer fo Pc aS a 11 days
PEP ETERS SU Gh aie ici cocci cle aie eetera a CRERTS ots cstesnticrs dal aiadal aie’ ake ele 12 days
Eat Senter eeeee ottawa nye cre trae ie loievaiere) Aiote afeln Gln verse ae vlasereze tele ajcjeva's 14 days

It will thus be seen that the incubation period is longer on the more
resistant varieties. These figures are, of course, not absolute, but vary
with the temperature, and, to some extent, with soil conditions. All
however, vary in nearly, but not absolutely, the same proportion.

The character of infection is distinctly different on the different
varieties. It is quite noticeable that the same phenomena are observed
as appear on various biologic forms. On Minnesota No. 163 the pus-
tules are large, varying from 2 to 6 millimeters in length. They rup-
ture the epiderm very readily and shed spores in great abundance.
Very rarely are smali, unruptured pustules developed. The host tissues
nearly always remain fairly healthy, a yellowing which gradually ap-
pears furnishing the external evidence that the fungous hyphae are in
the plant. On einkorn and Iumillo, which in the greenhouse are only
fairly resistant, the pustules are usually somewhat smaller. There is
a tendency in these two forms toward the development of small dead
areas. These areas are either very distinctly yellowed or sometimes
killed outright. The general appearance is, however, usually not
sharply different from that of infected Minnesota No. 163, except in
degree of infection as evidenced by smaller pustules on einkorn and
Iumillo. On emmer there were often long infected, yellow areas in
which there was a fairly large number of very small pustules, usually
less than one millimeter in length, many of which never ruptured.
Then again fairly large areas of host tissue were practically killed and
only a few small green islands developed. On both Arnautka and
Khapli, areas from one to two centimeters long were killed, the leaf
appearing white and dead (see Plate VI, A). In these areas there was
often a moderately large number of “green islands” with very small,
unruptured pustules in the centers. When the pustules did rupture they
were always very small, seldom, if ever, exceeding one millimeter in
length and often being mere dots. The large areas involved can be
explained rather on the basis of multiple infection than on the basis of
the spreading of the mycelium from a few infections. Histological
examination of diseased areas verifies this supposition.

The spores on the resistant varieties were smaller than on Minne-
sota No. 163. Spores of Minnesota No. 163 averaged 35.38 x 21.39 yu.
while those of emmer were 33.04 x 21.30 uw. The Khapli spores were
smallest, being only 29.69 x 20.68 uw. It was found that spores from
different pustules varied somewhat in average size. Therefore spores

32 A STUDY IN CEREAL RUSTS

from a number of pustules were measured in determining the averages.
The fact that pustules are produced only with difficulty and that the
spores are smaller on resistant varieties would seem to indicate that
the fungus is not vigorous and cannot develop extensively although it
may gain entrance into the leaf tissues.

COMPARATIVE VIRULENCE OF AECIDIAL AND LONG-TIME
UREDOSPORE INOCULATIONS ON RESISTANT FORMS

Various observers have remarked on the reinvigorating power of the
aecidial stage of Puccinia graminis. Plowright (1882, p. 234) was of
the opinion that much more damage was done by aecidial infections
than by infection by uredospores which had been reproduced for a
number of successive generations. Bolley (1889, p. 13) states that the
aecidium, being a sexual product, should be considered as functionally
reinvigorating. He also reasserts this principle in a later work (1909,
p. 182). Arthur (1902, pp. 68 and 69 and 1903, p. 17) observes that
primary uredospores have a greater disturbing effect on the host than
do long-time uredospores. Freeman and Johnson, on the other hand,
cite experiments (1911, p. 33) to show that when the aecidial and
teleutospore stages were excluded for 52 generations the fungus still
retained its power of infection. The fact that sexuality in the rusts has
been definitely established would make it seem reasonable to suppose
that there would be a reinvigorating power. However, Barclay (1892,
pp. 8 and 40) states that in India there are no barberries for “enormous
distances” from fields of wheat in which Puccinia graminis is quite
destructive. McAlpine (1906, p. 58) points out that Puccinia graminis
probably causes no more damage in any country in the world than it
does in Australia where barberries are practically absent and aecidia
have never been found. The rust is quite serious in South Africa, but,
according to Pole Evans (1911), the aecidial stage is absent.

Comparative trials were made with aecidiospores, primary ured-
ospores, and long-time uredospores. The varieties used were not in
all cases the most resistant, since no seed of some of the more resist-
ant forms was available when the aecidia appeared. Trials were made
on Minnesota No. 163, Kubanka 1516, Iumillo 1736, and einkorn 2433.
The two last-mentioned forms are fairly resistant. The long-time ured-
ospores used represented the twenty-fourth generation on wheat. A
number of trials were made and the results were not always uniform.
The incubation period of the fungus when developed from primary
uredospore or from aecidiospore inoculations was slightly longer than
when developed from long-time uredospores. The pustules developed
from long-time uredospores were apparently smaller and more numer-
ous, while those from aecidiospores and primary uredospores averaged

RUST-RESISTANT VARIETIES OF WHEAT 33

somewhat larger, were deeper brown, and seemed to be shedding
spores in greater abundance. On einkorn, in one experiment, the re-
sults were directly opposed to this. The differences were not especially
striking, the aecidial infections being perhaps slightly more virulent.
There is considerable evidence that the virulence of the rust attack
when carried by aecidiospores or primary uredospores is exceptionally
virulent. The results of these experiments, however, would not justify
such a conclusion in this particular case.

