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P33 @ NITED STATES DEPARTMENT OF AGRICULTURE
BULLETIN No. 759

Contribution from the Bureau of Plant Indusiry
WM. A. TAYLOR, Chief

Washington, D. C. PROFESSIONAL PAPER June 19, 1919

THE LEAF-SPOT DISEASES OF ALFALFA AND

RED CLOVER CAUSED BY THE FUNGI PSEU-

DOPEZIZA MEDICAGINIS AND PSEUDOPEZIZA
si TRIFOLI, RESPECTIVELY

. By :
FRED REUEL JONES, Pathologist, Cotton, Truck, and
; Forage Crop Disease Investigations
CONTENTS
Page Page
Scope of the Investigation . . 2... . 1 | The Fungi—Continued.
Che Diseases sy ohn relate Moet 2 Reported Conidial Stages of \These
Economic Importance . . . . . . 2 Fungi Siifepnte ini le went. ate ate ae 9
F Description of the Disease on Alfalfa 3 Physiology of the Fungi . . . . . ag
Description of the Disease on Red Pathogenicity of the Bunge Pees ie do
Clover: acs meal) ta putas NG 4 | Life History of the Causal Organism in
Host Plantsre ey Men lieu duok 5 Relation te the Host Plants . . . . 27
bre rane pray ne orem es ready ih ke 5 American Studies Bearing on Life
Synonomy of Pseudopeziza trifolii_ 5 RERSCORV OOH Siku, Wee Nt GRU aia 27
Synonomy of Pseudopeziza « medi- Method of Overwintering . . . . 28
Cagis yi aieciew Ra eee Nac CONA, i 6 Method of Distribution . . . . . 30
Comparative Morphology of the Fungi 6 | Summary ... PEN Re peas anette aay 35
Morphological Characters in Culture 8 | LiteratureCited ......2..., 36

e. 2
_ WASHINGTON
GOVERNMENT PRINTING OFFICE
1919

Contribution from the Bureau of Plant Industry
WM. A. TAYLOR, Chief

Washington, D. C. PROFESSIONAL PAPER June 19, 1919

THE LEAF-SPOT DISEASES OF ALFALFA AND RED
CLOVER CAUSED BY THE FUNGI PSEUDO-
PEZIZA MEDICAGINIS AND PSUEDOPEZIZA TRI-
FOLIT, RESPECTIVELY.

By Frep REvEL JoNEs. Pathologist, “~R2BaG
Cotton, Truck, and Forage Crop Disease Investigations.

CONTENTS.
Page. Page.

Scope of the investigation............-------- 1 | The fungi—Continued.

TAG GHSGHRE Sou canssce so ocoudesonErSeogceer ac 2 Reported conidial stages of these fungi... 9
Economicimportance....-.-..-.-------- 2 | Physiology of the fungi................-. 11
Description of the disease on alfalfa...... 3 Pathogenicity of the fungi............. a 19
Description of the disease on red clover. . 4 | Life history of the causal organism in relation
TE(OS HG OIENO ae oo cub obonesoresssseaoor 5 tothe host plantstess--sssace-seneaeas 27

PMH MUINED see eases ceive een sseitositee 5 American studies bearing on life history. 27
Synonomy of Pseudopeziza trifolii-......- 5 | Method of overwintering...............-. 28
Synonomy of Pseudopeziza medicaginis. 6 Method of distribution................... 30
Comparative morphology of the fungi... - Gee ouMmmManyeera eee poser stecce sense emer 35
Morphological characters in culture. ...-- 8 \Plotteraturecited: . s.-asnccsecenacteceeeeosece 36

SCOPE OF THE INVESTIGATION.

Among the diseases of the foliage of the alfalfa plant, the one
which is most widely known and is reported to cause the greatest loss
is the leaf-spot caused by the fungus Pseudopeziza medicaginis. The
disease is commonly called the alfalfa leaf-spot. This name is not
distinctive, and its continued use is open to the objection that it
promotes the present tendency to apply it inclusively to all the sev-
eral leaf diseases that may be present with the true leaf-spot. How-
ever, the usage is so prevalent that it appears likely to persist.

Although the importance of the disease has caused it to be men-
tioned widely and frequently, little careful study has been devoted
to it. A great number of scattered and conflicting observations have
left the life cycle of the fungus causing the disease in doubt; the

89950°—19——1

2 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE.

method of overwintering of the fungus has not been conclusively
demonstrated; and the oft-considered question whether this fungus
is identical with or merely similar to one which causes a correspond-
ing leaf-spot on red clover has never been decisively answered. It
is because of the last consideration that the two diseases have been
studied together. The leaf-spot of red clover caused by the fungus
Pseudopeziza trifolii occurs over a wide range of territory, but
usually not so abundantly as that on alfalfa. Mention of it occurs
frequently in literature. No distinctive common name has been ap-
plied to it. Much of the later interest in this disease is in connection
with the question whether or not red clover is a source of infection,
producing destructive outbreaks of leaf-spot on neighboring alfalfa
when this plant has been introduced into new localities.

Leaf-spots of a nature very similar to the two already mentioned
and commonly reported to be caused by the same fungi are known
on a long list of clovers, alfalfas, and closely related plants. All of
these diseases should be studied together and the relationship of the
causal organisms determined. However, most of them do not occur
in America or only in restricted localities, and none of them causes
great damage to the host plants. The only one of these of which any
living material has been available for study is that caused by
Pseudopeziza medicaginis on Medicago lupulina. The incomplete
notes on this disease have been included.

THE DISEASES.
ECONOMIC IMPORTANCE.

As has already been stated, the assertion has been made again and
again that leaf-spot is the most common and destructive of the
foliage diseases of alfalfa. That it is the most common is beyond
question. But in the estimates of the loss which it has caused it
appears highly probable that damage from other causes than the
conspicuous leaf-spot has been included. Nevertheless, even if proper
deductions for these inclusions could be made it might still be true
that leaf-spot causes greater loss than any other foliage disease.