METABOLISM OF THE HOST AND RUST RESISTANCE

There seems to be no question but that weather and soil condi-
tions, determining the metabolism of the host plants, exert an influence
on the prevalence of rust in the field. Little (1883, p. 634) states that
weather is the determining factor and adds that high manuring, espe-
cially with nitrogenous manures, predisposes wheat plants to rust.
Bolley (1889), Anderson (1890), and many others since have held that
this is the case. Bolley suggests as a possible cause the increased
evaporation and consequent raising of the relative humidity. Jones
(1905) shows that Phytophthora rot of potatoes tends to be more seri-
ous after a heavy application of nitrogenous fertilizers to the land.
Miss Gibson (1904) concluded as the result of experiments with the
chrysanthemum rust that in an almost immune form normal develop-
ment of rust does not depend on the state of health of the plant, but
that a luxuriant state of growth favors the development of the fungus.
Hennings (1903, pp. 41-45), on the other hand, states that in observ-
ing plants infected with perennial smuts and with rusts he found that
the disease disappeared when the plants were placed under the most
favorable cultural conditions. This is not in accord with Arthur’s gen-
eralization (1903, p. 13) that ‘“‘so intimate is the association of host
and parasite that as a rule the vigor of the parasite is directly propor-
tional to the vigor of the host.” Apparent discrepancies may, how-
ever, be explained by the fact that different plants and different para-
sites react quite differently. As far as the rusts of cereals are con-
cerned, Arthur’s generalization would seem to be correct. Biffen
(1912, pp. 421-429) shows that Puccima glumarum is most virulent
when a complete fertilizer is used and that the virulence decreases
with the decrease in amount of fertilizer.

Less work has been done to determine the exact manner in which
these causes operate. De Bary (1887, p. 359) says, “The physiological
reason for these predispositions cannot 1n most cases be exactly stated ;
but it may be said in general terms to lie in the material composition of
the host, and therefore to be indirectly dependent on the nature of its
food.” Marshall Ward (1902, p. 145), in experiments with Puccinia
dispersa on bromes, tried the effect of mineral starvation and concluded

34 A STUDY IN CEREAL RUSTS

that “lack of minerals in no way secured immunity from infection,
although seedlings deficient in phosphorus or nitrogen tended to show
retardation of infection.”’ The well-nourished plants produced more
spores than the underfed ones. This seems to be due not so much to
the presence or absence of any particular chemicals and a direct effect
on the fungus but rather to the effect on the host. However, attempts
have been made to prevent diseases, among them rusts of cereals, by
adding various substances to the soil. Anderson (1890, p. 84) recom-
mends the use of salt, iron sulfate, and lime as tending to decrease the
amount of rust. Galioway (1893, p. 208) tried the effect of flowers of
sulfur, potassium sulfid, ammonium carbonate, potassium bichromate,
calcium hydroxid, and iron sulfate when applied to the soil, but found
them of no particular value in preventing rust. Laurent (1902, pp.
1040-1042) concludes that potatoes can be immunized against Phytoph-
thora infestans by treating the soil with copper sulfate. Marchal (1902,
pp. 1067 and 1068) tried the effect of copper sulfate and iron sul-
fate of various strengths when added to Sach’s solution, on the se-
verity of attack of Bremia lactucae on lettuce. He found that by add-
ing 4 or 5 parts of copper sulfate to 10,000 parts of Sach’s solution
he was able to render the plants practically resistant to the fungus,
yet leave the vegetation normal. In experiments attempting to immun-
ize cereals to rusts he was unsuccessful. Massee (1903, p. 142) pre-
vented the development of fungi on tomatoes and cucumbers by water-
ing them with copper sulfate solution. This did not give the desired
results with Oidium on barley. He states that not all plants can be
ireated in this way without endangering their health. Chemical analy-
sis of treated and untreated tomatoes showed that there was no more
copper sulfate in treated and therefore immune plants than there was
in those which had received no treatment. Massee suggests therefore
that the copper sulfate reacts with certain substances in the soil, thus
indirectly conferring immunity. Freeman and Johnson (1911, pp. 69-
70) call attention to the complexity of the problem and the need for
differentiating results.

EFFECT OF WATER CONTENT OF SOIL

Statements to the effect that low-lying, wet soils predispose cereals
to rust are frequently made in the literature of the subject. Reference
has already been made to some of these. It was observed very fre-
quently in experiments mentioned in this paper that when the relative
humidity was high infection was not only surer to result but that it
was also more severe. It was therefore thought worth while to de-
termine whether a high water content of the soil would act as a pre-
disposing factor.

The varieties used were: Ejinkorn 2433, Kubanka 1516, Iumillo

RUST-RESISTANT VARIETIES OF WHEAT 35

1736, and Minnesota No. 163. There was no difference in the amount
of water until the plants germinated. Immediately after germinaticn,
however, the soil in one series was kept very wet while that in another
series was kept as dry as was possible without endangering the life of
the plants. The soil in the wet series had a water content of 31.35
per cent, while that of the dry series was 6.16 per cent at the conclusion
of the experiment. Repeated trials were made with substantially the
same resulis.

The number of einkorn and [umillo leaves which became infected
in the wet series was smaller than in the dry series. The percentages
for the others were practically the same. In virulence of infection,
however, there was considerable difference. The varieties also reacted
somewhat differently so each will be considered separately.

On Kubanka the virulence of infection, especially in the early
stages, is very markedly inferior on the plants in the wet series. The
pustules during the early stages are often small and on some plants
do not appear at all, the leaf merely becoming yellow. Later the plants
in dry soil were often completely covered with large, vigorous pus-
tules while those in wet soil, although producing a moderately large
number of pustules, were not nearly so badly affected. In both series
there were many. secondary infections along the leaf. There was a
distinct tendency in the wet series toward leaf-yellowing. It was at
first thought that the mycelium might be spreading through the tissues.
Histological examination, however, failed to confirm this supposition.
Apparently it was only a slight chlorotic condition due to excessive
water content. The infection was unquestionably more severe on plants
grown in dry soil (see Plate IV).

On einkorn the differences were not so sharp, although there ap-
peared to be a slightly more severe infection on plants in the dry series
than on those in the wet series. The rust appeared at about the same
time, the virulence of infection being at first quite distinctly greater
on the dry-soil plants. Later this difference was not quite so marked,
although still apparent.

The sharpest difference was on Iumillo. Only a few leaves in the
wet series were really badly infected, while those of the dry series
showed a surprisingly virulent attack. There is no question but that
the infections secured on plants in these dry series were more severe
than were those on any other Iumillo plants inoculated during the
various trials with this variety. It is not often that a really vigorous
development of the fungus occurs on Iumillo, but when the water con-
tent of the soil is very low, the infection at times shows surprising vir-
ulence (see Plate V).

The results on Minnesota No. 163 varied more than those of the
other forms. The pustules in nearly every case were large and vigor-

36 A STUDY, IN -CEREGL RUST S

ous with but little difference between the two series. There seemed to
be a tendency for the mycelium to spread more in the plants grown in
wet soil, but the pustules were not larger than those on dry-soil plants.
Whatever difference there was appeared as a slightly greater virulence
on the wet-soil plants.

It will be noticed that Iumillo and Kubanka, drought-resisting
plants, were more severely attacked when grown in dry soil. Minne-
sota No. 163, on the other hand, a mesophyte, did not show so much
difference. It is probable then, that, conditions having been favorable
for a rust infection, the water relation in the soil which is most favor-
able for the host plant’s development is also the most favorable for the
development of the rust. It seems probable also that in at least some
forms it is not the water content of the soil which predisposes grains
erowing in low places to rust but rather the increased relative humidity
which enables the rust spores to germinate and infect the plants. The
temperature in such places also probably exerts an influence. This is
pointed out by Freeman and Johnson (1911, p. 65) in connection with
the rust epidemic of 1904.

EFFECT OF FERTILIZERS

In the first series the varieties used were: Minnesota No. 163,
Arnautka 288, Khapli, emmer 1522, Iumillo 1736, einkorn 2433, and
Kubanka 2094. Some were planted in ordinary rich loam, others in
rich loam plus fresh barnyard manure, and a third series in rich loam
io which barnyard manure and bone meal had been added. Especial
care was taken to keep all plants under the same conditions of tempera-
ture, moisture, and light both before and after inoculation.

On the wheat plants which were grown on very heavily fertilized
soil the infection was clearly more severe. The infected areas were
very large as were also the individual pustules. The most severely
attacked plants were in one pot which had been fertilized with manure
and bone meal. Aside from this one pot, however, there was but little
variation among the fertilized pots. The infection on the check plants
remained inferior, although it was very vigorous.

Similar results were obtained from einkorn and Iumillo plants. In
the’ case of these two forms the plants in the manure-and-bone series
developed the worst rust attack, the manure was next, and the plants
in ordinary soil were more lightly attacked. It should be remarked that
the differences were not strikingly sharp, although they were quite
apparent. Emmer and Arnautka gave no distinct results. There was
a great deal of variation in the individual pots and no one series stood
out clearly from the other two.

The Kubanka and Khapli plants showed some differences. The
character of infection is very different from that of the other forms.

RUST-RESISTANT VARIETIES OF WHEAT 37

In the case of the former the plants grown in pots to which barnyard
mauure had been added were most severely attacked, while there was
little difference between those grown in loam and those manured with
both barnyard manure and bone meal. Khapli plants grown in soil
fertilized with both manure and bone were fairly successfully infected.
Areas of the leaf, one centimeter in length, were sometimes infected,
and pustules as big as a pin head were developed. There was little dif-
ference between the other pots, the infection being somewhat milder
than in the heavily fertilized ones

It is quite probable that there was available in the rich loam very
nearly all of the plant food the plants were capable of utilizing. This
would account for the fact that the differences were not always greater.
On the whole it might be concluded that very heavy fertilization is
somewhat conducive to increased severity of attack on very resistant
varieties as well as on susceptible forms.

Since the check plants in the trials just described were grown un-
der such favorable conditions, it was determined to grow the checks in
poorer soil in the next series. Therefore they were planted in mod-
erately fine sand (S) (see table below) to which but a very slight
amount of leaf-mold had been added. Nitrogen (N) was added to
another series in the form of calcium nitrate, to a third was added cal-
cium phosphate (P), and the fourth received both calcium phosphate
and calcium nitrate (P and N). The salts were applied in water. The
plants were watered three times with distilled water containing the
proper salt or salts, at the rate of 3 grams per 500 cc. The same varic-
ties were used as were used in the preceding series.

Observations on results gave the following order of virulence, the
first being most virulent and the others arranged on the same basis.
Two observers took notes with the following results:

EFFECT OF FERTILIZERS ON VIRULENCE OF Rust ATTACK

Order of Virulence

Grain

1 ava 3 4
Einkorn N | PandN 12 S
Emmer Pand N heal] P S
Kubanka PandN ea | P S
Khapli PandN ) aN P ees)
Arnautka N S ae | PandN
Tumillo N pile S PandN

The somewhat conflicting results suggested the desirability of aa-
other trial. Four series were arranged as follows: Pure sand (S),
ordinary field soil (O), sand plus nitrogen (N), and sand plus phos-
phorus (P). The nitrogen and phosphorus were added as in the pre-
ceding experiment. Three persons working independently made ob-

38 A STUDY IN CEREAL RUSTS

servations, but there was no great difference of opinion in any case.
The order in which different observers placed them was sometimes dif-
ferent, showing that there was sometimes little choice among the
various pots. The results are given in the following table:

EFFECT OF FERTILIZERS ON VIRULENCE OF Rust ATTACK

Order of Virulence
Grain i , 3 mae Remarks
Emmer N 18 S O
Kubanka S Pp N O
Tumillo N O Ie S
Khapli S N 1B) O
Arnautka 12 Ss N O
Wheat S N 12 O
Einkorn N P S O
Einkorn 13 N S O ‘Little difference between N and P
Emmer N S 12 O Little difference between P and S
Kubanka S 12 N O Distinct
Tumillo N lu O S Little difference between P and N
Khapli Ss N 12 O Little difference between P and N
Arnautka 12 N S O Little difference between N and S
Wheat N S iP O Little difference between S and P

In this experiment the two most resistant forms available for study
were watched very carefully. Both Kubanka 2094 and Khapli proved
to be very resistant even when very highly fertilized. It is a rather
striking fact that in both cases plants grown in sand showed a slightly
more virulent infection. The differences were not great. In fact, it
was often hard to decide which plants were most severely affected. It
was observed that plants which had been under the most favorable con-
ditions for infection were most severely attacked regardless of the fer-
tilizer used. The difference in conditions was due to the fact that there
were not enough bell jars to cover all the plants, so sorne were placed
in tubs containing water on the bottom. They were then covered with
pieces of glass. The films of moisture were not so persistent here as
under the jars, and the difference in the amount of rust was quite
marked. This was taken into account in determining results and mak-
ing comparisons.