The highest estimate of loss from this disease is that of Pammel
(1891) + from Iowa. In 1890 he attributes to this cause a loss of
half the crop. Stewart and others (1908, pp. 384-387) report from
New York that young stands are often ruined and that old stands
are killed outright. Chester (1891) reports that some plats at the
Delaware station in 1889 were attacked severely before the plants
were large, and some of them were completely destroyed. Voges
(1909) in Germany and Ivy Massee (1914) in England note the
sickly appearance of diseased fields.

1 The dates in parentheses refer to * Literature cited”’ at the end of this bulletin.

LEAF-SPOT OF ALFALFA AND RED CLOVER. 3

The amount of damage which the fungus may cause appears to de-
pend on several circumstances relating to the development of the
crop and the weather. Under ordinary conditions the incubation
period of the disease is more than a week. If for any reason the
plant is growing slowly, the stand is thick, and the weather is fre-
quently wet, only a few of the upper leaves reach full development
before they are covered with the disease. Thus young stands which
grow slowly before becoming firmly rooted and old stands which are
retarded for any reason are likely to show bad attacks, while stands
which are growing rapidly keep most of the upper leaves well above
the rising invasion of the fungus and show little harm. Thus, in
most cases where the fungus is found in great abundance, apparently
defoliating plants, it will be found that some condition has reduced
the normal rate of growth of the plants and is in part résponsible
for the resulting damage. When plants are vigorous, infection must
_ be heavy indeed to cause extensive yellowing and falling of leaves,
though this may occasionally occur.

Nevertheless, the fungus is present in almost every alfalfa field,
if not in all fields, taking a small toll of the foliage under even dry
conditions and a large toll under more humid conditions. Since it
rarely produces great loss at one time it has come to be regarded as
one of the unavoidable evils to which the alfalfa plant is subject.

The leaf-spot of red clover caused by the fungus Pseudopeziza
trifolz has not frequently been reported as occasioning great loss. In
Russia Jaczewski (1912, p. 98) speaks of it as causing appreciable
damage. Blasdale (1902, p. 75) states that it injures nearly all the
clovers of the stock ranges of northwestern California. Freeman
(1905, p. 309-310) notes that it causes local epidemics in clover fields
in Minnesota. In fields in northern Wisconsin and in Maine in the
summer of 1915 it was observed by the writer to be so abundant as to
cause appreciable loss of foliage. From the evidence at hand it
appears that the disease is not of great significance to clover and
that this significance is only in northern regions. However, the
destroyed foliage is so much less conspicuous than that on alfalfa
that the amount of damage is more likely to be underestimated than

overestimated.
DESCRIPTION OF THE DISEASE ON ALFALFA.

There are two characteristics of the leaf-spot caused by Pseudo-
peziza medicaginis which usually serve to distinguish it from spots
caused by other parasitic fungi. The first of these is the circular
shape and limited size of the spot. (Pl. I, A.) The second is the
presence of a small raised disk (Pl. II, B) that appears in the center
of the spot when it has reached full development. The edge of the
spot may be smooth and definite, especially if the leaf has been much

4 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE.

exposed to the sun, or it may be more or less dendritic, with a fringe
of olive-colored rays. No marked killing or sinking of the leaf
tissue occurs. In size, the spot rarely exceeds 2 or 3 millimeters in
diameter. !

The disk at the center of the spot is the fruiting structure of
the fungus and consists of a mass of asci which discharge large
numbers of spores when sufficient moisture is present. These disks
usually occur on the upper side of the leaf, sometimes on the lower
side, and rarely on both sides from the same spot. Typically they
are 1 to 14 millimeters in diameter, slightly raised, and when fully
developed surrounded by the torn edges of the epidermis of the leaf.
Rarely the central disk is found surrounded with several smaller
disks at its margin. Under very moist conditions the disk may ap-
pear as a‘jellylike drop of exudate at the center of the spot. Under
arid conditions it becomes very dark in color, often almost black.

There is not usually a striking difference in color between the dis-
eased tissue and the disk at the center. This color varies from dark
brown to almost black. If the leaf has begun to yellow, the green
color is sometimes retained longest around the diseased area.

The disease often occurs on succulent stems, where it has an ap-
pearance so characteristic that it can hardly be confused with injury
from any other cause. The spot is elliptical in shape, with perfectly
smooth edges. In size it is about 14 by 3 mm. It is not abundant,
and rarely bears a fruiting disk.

DESCRIPTION OF THE DISEASE ON RED CLOVER.

The leaf-spot on red clover caused by Pseudopeziza bears a very
close resemblance to the similar disease on alfalfa. The spots are
limited in size, usually slightly larger than on alfalfa, in early
stages tending to be angular (Pl. I, C). The border of the spot is
more frequently dendritic in outline. In early stages the color is
dark olive, becoming brown or almost black in later stages. After
the death of the entire leaf, the spot usually becomes almost indis-
tinguishable. Fruiting disks are not as frequently found abundantly
on the spots while the leaf is still alive as in the case of the leaf-spot
of alfalfa, but they may develop abundantly after the death of the
leaf. In early stages they are brownish or dirty yellow, but later
they become almost black. They are more frequently found on the
under side of the leaf than on the upper side, and occasionally occur
on both sides from the same spot. On dead leaves they appear as
amber drops of jelly in wet weather, but when dried they shrink to
bodies so small and inconspicuous that it is practically impossible
to find them. The disease has not been noted on any other part of
the plant than the leaves.

et theca

—

ee a ee

Seer.

Bul. 759, U. S. Dept. of Agriculture. PLATE I.

PSEUDOPEZIZA ON Host PLANTS AND PURE CULTURES.

A, Leaf-spot of Medicago lupulina caused by Pseudopeziza medicaginis (Lib.) Sace. B, Pseudopeziza
medicaginis developing in pure culture from spores discharged on plaster of Paris (right) and filter
paper (left). C, Leaf-spot of red clover caused by Pseudopeziza trifolii (Biv.-Bern.) Fckl. at an early
stage of development. Apothecia have not yet appeared.