If these plants had been allowed to grow longer it is quite prob-
able that those fertilized with nitrogen would have become more se-
verely rusted while those fertilized with phosphorus would have been
slightly less severely affected. This would seem to be due not to the
specific action of the chemicals on the rust fungus but rather to their
effect on the general condition of the plant, and, in the field, on the

RUST-RESISTANT VARIETIES OF WHEAT 39

immediate atmospheric conditions. The direct effect of chemicals in
the soil on the amount of rust on resistant varieties is not great, only a
slight quantitative difference being apparent.

In order to determine by more accurate methods whether or not
there was a direct effect of substances in the soil on the amount of
rust (1) fungicides were put into nutrient media, and (2) certain
nutrient salts were used in amounts varying from deficiency to excess.

Sach’s modified culture solution was used and to this one per cent
of agar was added. The series was arranged as follows, the amounts
of nitrate and phosphate being the variables:

I. Potassium nitrate; 2 grams per 1,000 cc.
II. Calcium phosphate; 3 grams per 1,000 cc.

III. Potassium nitrate; .05 grams per 1,000 cc.

IV. Calcium phosphate; .075 grams per 1,000 cc.

Minnesota No. 163 wheat was used. After inoculation plants were
all placed under bell jars and kept under uniform conditions. Arranged
according to virulence, the most severely affected being placed first,
they would be arranged as follows: III, II, IV, I.

This was tried a second time with exactly the same results. The
plants appeared about the same, all growing fairly well in the agar.
All were well infected, producing a fairly large number of large,
healthy pustules. They were kept three weeks after inoculation and
by this time there was not much difference between II, IV, and I, but
III was still much more virulently attacked. It seems, therefore, that
an excess of nitrogen does not necessarily, in itself, cause an increase in
the amount of rust and an excess of phosphorus does not affect it very
appreciably.

The effect of excluding nitrogen and phosphorus was next tried.
Sach’s modified medium plus one per-cent of agar was again used and
in I no potassium nitrate was added while calcium phosphate was ex-
cluded from II. The plants in I were lighter colored from the first
than either those in II or the checks. They were inoculated six days
after planting. A good, vigorous infection resulted, the plants in I
being slightly more severely attacked than those in II. The leaves of
I began to turn yellow after three weeks, and the rust did not spread
farther. The check plants were more severely attacked than those in
either I or IJ. Here again, however, the differences were not very
great. There was a slight quantitative difference but qualitatively there
was scarcely any difference. This is in keeping with Ward’s conclu-
sions reached after his work on mineral starvation, reference to which

has already been made.
An attempt was then made to determine whether it was possible

to confer immunity by means of various salts. Copper sulfate, copper
carbonate and iron sulfate were used in strengths varying from 1 to

40 A STUDY IN CEREAL RUSES

10,000 to 1 to 2,000. They were added to Sach’s medium in these pro-
portions. Minnesota No. 163 was used for all the trials. Copper sul-
fate could not be added in larger amounts than 1 to 5,000, since it
stunted the plants when more was added. Copper carbonate could not
well be used in greater concentration than 1 to 2,000. Iron sulfate
did not dwarf the plants when used at the rate of 1 to 2,000. None of
the solutions used diminished the amount of rust very appreciably when
used in such concentration as to permit of normal development of the
host plant. There was a slightly smaller amount of rust on plants
grown in the medium to which copper sulfate had been added in
amounts of 1 to 4,000 and 1 to 2,000. However, a very good infection
was secured on all of them, even on those which never grew to a greater
height than one inch. Neither was there any less mildew on any of
the plants. None of these substances, apparently, can immunize wheat
against rust, even when used in such concentration as to dwarf the
plants to one-sixth their normal size.

These experiments show that in the case of Puccimia graminis,
since it is a very highly specialized, obligate parasite, there is a very
intimate relationship between host and parasite, and whatever is con-
ducive to the health of host is ordinarily conducive to the vigorous de-
velopment of the parasite also. This applies not only to susceptible
forms but also to forms uncongenial to a biologic form and to resistant
varieties.

THE NATURE OF RESISTANCE

The work of Cobb, Eriksson, Ward, and others on the nature of
resistance has already been mentioned. The theory which Ward de-
duced from his extensive work on parasitism was that there are en-
zymes or toxins and antitoxins produced by host or parasite or both.
His work on “A Lily Disease” (1888) showed that in all probability
Botrytis secretes an enzyme which enables it to live more successfully
on the host. Pfeffer had already given the name chemotaxis to the at-
traction certain substances seemed to have for certain growing plant
parts. Miyoshi (1894, p. 21) claimed to have been able to observe a
very definite chemotropism when a Tradescantia leaf was injected
with a wheat-leaf decoction and then inoculated with Uredo linearis
(Puccimia graminis). The same author decided (1895) that a large
number of fungi responded to chemical attraction. Massee (1904,
p. 7) attached a great deal of significance to chemotaxis. He asserted
that infection depended on the presence in the plant cell of positive
chemotactic substances and further that “in the future we shall be justi-
fied in defining an immune plant as an individual in which the positive
chemotactic substance, necessary for facilitating the entrance of the
germ-tubes of a given parasitic fungus into its tissues, is absent.”’ On

RUST-RESISTANT VARIETIES OF WHEAT 41

the other hand Errera (1892, p. 373) contends that so-called chemotro-
pism is in many cases merely positive or negative hydrotropism. Ful-
ton (1906, pp. 81-107) says that there is no definite chemotropic re-
sponse on the part of fungi. Nutrient solutions cause marked growth,
in his opinion, but cause no definite turning of hyphae in their direc-
tion. Hydrotropism, however, was observable.

The behavior of the germ tube of Puccinia graminis and its en-
trance into the host plant has been described and figured by various
authors. Ward (1881-1, p. 217) figures it as forming a slight swell-
ing and then growing directly into the tissues of the host. Bolley
(1889, p. 14) shows the germ tube growing directly through the stom-
atal opening and branching out between the mesophyll cells. This has
never been seen by the writer. An appressorium always formed in all
cases of infection observed. Pole Evans (1907, p. 445) describes and
figures normal infection quite completely.

A considerable amount of work has also been done on determining
the fate of germ tubes when they are produced on immune host plants.
Klebahn noted (1896, p. 263) that sporidia of Puccinia convallariae-
digraphidis could bore through the epidermal walls of Polygonatum
multifiorwm, an uncongenial host, but that the germ tubes developed no
further. He concludes that infection is of the nature of a conflict be-
tween host and parasite. Ward (1901 and 1902) showed in connection
with Puccinia dispersa on bromes that the germ tube might enter and
cause normal infection, the mycelium might develop and never produce
pustules, or the tissues of the host plant might be killed very early,
thus precluding the possibility of much development on the part of
the fungus. The same author further shows (1904, p. 29) that in
normal infection of a susceptible species of Bromus the host cells
retain life for a surprisingly long time. Miss Gibson (1904) examined
the leaves of a large number of plants, widely separated taxonomically,
which had been inoculated with spores of Uredo chrysanthemi. She
- found that the germ tubes might enter the plant tissues very readily
but never formed any haustoria and consequently no pustules.
Furthermore the hyphae usually Gied when they came in contact with
a cell. On resistant varieties of Chrysanthemum ii was found that
haustoria might develop, but areas of host tissue in the neighborhood
of the hyphae were killed, thus preventing the further spread of the
fungous mycelium. Her conclusion is to the effect that when the
germ tube of a uredine fungus enters any but its proper host plant a
struggle goes on, resulting in the death of the host locally and of thc
parasite. The closer the relationship between the plant and the proper
host of the rust the longer and more extensive will be the struggle.
Salmon (1905) found that when barley was inoculated with spores
of Erysiphe graminis from wheat incipient haustoria might be formed

42 A STUDY IN CEREAL RUSTS

in the cells, but that they became disorganized within a very few days.
He attributed this to defective symbiotic relations between host and
parasite. Miss Marryat (1907) showed that Puccinia glumarum when
grown on a semi-immune host plant killed local areas of the host, sent
out but few haustoria, and never developed any but small or abortive
pustules.

It is a matter of common observation that in dealing either with
cereals uncongenial to a given biologic form of Puccinia graminis or
with varieties of wheat resistant to Puccinia graminis tritici flecks are
often visible after inoculation, but no pustules, or only small ones, are
produced. Examples of this are shown in Plate I, A and Plate VI, A.
All degrees of this killing can be observed. The more readily the rust
infects a plant the less likely are these dead areas to appear. When
Puccinia graminis tritici is put on Minnesota No. 163 wheat, pustules
are formed in great abundance, but the leaf tissues remain alive for a
long time. When, on the other hand, resistant forms such as. Kubanka
2094 or Khapli are inoculated, areas of the leaf are killed outright; and
if pustules are formed at all they are very small. In extreme cases of
incompatibility such as is found between Puccinia graminis avenae
and wheat, the leaf area involved is usually so small that no indications
of it can be seen with the unaided eye.

HISTOLOGICAL DETAILS OF INFECTION

In order to determine the behavior of germ tubes in susceptibie
and nearly immune forms, leaves of Minnesota No. 163 and of Khapli
were inoculated with Puccinia graminis tritici. Minnesota No. 163 was
also inoculated with Puccinia graminis avenae. They were then placed -
in a pan of water under bell jars for 48 hours. Leaves were selected
and killed every 24 hours, beginning with the first day.

For killing, aceto-alcohol, medium chromo-acetic acid, and Flem-
ming’s weaker killing-fluid were used. The leaves were embedded
in the usual manner and sectioned from five to ten microns thick.
For staining, Haidenhain’s iron-alum haematoxylin and orange G,
Erlich’s haematoxylin, Gram’s stain and eosin, Delafield’s haematoxylin
used according to Durand’s method, fuchsin and methyl green and the
safranin, gentian violet, orange G. combination, used according to
Uarper’s modification of Flemming’s method, were used. The last
named gave the best results.

INFECTION OF MINNESOTA NO. 163

At the end of 24 hours many of the spores have usually germ-
inated, sending out long germ tubes, although some apparently
germinate later. Two tubes may be sent out from the same spore,
but usually one develops more vigorously than the other. The tube
usually follows the epiderm closely; swellings are often found above

RUST-RESISTANT VARIETIES OF WHEAT 43

the wall between two epidermal cells (Plate VII, 3). In these swell-
ings, which appear very much like very young appressoria, the pro-
toplasm often aggregates more densely than in the other parts of the
tube. The germ tube may often follow the epiderm for considerable
distances, sometimes for the length of 15 epidermal cells, before a
definite appressorium is formed. Nearly always when a stoma is
reached the tube forms a very definite swelling (Plate VII, 1) which
constitutes an appressorium. The protoplasmic contents of practically
the entire germ tube are concentrated in this appressorium. It dips
down into the stomatal opening and a fine protoplasmic process is sent
through to the substomatal space. Here a definite swelling takes
place, forming the substomatal vesicle (Plate VII, 4 and 5). Appar-
ently, in some cases, the vesicle develops no further. In the great
majority of cdses, however, the protoplasm aggregates in it and
infection-threads are sent out. Very often these infection-threads
follow closely along under the epiderm cells and send small knoblike
or sometimes flattened haustoria into the host cells. Branching then
takes place among the cells of the leaf, many haustoria being sent out
and the hyphae developing very rapidly. The threads may grow
directly across the substomatal space and branch between the mesophyll
cells (Plate VII, 6). This does not seem to be the usual method,
however. Nuclear division takes place rapidly to keep pace with the
growth of the hyphae. Frequently a number of nuclei are found in a
single cell. The entire hypha often retains its protoplasm for a con-
siderable length of time. Ordinarily the tip only remains densely
protoplasmic while the rest of the hypha becomes much vacuolated.
Very long hyphae are often found growing very vigorously but appar-
ently not sending out haustoria. They seem to be in the nature of
distributive filaments. The fungus does not seem to spread very far
from the point of infection. When large areas of the leaf are involved,
a number of points of entry can nearly always be found. On the
fourth day an infected area is usually well filled with much branched
hyphae. About this time wefts begin to be formed, the protoplasm
ageregating in the tips of the hyphae. A dense mass of filaments is
formed just beneath the epiderm and the epidermal cells are wedged
apart. The large, upright fungus cells, which show the binucleate
condition very clearly, begin to form uredospores. This is well under
way on the fifth day and by the sixth or seventh day the epiderm has
been completely ruptured while spores are being shed in great pro-
fusion. It is interesting to note that the host cells, which are often
half filled with large knoblike, filamentous or coiled haustoria, are
usually still quite healthy at the time pustule formation begins.