Bul. 759, U. S. Dept. of Agriculture. PLATE II.

RE

PSEUDOPEZIZA ON ALFALFA.

A, Apothecia of Pseudopeziza developing on old leaf-spots on overwintered leaves. In many cases
the apothecia are clustered. XX 3, approximately. B, Apothecia of Pseudopeziza on an alfalfa
leaflet. The leaf has been decolorized to show the fungus more clearly. The small dark circles

near the lower right-hand edge of the leaf are early stages of apothecia of Pyrenopeziza medicaginis.
X 5, approximately. ;

LEAF-SPOT OF ALFALFA AND RED CLOVER. 5

HOST PLANTS.

Although these fungi occur extensively in America on alfalfa and
clover only, each of them has been reported in mycological literature
on a number of related plants.

The names of these host plants are shown in Table I.*

Taste I.—List of-host plants of Pseudopeziza trifolit and Psecudopeziza
medicaginis.

Hosts of Pseudopeziza trifolii.

Hosts of Pseudopeziza medicaginis.

Name of host plant.

Trifolium:
alpestre ly) 32 -------=-
arvens@l.-. 23-2 ses2
cyathiferum Lindl ..
fragiferum L........
hybridum L -........

incarnatum L......-

medium L........--

macrodon Hook and
Arn.

nigrescens Viv..----

pallescens Schreb-....

_ pratense L..........

pallidum W. and K.
Tepens ease seo.
resupinatum L...-.-.
spadiceum L......-.

Authority cited.

Name of host plant.

Authority cited.

Saecardo, D., 1903,n0.526,
1319.
Krieger, 1892, no. 781, 794

Blasdale, 1902.

Massee, 1914.

Bivona- Bernardi, 1816,
man. 4, p. 27, pl. 6, fig. 5.

Saccardo, P. A., 1889, pp.
723-724.

Lambotte, 1880, p. 264.

Blasdale, 1902, p. 75.

Briosi, 1888.
Massee, 1914.
Saccardo, P. A.,
pp. 723-724.
Maire, 1913, no. 119.
Briosi, 1888.
Massee, 1914.
Saccardo, P. A., 1897, p.
623.

1889,

Medicago:
carstiensis Jacq.-.---

fal catalase aes eee
hispida denticulata
( Wild.) Urban.
lupulinaje.- 325-2
lupulina wildenowii
(Boenn.) Aschers.
minima Link.......-.
prostrata Jacq...----
Sativarluso cases acer:

Melilotus alba Desv-.---

Onobrychis sativa Link -
Trigonella:
coerulea (L.) Ser... .

corniculata L....-..-
foenum-graecum L..
Vicia villosa Roth....---

Saccardo, D., 1903, no.
526, 1319.

Cavara, 1892, p. 243.

Massee, 1914.

Libert, 1832, fase. 2, no.
176.

Fuckel, 1870, p. 290, 236.

Jaap, 1916.

Desmazieres, pp.
182-183.

Tracy and Earle, 1895,

1847,

p. 106.
Berthault, 1913.

Mazerius, 1875, fase. 33,
no. 1645.

Jaap, 1916.

Massee, 1914.

Tracy and Earle, 1895,
p. 106.

THE FUNGI.
SYNONOMY OF PSEUDOPEZIZA TRIFOLII.

Pseudopeziza trifolii was first described by Antonio Bivona-
Bernardi (1816, Mar. 4, p. 27, pl. 6, fig. 5) on Trifolium hybridum
from Sicily under the name of Ascobolus trifolii. When Boudier
(1869) revised the genus Ascobolus he listed this species among
those which he believed should be excluded, and suggested that it
be placed in the genus Phacidium. The following year Fuckel
(1870) made this species the type of his new genus Pseudopeziza.

Other early synonyms as given by Rehm (1892, p. 597-598) are
as follows: Peziza trifoliorum Uibert, Trochilia trifolii DeNot.,
Molliscia trifolii Phill., Phyllachora trifolti Sace.

The following three names have been included in the synonomy
by Ivy Massee: Pseudopeziza (Phacidium) divergens (Desmaz.)
Sace.; Peziza dehnii Rab., a common parasite of Potentilla; Pyreno-
peziza medicaginis Fckl. The last of these three species has already
been shown by the writer (Jones, 1918) to be the ascigerous stage of

1In compiling this host list, the fungus found on species of the genus Trifolium is
regarded as Pseudopeziza trifolii (Biv.-Bern.) Fckl., while the fungus on species of the
genus Medicago or closely related genera is regarded as P. medicaginis (Lib.) Sace.
Owine to the fact that the two fungi have frequently been regarded as one, the fungus
on Medicago and its relatives has often been reported as P. trifolii.

6 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE.

Sporonema phacidioides Desmaz. The writer has not been able to
discover any adequate reason for the inclusion of the other two

names.
SYNONOMY OF PSEUDOPEZIZA MEDICAGINIS.

The first collection and description of Pseudopeziza upon a species
of the genus Medicago were made by Madam Libert (1832, fase. 2,
no. 176) under the name of Phacidium medicaginis. The host was
Medicago wildenowii, now known as Medicago lupulina wildenowii.
Later, when Desmazieres (1841) found Pseudopeziza upon alfalfa he
assumed that it was identical with the species described on I/edicago
wildenowii. His assumption has not been seriously questioned.

Tn 1883 Saccardo (1883, no. 1390, 1391) transferred this species
to the genus Pseudopeziza which Fuckel (1870) had established
with Pseudopeziza trifolii as the type species. As soon as the two
fungi were brought together in the same genus their similarity
raised the question whether they were not identical. Briosi (1888)
compared the fungi as they occurred on several species of Trifolium
and Medicago and failing to find sufticient morphological difference
between them to justify retaining them as distinct species advised
that Pseudopeziza on alfalfa be called Pseudopeziza trifolii forma
medicaginis. This usage has been followed by Rehm (1892, p. 597-
598) and appears to have been generally accepted by mycologists,
many of whom drop the form name altogether. Plant pathologists,
on the other hand, have found it more convenient to retain the two
names, though in most texts it is noted that possibly or even prob-
ably the two species are identical. The writer believes that the fol-
lowing pages present adequate evidence that the fungi on the two
hosts are separate and distinct species.

COMPARATIVE MORPHOLOGY OF THE FUNGI.

The apothecia of both these species of Pseudopeziza arise in a deli-
cate stroma beneath the epidermal layer of the leaf. The apothecia
on alfalfa are usually solitary, except on overwintered leaves, where
several clustered apothecia may develop on a stroma. On red-clover
apothecia are sometimes clustered. The hymenial layer when first
developed is covered with a thin stromatic stratum of small rounded
cells, the outer layer of which may develop thick dark-colored walls.
This stroma usually remains adherent to the epidermis when this is
ruptured by the developing asci.

As the hymenial layer develops, the stroma from which it arises
becomes thicker, forming in and among the collapsing leaf celis. The
epidermis is ruptured, the hymenium is raised above the surface of
the leaf, and after the spores have been largely discharged and the
hymenium has shrunken the recurved flaps of the torn epidermis
become conspicuous around the apothecium.

LEAF-SPOT OF ALFALFA AND RED CLOVER. Tl

Under favorable conditions apothecia may reach 14 mm. in di-
ameter, but are usually 1 mm. or less. Asci are 60 to 70 microns
long, and about 10 microns in diameter. Paraphyses are slightly
longer than the asci, nonseptate, and swollen at the ends.

Ascospores of the two species (figs. 1 and 2) show slight differ-
ences in size, those of Pseudopeziza trifolii being larger. The
spores of each species have shown small variations in measurement
when they were obtained under different conditions affecting their
discharge. The most important of these variations has occurred
when spores are obtained from apothecia which are drying rapidly.
Under these conditions discharge is greatly accelerated, and. the
number of spores of smaller size is increased. Therefore, in order to
obtain comparable measurements certain precautions were always
taken to obtain spores of the same degree of maturity. Fruiting cul-
tures of the fungus or leaves bearing apothecia were placed in the
cover of a Petri dish over a layer of clear agar. After about 10
hours, when the discharge
of spores was apparently “a S
proceeding at a uniform 00) O
rate, the cover of the dish ae 76) )
was turned about so that pre, 1.—Ascospores of Fic. 2.—Ascospores of
the spores now fell on a new Pseudopeziza trifolii. , Pseudopeziza medica-

C x 600. ginis. X 600.
portion of the agar surface.
After half an hour a considerable number of spores were usually
found on the agar. The cover was then removed from the dish, a
small drop of water and a cover glass were placed on the area on
which the spores were scattered, and measurement was made as
rapidly as possible.

When a large number of spores have been measured to the nearest
micron and the spore lengths arranged, as shown in Table IJ, it has
always been found that the number of spores of Pseudopeziza medi-
caginis which measure 10 microns and less constitute more than
half the total, while in the case of P. trifolii the number of spores
which are 11 microns and longer constitute more than half the total.
Table II presents a typical comparison of the measurement of 100
spores of each species.

TABLE II.—Comparison of the lengths of 100 spores each of Pseudopeziza medi-
caginis and Pseudopeziza trifolii, measured to the nearest micron.

Length (microns).

a

Spores of—

a | ee Ss ee ee eS ee eee

Pseudopeziza medicaginis........... number... 7 26 33 24 10 Reeec dae eso oce
Pseudopeziza trifolit-s 222) 2-3... Goss: sleaceceee 3 26 46 18 6 1

8 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE.

By careful comparison in this manner it has been found possible to
distinguish between the two species on the basis of spore measure-
ment alone.

In addition to the difference in size, there is a difference in shape
that is discoverable by the examination of many spores—a difference
that does not significantly appear in measurement. Some of the
spores of Pseudopeziza trifolii are slightly flattened on one side.
When the flattened side is seen in profile the spore has a somewhat
pointed appearance. The occurrence of occasional pointed spores
(fig. 1) is a distinguishing feature of this species.

Spores from dried specimens have not been found satisfactory for
comparative measurement. Unless the collection is made just before
the apothecium is completely mature, nearly all of the spores are un-
avoidably discharged during drying. The few remaining are likely
to be found much shrunken.

MORPHOLOGICAL CHARACTERS IN CULTURE.

MYCELIUM.

In culture these fungi preserve the same general characteristics
that they show on the host plant. The mycelium radiating from the
germinating spore or group of spores soon produces a stroma at the
center. Thereafter this stroma is surrounded with a narrow fringe
of hyphe, which never advance far beyond the stroma. When the
fungi are grown from spores on the same nutrient substratum, differ-
ences in the character of the mycelium can be noted. That from
Pseudopeziza medicaginis branches earlier than that of P. trifolii;
most of its branches gome off at an acute angle, while those of P.
trifolii come off somewhat regularly at a right angle, or occasionally
at an obtuse angle. ‘

CONIDIUMLIKE STRUCTURES.