The whole appearance of both fungus and host during the first
few days after infection indicates a fairly perfect relation between

44 A STUDY IN CEREAL RUSTS

the two. The fungus flourishes vigorously while for a considerable
length of time the host cells, even in the infected area, are apparently
quite healthy. In no case does there seem to be an extensive killing of
host tissue.

INFECTION OF KHAPLI

Spore germination of course occurs normally. The germ tubes
grow along the surface of the host epiderm cells in the same manner
as do those on Minnesota No. 163. The formation of appressoria
takes place in an entirely normal manner. The stimulus causing entry
into the stomatal slit is present, the method of entrance being sub-
stantially the same as in Minnesota No. 163. Apparently the vesicle
sometimes fails to send out infection threads but merely remains
directly beneath the stomatal slit and becomes vacuolated. It may send
out numerous, short, club-shaped branches all of which soon become
vacuolated and never send out any haustoria into the host cells. From
the beginning of growth in the host it is easily discernible that the
vigor of the hyphae is not nearly so great as is the case with those
crowing in Minnesota No. 163. There is a greater tendency for the
tips of infection threads to round up, become vacuolated, and never
develop further.

Fairly successful infection, however, may take place. Infection
threads may be sent out just under the epiderm or directly across the
substomatal space (see Plate VIII, 3 and 5). MHaustoria, attached to
the hypha by delicate sterigmata, are sent into the cells and the hypha
grows fairly well. Sometimes many incipient infection threads are
formed from a single vesicle, only one developing (Plate VIII, 5).
Shortly after infection threads are sent out the vesicle usually dies.

The infection threads are not always successful in sending
haustoria into the host cells. When the hypha comes into contact
with the cells the protoplast of the latter often shrinks back from the
wall, the nucleus shows definite signs of disintegration, the chloroplasts
are apparently lost, and the entire cell dies. The hypha may die also,
or it may grow and kill other cells. However, it usually eventually
succumbs. Typical examples of this will be seen on Plate VIII, 6 and
7. It will be noticed that at 6 the cell on which the hypha is abutting
is apparently dead, the chloroplasts have disappeared, and the nucleus
is disintegrating. At 7 this has taken place in only a part of the cell.
It sometimes happens that one branch of a hypha is fairly successful
while another may never develop to any extent at all.

Whether or not the host cells are killed within a short time after
the hyphae come in contact with them, infection does not appear to
be normal. The hyphae may grow fairly well, but never as vigorously
as in Minnesota No. 163. Haustoria may be sent out in fair abundance

RUST-RESISTANT. VARIETIES OF WHEAT 45

(Plate IX, 1, 2, and 4). The hyphal tips may branch very profusely
but they become vaculoated very early in most cases and appear un-
thrifty. It requires 8 or 9 days for the stage of infection to be
reached on Khapli that is reached in 3 or 4 days in susceptible varieties.
Short, thick, or rounded hyphal segments are quite common, those
at the end of a branch often containing as many as 5 or 6 nuclei, some
of which appear to be distintegrating.

The tips of hyphae naturally die when a group of host cells among
which they are growing is killed. However, they may disintegrate
without having first killed host cells (Plate [X, 1, 2, 3, and 4). There
may be many variations. The hyphae may not send out haustoria and
die in consequence, or, even if they. do send them into the cells, death
may occur. Branches of hyphae which have sent haustoria into host
cells frequently become vacuolated and gradually die, or the pro-
toplasmic contents may change to granular, deep-staining masses.
The whole appearance suggests fungous hyphae growing in an un-
favorable nutrient solution.

In about 11 or 12 days a distinct tendency toward the formation
of hyphal wefts can be observed. These vary greatly both in size
and position. They may be mere aggregations directly under the
epiderm or deeper down in the tissues; or they may begin to wedge
the epiderm cells apart after the manner of young pustules. Often
small, unruptured pustules are formed in which there is a fairly large
number of abortive spores. The pustules may rupture the epiderm
but they are always extremely small, and as a rule the spores are
small. The average size of a large number of spores measured was
29.69 x 20.68 4, whereas the average size of the wheat rust spores
which were used in making the inoculations was 35.38 x 21.39 pm.

After about 20 days practically all the host cells and a large
number of the hyphae in an infected region are dead. Haustoria may
be present in the host cells in fairly large numbers, but most of them
are dead, their protoplasmic contents having broken up into granular,
deep-staining masses. The nuclei of the host cells are often dis-
integrating also. The infection by. this time, and usually earlier, has
completely run its course. Comparatively very few spores have been
produced, and, under natural conditions, secondary infections would
probably not occur to any extent.

THE COURSE OF INFECTION IN OTHER RESISTANT FORMS

Substantially the same sequence of events occurs in other resistant
forms, such as Arnautka 288, Kubanka 2094, emmer 1522, einkorn 2433
(sometimes), and in such cases as the infection of rye by barley rust.
The differences seem to be in degree rather than in kind. In the cases
of emmer and einkorn the killing of host cells is rarely found but

46 A SEUDY IN CEREAL ROSES

unsuccessful attempts to form pustules are often noticed. The
sequence of events in Arnautka 288 and Kubanka 2094 is quite similar
to that in Khapli. These are semi-immune forms in which the contest
between host and parasite is somewhat prolonged. Fatalities occur on
both sides but not in sufficient number to render infection absolutely
unsuccessful. It is, therefore, essential to note the sequence in such
extreme cases of almost total immunity as are furnished by wheat
when inoculated with Puccinia graminis avenae.