Although no conidia have been found in nature, conidiumlike struc-
tures occur regularly in culture and are a feature by which cultures
of the two species can be most easily distinguished. They arise from
the ends of branches or from the distal ends of somewhat swollen
cells. They measure 5 to 8 by 3 to5 microns. They occur most abun-
dantly when the ascospores are germinated on clear agar to which
no nutrient has been added, appearing in about three days in the
case of Pseudopeziza medicaginis and somewhat later on mycelium
of P. trifolii. On mycelium of the first fungus they are produced in
great abundance before the end of the first week, though the mycelium
from different spores or groups of spores produces them in varying
amount. (Fig. 3.) The mycelium may grow but little, becoming
thickly covered with the conidia, or it may grow more freely with
but a few conidia at the ends of short branches. Rarely are they

LEAF-SPOT OF ALFALFA AND RED CLOVER. 9

absent. In striking contrast is the scarcity of these spores on my-
celium from spores of P. trifolii. (Fig. 4.) Never are they pro-
duced in great numbers, and frequently they are entirely absent from
all but a few fungous colonies. Thus, the striking abundance of these
structures on mycelium of P. medicaginis and their scarcity on

Fie. 3.—Mycelium and conidiumlike structures developing from ascospores of Pseudo-
pesiza medicaginigs on agar agar. X 400.

mycelium of P, ¢rifoli furnish, an easy morphological distinction
between the two species.

REPORTED CONIDIAL STAGES OF THESE FUNGI.

It is a matter of some interest to note that all the studies of
Pseudopeziza on alfalfa and clover

which have been made by European
mycologists and pathologists with
but a single exception (Briosi,
1888) have contained a discussion
of an associated conidial stage.
Thus at least three, perhaps four,
imperfect fungi have been assigned
to this role in addition to the coni-
diumlike structures which are pro-
duced in culture. A summary of
the evidence on the basis of which
the association of these conidial — Fic.-4.—Mycelium developing from an
stages has been made is here given. sili eae} earners at Dot
The first of these fungi to be
regarded as a conidial stage of Pseudopeziza medicaginis was Sporo-
nema phacidioides Desm. Since the writer has a previous article
(Jones, 1918) traced the development of the purely observational
evidence on which this association was based and has shown that this
Sporonema is the conidial stage of Pyrenopeziza medicaginis Fckl.,
no further discussion is necessary here,
89950°—19

Gb)
ad

10 BULLETIN 759, U. S.. DEPARTMENT OF AGRICULTURE.

The second suggestion that Pseudopeziza produces conidia comes
from the work of Brefeld (1891, p. 325). In the course of his study
of Pseudopeziza on both clover and alfalfa he germinated the asco-
spores. The cultures thus obtained of these fungi behaved alike.
Very little mycelium was produced. After about 14 days conidia
were cut off laterally from certain threads and from the ends of side
branches. These conidia were not observed to germinate. After
describing them, Brefeld refers them to the conidia described by
Tulasne (probably referring to the conidia of Sporonema phacid-
zoides), but he does not state whether he regards his conidia iden-
tical with those described by Tulasne or not. The structures which ~
Brefeld describes and figures as conidia appear to be identical with
those already described as occurring in culture.

The third and most extended reference to a conidial stage of
Pseudopeziza medicaginis is that of Voges (1909). In the course of
a study of an outbreak of the disease in fields under his observation
in Germany, he reports that he finds closely associated with the fruit- _
ing disks of Pseudopeziza on living leaves the pyenidia of a Phylio-
sticta which does not appear to him to belong to a previously de-
scribed species. Unfortunately, his’own description of this Phyllo-
sticta is so meager that it does not serve to identify it. He states that
the spores are differentiated into two forms—a smaller 1-celled spore
and a larger, often 2-celled spore. No mention is made of any
attempt to determine whether or not the two types really belong to
the same fungus or not, nor does he explain why such a fungus should
not be called Ascochyta rather than Phyllosticta.

Voges next attempts to identify the Phyllosticta with Pseudope-
ziza by cultural methods. He places fragments of Pseudopeziza fruit
disks on a nutrient substratum. When this is done in March and
October, no results are obtained; but in June he gets a fungus on his
plates which first produces aerial conidia and later pyenidia like
those previously found on the leaves. Inoculations made on alfalfa
leaves with these leaf-spot cultures produced typical Phyllosticta
spots. Inoculation of alfalfa leaves with fragments of Pseudopeziza
fruiting disks gave like results. Consequently he concludes that the
Phyllosticta and Pseudopeziza are identical and that Pseudopeziza
has three spore forms—aerial conidia, conidia in pyenidia, and asco-
spores. Finally he inoculates clover leaves with fragments of his
Phyllosticta culture and finds that typical spots bearing Phyllosticta
spores are produced. Hence he concludes that the Pseudopeziza on
alfalfa must be identical with that on clover.

Even if these results of the few experiments which he performed
are accepted at their full value, the conclusions which he draws are
manifestly not justified. In the first place, the fact that he was
unable to get cultures of his Phyllosticta from Pseudopeziza spots

LEAF-SPOT OF ALFALFA AND RED CLOVER. 11

except at a certain period in the summer is not adequately explained
by his extraordinary theory of a periodicity in the vegetative vigor
of the fungus. In the second place, inoculations with this Phyllo-
sticta whether upon alfalfa or upon clover produced lesions which
bore only the pycnidia of the Phyllosticta, never the apothecia of the
Pseudopeziza. These cultures, obtained under doubtful conditions
produced ascospores neither in culture nor as a result of inoculation.
Thus, the evidence which Voges presents, judged entirely by itself,
does not prove or even clearly indicate that he ever had Pseudopeziza
in culture. It does not appear, however, that the work of Voges
has been widely accepted, at least not in America, even though the
report of his work as presented in the Experiment Station Record 1
is incorrect or misleading in almost every detail, causing his conclu-
sions to appear much more justifiable than when they are read in
the original article.

In the same year that the article by Voges ‘was published Voglino
(1909, pp. 226-228) in Italy presented evidence which he believed
indicates that Gloeosporium caulivorum Kirch. or G@. trifolii Pk.,
which in his opinion may be identical with it, is the conidial stage
of Pseudopeziza on Trifolium pratense. His evidence was obtained
both from observation and from cultures. In a certain field consid-
erably injured by Gloeosporium he finds apothecia of Pseudopeziza
trifolii developing in close association with the acervuli of the
Gloeosporium. Later he makes cultures from conidia obtaineetl from
stems on a clover decoction with gelatin, and in a single culture he
found after 30 days three apothecia of a fungus which he assumes
to be a Pseudopeziza. On the basis of this evidence he decides
that the Gloeosporium must be the conidial stage of Pseudopeziza.