MINNESOTA NO. 163 INOCULATED WITH PUCCINIA GRAMINIS AVENAE

It will be recalled that very rarely indeed does successful infection
follow inoculation of wheat with Puccinia graminis avenae. Long
germ tubes are sent out by spores germinating on the surface of the
leaf. These follow the epiderm, dipping into depressions in an entirely
normal manner. Appressoria are found, a small neck grows through
the stomatal slit and the substomatal vesicle is formed. This vesicle
sometimes sends out only very small knoblike branches which soon
die, or branches may be sent out and very few hausteria produced.

The vesicle often sends out many short, knoblike branches,
appearing almost like elongated amoebae. These do not appear
vigorous after a few days. The epiderm cells in the immediate vicinity
appeared to be killed. If definite infection threads are sent out they
never grow very long but kill two or three host cells and then stop
growing.

The difference between conditions in such a case as this and
such a one as Khapli is apparently in degree only. Whereas in Khapli
the fungus might develop to a certain extent, thus involving fairly
large leaf areas, in such an extreme case of immunity as is presented
by wheat to oat rust only a few host cells are involved and the contest
between host and parasite is short and decisive, only a very few host
cells being killed; and the hyphae seldom develop sufficiently to give
any external evidence that the germ tube has even entered.

As to the fundamental causes for these facts, only speculation is
possible. It would seem reasonable to suppose either that there was
a lack of an attracting substance or the presence of a deleterious sub-
stance. Massee’s chemotaxis theory has already been mentioned.
Chemotaxis or its absence would hardly explain the phenomena men-
tioned, since the fungus succeeds in effecting an entrance into even the
most immune forms. The evidence would rather seem to favor the
view that the whole problem is one of toxins in host or parasite or,
very probably, in both. In some cases the host is apparently hyper-
susceptible, while further study may prove that there is in other cases
a real resistance. Careful biochemical investigation alone can settle
this question definitely.

RUST-RESISTANT VARIETIES OF WHEAT 47

Certainly, however, there can be no question of a certain antagon-
ism between host and parasite when one observes phenomena such as
are illustrated in Plate VIII, 6. This is in remarkably sharp contrast
with the apparent congeniality exhibited in such cases as are shown
at 6, 7, and 8 in Plate VII. Antagonism would seem to be explicable
at present only by the toxin or enzyme theory. The recent work of
Bolley (1908 and 1909), Pole Evans (1909 and 1911), McAlpine
(1910), Freeman and Johnson (1911), and Biffen (1912) indicates
clearly that immunity and resistance are concepts which, from the
very nature of their variability and sometimes apparent capriciousness,
must be cautiously discussed. At least one substance, commonly
found in plants, has been found by Cook and Taubenhaus (1911, pp.
40 and 43) to be toxic to certain rusts of the genus Uromyces.

Whatever the immediate instruments governing congeniality or
antagonism, the fundamental facts brought out quite clearly in the
results described in the present investigation have a bearing on the
practical and theoretical questions involved in the problem of pre-
venting cereal rusts and the breeding of rust-resisting varieties. Ex-
ternal morphology as pointed out by Ward (1902-1) for brome rusts,
Salmon (1905-2) for mildews, and Biffen (1907) for yellow rust is
also of very slight importance in the immunity of cereal varieties to
stem rust. No observed facts in intimate histology, moreover, give
any clue to resistance. In the absence of biochemical information
concerning the activities of invading hyphae and invaded host tissues,
actual performance alone can be depended upon as a safe criterion
in the development of resistant forms or immune varieties. This is
all the more true since Pole Evans (1911) has found that a hybrid
wheat produced by crossing rust-immune and rust-susceptible wheats
may rust quite badly and be capable of causing infection of the immune
parent and a more severe attack of rust on the susceptible parent
variety than rust from that variety itself will cause. The production
of flecks and dead areas on an inoculated plant is a character of
possible use in indicating at least a semi-immunity.

There does not seem to be any obvious constant correlation be-
tween immunity and other observable characters, as for instance
drought-resistance. Although the immune varieties of wheat used
in this investigation are drought-resistant, it is also a well-estab-
lished fact that other drought-resistant wheats such as Kubanka 1516
are very susceptible. Moreover, the antagonism exhibited by Minne-
sota No. 163 wheat toward Puccinia graminis avenae and by rye
toward Puccinia graminis hordei does not differ fundamentally from
that exhibited by Khapli toward Puccinia graminis tritici and in the
first two cases a correlation with drought-resistance is out of the
question.

48 A STUDY IN CEREAL RUST S

That the important scientific questions involved in the specializa-
tion of biologic forms and that of rust-resistant varieties of wheat
are essentially the same seems obvious. The same phenomena can be
observed in both; there are various degrees of resistance and suscep-
tibility in both and a thorough investigation with refined biochemical
methods will probably not only prove the similarity, but show the real
reason for resistance and susceptibility.

SUMMARY OF PART II

1. In making inoculations in the greenhouse on wheats resistant
to Puccinia graminis tritici it was found that only two, Khapli and
Kubanka 2094, especially the former, possessed a very marked degree
of real resistance, although a number of others were fairly resistant
in the field.

2. It was observed that the more resistant a form proved, the
more pronounced was the tendency of the rust to kill small areas of
the leaf. The pustules developed in these areas were always very
small.

3. The length of the incubation period of the rust is correlated
to a certain extent with the degree of immunity, the most nearly
immune forms, as a rule, having the longest incubation period.

4. On the most resistant varieties, such as Khapli, the spores
are often small in size and sometimes abortive.

5. Infection secured on partially resistant varieties as a result
of inoculations with aecidiospores and primary uredospores proved
only slightiy more virulent than did that secured by means of inocula-
tion with long-time uredospores.

6. Drought-resistant durum wheats grown in very dry soil
rusted more severely than those grown in soil with a higher moisture
content. Minnesota No. 163 did not show much difference, the plants
in wet soil being slightly more severely attacked. The conditions
normal for the host plant are probably also the conditions under
which the rust develops best.