The account which Voglino gives of his work is very brief and
bare of details. No mention of inoculations is made. No descrip-
tion of the Pseudopeziza which he regards as Pseudopeziza trifolii
is given. It is not clear from his account that he obtained a pure
culture. Therefore his results can hardly be regarded as having
more than a suggestive value.

This review of European literature brings us to the conclusion
that, with the possible exception of the description by Brefeld of
conidia in culture, there is no conclusive observational or experi-
mental evidence that either of these Pseudopezizas has an associated

condidial stage.
PHYSIOLOGY OF THE FUNGI.

ISOLATION OF THE FUNGI.

Efforts to isolate these fungi by ordinary méthods were continued
for a long time without avail. The first success was obtained by

1Experiment Station Record, v. 22, no. 7, p. 648.

12 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE.

taking advantage of the fact that when the ascospores are dis-
charged from the ascus they are thrown several millimeters verti-
cally. Repeated trials showed that if fresh leaves were used, spores
could sometimes be obtained in considerable numbers on an agar
surface placed over the apothecia without bacterial or fungous con-
tamination. This work of collecting spores was best carried out in
a Petri dish in which a layer of very clear agar had been poured.
The dish was inverted and the leaf bearing the apothecia supported
2 or 8 millimeters below the agar. After a short period the area over
the apothecia was marked, the dish turned, and examination made
with the low power of the microscope to determine whether the
requisite number of spores were present. The difficulty would have
been lessened had a few spores been sufficient for the development
of a culture, but experience soon showed that a large number of
fungous colonies crowded together developed better than a few.
When several areas on the plate had been scattered with spores, the
leaf bearing the apothecia was transferred to another plate. By using
the utmost care and exposing a large number of plates a few could
be obtained without contamination or with so small a number of
foreign organisms that they could be cut out with a sterile needle.
After a plate had been observed until it appeared certain that no
foreign organism was present, it was found advisable in order to
prevent drying to cut out the area bearing the developing fungous
colonies and transfer them to agar slopes in test tubes.

In the course of experiments with cultures made in this way the
first culture of Pseudopeziza from alfalfa to produce apothecia was
obtained. The spores were discharged on an alfalfa-agar plate on
October 5, and the agar was transferred to a water-agar slope on
October 22. The ascospores were being produced on November 6.
At first it was assumed that the fungus had been starved into fruiting
by this process, but later work does not indicate that this was the
case. Fruiting cultures can be obtained most readily by transferring
the developing fungous colonies as soon as they become macroscopic
from the water-agar plates to oatmeal-agar slopes. In this way the
fungus was isolated six times in the autumn of 1914 and once in the
autumn of 1916.

Pseudopeziza was isolated from red clover in the same way as
from alfalfa. Two isolations were made of this fungus in 1915, one
from clover leaves collected by Prof. H. H. Whetzel at Ithaca, N. Y.,
and one from clover collected in Door County, Wis.

A later successful reisolation of this fungus from plants inoculated
in the greenhouse suggests that it may not always be necessary to
employ this tedious process. In the instance referred to, diseased
leaf fragments were cut from clover leaves two weeks after inocula-
tion. ‘The fragments, each bearing from one to three infections, were

LEAF-SPOT OF ALFALFA AND RED CLOVER. 13

dipped in 50 per cent alcohol and placed in a solution of 1 part of
bichlorid of mercury in 1,000 parts of water for 1 to 14 minutes.
After washing, the fragments were placed separately on slopes of
2 per cent water agar. After these cultures had been kept three
weeks at 17° to 19° C., small tufts of mycelium emerged from the
fragments which had remained free from contamination. The frag-
ments were then transferred to oat agar. Apothecia appeared two
weeks later, and cultures were started. Success in this instance seems
to be due to the relative freedom of these greenhouse plants from
fungi which quickly enter the host tissue that has been killed by the

parasite.
CULTURAL CHARACTERS OF SPECIAL MEDIA.

Only a few of the more common media on which the fungi grow
most readily and show the most striking differences are selected.
In connection with these descriptions the following facts regarding
the method of making cultures and their habit of development should
be kept in mind:

(1) New cultures are started by placing near the top of an agar slope a
fragment of a culture which is producing and discharging ascospores abun-
dantly. The position of the slope should be changed from time to time to insure
a somewhat uniform distribution of the spores over its entire length. After
from one to four days the original transfer may be removed to another slope
and thus serve to start a number of cultures successively.

(2). The small fungous colonies which arise fruit better and earlier when
closely crowded together. Yet excessive crowding may delay fruiting.

(3) Apothecia appear in three to five weeks at favorable temperatures.
After a period of active spore production lasting from one to two weeks, fur-
ther spore production takes place only occasionally. Transfer of the stroma
to new media increases the likelihood of its occurrence but does not insure
it. The stroma itself continues to grow very slowly.

Oatmeal agar—As a culture medium oatmeal agar has proved to be
the most useful for general culture work, because upon it ascospores
are produced in greatest abundance. The following description
applies to cultures kept at 20° to 22° C.

Pseudopeziza medicaginis: The first evidence of growth appears
about one week after spores are discharged upon the medium. At
this time the surface of the substrate appears roughened as though
pushed up into a multitude of minute flat cones. In about two
weeks the cones are increased in size and show a rusty brown color at
the center. The color becomes darker at the center of the cone,
surrounded by a rusty rim. Soon the dark-brown color covers the
entire slope. If the colonies are not sufficiently close to touch each
other in three weeks, the color is darker than if the colonies merge.
At the end of three or four weeks apothecia appear at the center of
the stromata as small grayish white, often glistening gelatinous

14 BULLETIN: 759, U. S. DEPARTMENT OF AGRICULTURE.

masses, very small at first, but often extending until they merge.
The apothecium is merely a rounded, sometimes flattened, firm mass
of asci and paraphyses which crumbles when crushed (Pl. III, B).
The gray color of the mass becomes darker with the increased age
of the culture, and finally is dark brown or almost black. The rusty
brown color that is seen early in the development of the culture is
usually found to have permeated the agar slightly by the time the
fruiting stage is reached. A greater amount of color is usually
found in cultures which have relatively few colonies and which
fruit but little. Retardation of the growth of the cultures by low
temperature appears to allow the color to diffuse farther through the
agar. In cultures which grow rapidly, the yellow color may be
diffused only in the upper part of the slope where the layer of agar
is thin.

Pseudopeziza trifolii: The development of Pseudopeziza trifolii
on this culture medium differs from that described for P. medicaginis
only in the details here stated. The color which develops in the stro-
mata as they develop is dark gray, becoming black, with no trace of
brown. The fungous colonies appear a little more vigorous and co-
alesce into a more solid crust on the surface of the medium than those
of P. medicaginis. The fruiting structures appear more typical (PI.
IIf, A), being flat on top, but are not surrounded by a wall. The
substrate never becomes discolored.

Potato-dextrose agar.—Cultures of both fungi grow very rapidly
on potato-dextrose agar. When the colonies are much crowded the
leathery surface growth becomes crumpled.

Pseudopeziza medicaginis: Color at the end of four weeks brown,
sprinkled with a few black stromata; substrate decidedly colored;
apothecia produced in small numbers.

Pseudopeziza: Culture coal black, with slight amount of frosty
mycelium. There is a slight staining of the substrate.

Lima-bean agar.—Pseudopeziza medicaginis: Growth yellowish in
varying degree; no fruiting observed; substrate shows discoloration.

Pseudopeziza trifolii: The growth is a rough black mat with a little
white mycelium. The substrate shows slight discoloration.

Corn-meal agar.—Pseudopeziza medicaginis: Growth not vigor-
ous; colonies scattered, black, and distinctly raised from the sur-
face, which appears as though sprinkled with coarse black pepper;
apothecia minute and scattered.

Pseudopeziza trifolii: Growth black, almost submerged in the sub-
strate; colonies tend to remain separate, though the slope appears a
solid black color; scattered minute apothecia occur.

Liquefaction of gelatin—Both fungi cause rapid liquefaction of
gelatin.

Bul. 759, U. S. Dept. of Agriculture. PLATE III.
> ee

ied we od

we pe we 2 2

FRUITING CULTURES OF PSEUDOPEZIZA.

A, Fruiting culture of Pseudopeziza trifolii. 15, approximately. B, Fruiting culture of Pseudopeziza
medicaginis from alfalfa. > 15, approximately.

LEAF-SPOT OF ALFALFA AND RED CLOVER. 15

SPORE DISCHARGE,

The discharge of ascospores has been observed frequently under the
microscope when apothecia have been crushed in water. The ascus
slowly becomes longer and of greater diameter, forcing the spores
forward in a more or less oblique biseriate position. The increase in
diameter of the ascus of Pseudopeziza medicaginis appears to be
somewhat greater than in the case of P. trifold, thus allowing
the spores to come more nearly into a biseriate position. When
the limit of resistance of the ascus wall has been reached, the end rup-
tures, allowing the spores to be expelled in a column. When dis-
charge takes place under water the spores show no tendency to re-
main together, but when they are discharged in air they show a ten-
dency to remain in pairs. This pairing of the spores is probably due
to the adhesive quality of the spore wall, a quality which is also
shown by the tenacity with which the spores adhere to the cuticle of
a leaf.

It is interesting to note that the tendency to remain in pairs is not
shown equally by spores of the two species of Pseudopeziza. It is
somewhat more marked in the case of Pseudopeziza medicaginis.
This may be due to the fact already cited that the spores of this
species are brought more clearly into biseriate position before dis-
charge takes place. In order to determine this difference, spores were
collected on water agar as though for measurement. The spores in
each group in selected miscroscopic fields were counted until, the
grouping of 1,000 spores had been determined. The results obtained
at various times are shown in Table ITT.

TABLE III.—Grouping of ascospores of Pseudopeziza medicaginis and Pseudo-
peziza trifolii when caught on an agar surface after being discharged
normally from cultures.

Spores in group—

Spores of—
yl ie 3 4 5 6 7 8
Pseudopeziza medicaginis:
HES bill O Gerace ew vcr ie bs aes oeterciorsd oo| MOON eezoS 19 43 7 19 2 9
Second lot... Goesaa|)) 66n|—342 8 30 4 el bo Goes 4
Third lot... 58 | 356 6 33 6 yd Seton 1
BOte. Se M2 aes sss. 245. ISS S. eth. dk 184 | 936 | 33 | 106 17 32 2 14
Pseudopeziza trifolii:
Rirsiglobe-cee cs eas uae See number..| 133] 215 39 28 16 11 2 6
Seconds ae! Sk. se scenes. ease Gos aie 2720) 5189 48 22 7 4 5 3
Aviaries seen Sarat ae eerie intact Be erawtera Gos.ce |e 242 185 54 19 10 9 2 f
THELESS es AR aR I ihc laa 647 | 589 | 141/60 |e ga eg 13
}

Tt will thus be seen that while in the case of Pseudopeziza medi-
cagimis each spore group containing an even number of spores is
greater than the preceding or following group, in the case of P.

16 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE.

trifolit there is almost a regular decrease of the number of groups
from those containing one to those containing eight. Sufficient evi-
dence has not been collected to determine whether the same clean-cut
difference in the behavior of the two species appears when the
spores are discharged from apothecia on living leaves.

SPORE GERMINATION.