7. It was found that in general the absence or presence, in
excessive amounts, of various nutrient substances, such as nitrogen
and phosphorus salts, did not directly affect the immunity or suscepti-
bility of wheats. Conditions favoring a normal development of the
host were conducive to vigorous development of the rust. The action
of fertilizers, either natural or artificial, is probably indirect. Tem-
perature conditions and: relative humidity of the atmosphere are prob-
ably more important than soil conditions.

8. The addition of copper sulfate, copper carbonate, and iron
sulfate to nutrient media in which plants inoculated with rust were
grown did not markedly diminish the amount of rust when they were

RUST-RESISTANT VARIETIES OF WHEAT 49

used in such concentration as to permit of the normal development
of the host plants.

9. A careful comparison of the sequence of infection in such
a susceptible form as Minnesota No. 163 with that in such an immune
form as Khapli reveals the fact that the fungus gains entrance in
the same manner in both cases. The rust mycelium is able to grow
luxuriantly in Minnesota No. 163 and produce spores in great abun-
dance. In Khapli, however, it does not thrive. The reason seems to
be a physiological incompatibility as evidenced by the killing of host
cells by the fungus and the more or less sudden death of the fungus
itself. Infection may occur and pustules may be developed, but it is
evident that the fungus is not in a congenial environment. The
conditions seem to be essentially similar when examination is made of
a cereal almost completely immune to a biologic form, such as Min-
nesota No. 163, inoculated with Puccinia graminis avenae. Here,
however, the host cells and rust hyphae are killed earlier and the leaf
area involved is consequently smaller. This, however, requires further
study.

10. The question as to the immediate instruments of immunity
can probably only be answered by means of biochemical investigations.
In the meantime, morphological and histological characters being
clearly of minor importance in determining immunity, only the per-
formance of a supposedly resistant variety under varying conditions
can be depended on for a criterion of its value in this respect.

ACKNOWLEDGMENTS

The writer takes pleasure in making acknowledgment to E. C.
Johnson for suggestions and material, and especially to Dr. E. M.
Freeman, under whom the work was done, for many suggestions and
much criticism during the progress of the work.

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54

A STUDY IN CEREAL RUSTS

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EXPLANATION OF PLATES

The drawings were all made with the aid of the camera lucida. The Zeiss
3 mm. N. A. 1.3 homogeneous oil immersion and compensating ocular No. 4

were used for all except Figures 8 and 10, Plate IX, where compensating ocular
No. 6 was substituted.

Plate I A. Rye after inoculation with Puccinia graminis horde1, showing dead
leaf areas and a very few minute pustules.
B. Normal but rather light infection on rye by P. graminis secalis, showing
absence of dead areas.

Plate II A. Einkorn 2433 inoculated with P. graminis originally obtained from
wheat and confined to Einkorn for twenty-five successive generations.
B. Einkorn 2433 inoculated with P. graminis from wheat.

Plate III A. Wheat, Minnesota No. 163, inoculated with rust which had been
confined to Einkorn for twenty-five generations.
B. Normal infection of wheat following inoculation with P. graminis tritici
from wheat.

Plate IV A. Kubanka 1516 grown in very wet soil after inoculation with
P. graminis.
B. Kubanka grown in very dry soil after inoculation.

Plate V A. Iumillo 1736 grown in very wet soil after inoculation with P.
graminis tritici.
B. The same variety inoculated under same condition but grown in very
dry soil.

Plate VI A. Khapli, showing characteristic infection following heavy inoculation
with P. graminis.
B. Normal infection on Minnesota No. 163.

Plate VII. Infection of Minnesota No. 163 wheat by P. graminis tritict.

1, 2 and 3—48 hours after inoculation.

4 and 5—72 hours after inoculation.

6 to 9—5 days after inoculation.

1. Surface view showing appressorium forming over a stoma.

2. Appressorium being formed directly, without germ tube development.

3. Germ tube apparently passing over stoma and forming a swelling—an
unusual occurrence.

4. Part of a germ tube, appressorium, and neck connecting the appressorium
with the substomatal vesicle which has been cut at one side.

5. Substomatal vesicle, cut at one side, beginning to branch.

6. Infection thread growing from vesicle directly across substomatal space;
remains of appressorium outside. (Section slightly torn.)

7. Part of an infection thread showing haustorium in epidermal cell.

8 and 9. Later stages showing development of long hyphae.

Plate VIII. Infection of Khapli with P. graminis tritici.
1 and 2—72 hours after inoculation.
3 and 4—6 days after inoculation.
5, 6 and 7—8 days after inoculation.

55

56

Plate

6

i

Z:
SI

4.

On

ES)

Ay SLUDY VIN CEREAL TOS IS:

Part of a germ tube and an appressorium.

Germ tube, appressorium, and substomatal vesicle into which the nuclei
have passed.

Infection thread, with a few short branches, killing the host cell. The
protoplast has shrunk and the nucleus is disintegrating.

Infection thread in contact with a cell which it is apparently killing.
Substomatal vesicle and a number of somewhat abortive infection
threads. In the epidermal cell on the left two haustoria, deeply stained,
and possibly dead. Below, fairly successful infection.

Empty appressorium and vesicle. On the left infection threads, one
of which has sent a haustoriim into an epidermal cell.

Infection threads growing toward leaf tissues.

IX. Same as Plate VIII, except Figs. 6 to 10, which are of Arnautka.
to 5—10 days after inoculation.
to 10—23 days after inoculation.
Short hyphal segment containing four nuclei. Haustorium in epidermal
cell. Hyphae sent out from segment disintegrating.
Disintegrating hypha with haustorium in host cell.
Long hyphae, the tips of some becoming much vacuolated and apparently
dying.
Typical appearance of hyphae under epiderm, showing haustoria and
some dying hyphal tips. :
Hyphae deeper down in leaf tissues showing tendency to aggregate.
Dead host cells and practically dead hyphae.
Hyphae typical of those in subepidermal wefts showing knoblike branches
which are often quite characteristic.
Small, partly ruptured pustule.
Single uredospore. ;
Subepidermal weft showing unsuccessful attempt at pustule formation
and a number of abortive spores.

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