In all the more obvious features of germination, the ascospores of
the two fungi behave alike. They germinate readily when they have
been discharged naturally upon a suitable moist surface; but germi-
nation is infrequent if the spores have been crushed out of the asci
or if they are submerged in water. A single germ tube emerges from
any point in the circumference of the spore, except that germination
from the end has been observed only in the case of a few spores of
Pseudopeziza trifolii. Under the best conditions that have been
found, the proportion of germination is usually from 30 to 50 per
cent. The vigor of germination varies greatly. Many of the spores
which push out short germ tubes cease growth promptly, while a few
develop vigorous germ tubes.

TaptE 1V.—Time required for the germination of ascospores of Pseudopeziza
medicaginis and Pseudopeziza trifolii and rate of growth of the germ tube
for three days at constant temperatures.

[A plus sign indicates that the germ tube could be seen emerging from the spore, but that it did not reach

a length equal to half the length of the spore. The figures in the body of the table represent the estimated
length of the germ tube in terms of the length of the spores. |

Pseudopeziza medicaginis, Pseudopeziza trifolii.

Temperature (°C.),
4 8 12 24 48 72 4 8 12 24 48 72
hours, |hours.|hours.|hours;|hours, hours, hours, |hours.|hours,|hours,|hours,|hours.

1 PH psoor scsdce bossa + 1 2
On|) ko Al cco | arerete pie + | 0.75 | 1.5 2
5] 1.5 il eestor oF ap -5 1.5 2
3 Be eee + O55 U2e5 3 4
3 4/ + 0.5] 1 1.5 3.5 5
UT escad + Dae 2 Oia | aati
5 Sn04 + sah | al 2.5 by Wea so
7rd eae oy Jae + |1 Q) | @)
2 (2) | Steateictall Sstote c | mreratereta me ct ae Sietereteral terete
OT OW OD Oy Oy Peery ep ©) |)
1 Spores disintegrate. 2 No germination.

The one significant difference that they show is found in the fact
that the spores of Pseudopeziza medicaginis will continue to germi-
nate at a slightly higher temperature than those of P. trifoli. In
order to test this relation of temperature to germination, spores were
discharged naturally upon an agar surface with all the precautions
necessary to secure mature spores previously described in obtaining
spores for measurement. The agar used in all cases consisted of 2
per cent of agar-agar in water carefully cleared and filtered. In
order to insure identical conditions for the two species in each test,

LEAF-SPOT OF ALFALFA AND RED CLOVER. LG

transfers of both were made to the cover of the Petri dish contain-
ing the agar. The spores were discharged simultaneously for 1 to 1}
hours. If at the end of this time sufficient spores were present, the
cover containing the cultures was transferred to another dish and the
spores placed at the desired temperature. Petri dishes to the number
of 72, nearly all of them containing spores of the two fungi, were
incubated in this way. The results are given in Table IV.

This table shows that the two species germinate with almost ex-
actly the same degree of vigor at each temperature, except that the
spores of Pseudopeziza trifolii cease to germinate, or, perhaps more
exactly, to make growth after the initial stages of germination, at a
slightly lower temperature than those of P. medicaginis. Although
the difference here may seem slight—almost within the limit of ex-
perimental error—it has been found constant and definite in repeated
tests. The possible significance of this fact in connection with the
difference in geographic range of the two fungi will be considered

later.
EFFECTS OF TEMPERATURE ON SPORE PRODUCTION.

When cultures of the two fungi are placed at a series of constant
temperatures they appear to show constant differences in the time
required for spore production and in the abundance of spore pro-
duction at each temperature. At the outset it must be stated that
limitations inherent in the method of starting cultures cause wide
variation in the behavior of the cultures of each species. This diffi-
culty is partly overcome by using several cultures and by repeating
the work. Even then the results must be regarded as being sug-
gestive rather than accurate. The time required for the production
of ascopores as obtained in two trials is given in Table V. Three
cultures of each fungus were used, and the earliest time at which
the discharge of ascospores could be demonstrated in any of the three
cultures was recorded. The three highest and the lowest temperature
rarely varied more than one degree, and the remaining temperatures
varied only half a degree.

TABLE V.—Time required for the production of ascospores of Pseudopeziza
medicaginis and Pseudopeziza trifolii at constant temperatures.

Temperatures (°C.),

Ascospores of—

16 | 18.5

20 | 21.5 | 24

Pseudopeziza trifolii.......... days.. 36 | 30 20} 16 16 21
Pseudopeziza medicaginis...-. GOeese|L 182) acces |seieere te POS: | tate nte 49 | 36 16] 14 14 20

Table V shows that below 14° C. the behavior of Pseudopeziza
medicaginis was erratic, rarely fruiting at all. Only one culture
89950°—19——3

18 BULLETIN 59, U. S. DEPARTMENT OF AGRICULTURE.

fruited at 8° C. in one of the trials. At and below 16° P. trifolit
requires a shorter time for the production of spores than P. medi-
caginis, while above this temperature the condition appears to be
reversed. But the most striking differences in the behavior of the
two fungi are not shown in the table. These differences lie in the
length of the spore-producing period and in the abundance of the
spores produced. Although P. trifolii is under most circumstances
the more prolific spore producer, this preponderance is greatly in-
creased at and below 18° C. Here abundant fruiting may occur for
two weeks and longer, while cultures of P. medicaginis fruit very
meagerly and only for a short period at these temperatures. Above
16° P. medicaginis begins to fruit more abundantly, reaching its max-
imum at temperatures a little above 20° C. Thus, the optimum tem-
peratures for the fruiting of these fungi may be judged roughly as
about 13° to 22° C. for P. trifoléi and 16° to 24° for P. medicaginis.

RESISTANCE OF THE SPORES TO DESICCATION,

In order to determine whether ascospores discharged from the
ascus might be able to live over winter on seed or débris carried with
the seed, it was desirable to test the resistance of discharged asco-
spores to periods of drying of such duration as they would be obliged
to endure on the seed. Owing to the slow growth of the fungus after
germination and the limitations of conditions under which spores
will germinate at all, it is obvious that the spores must be dried and
germinated under such conditions that all other organisms will be
excluded; that is to say, the entire process must be carried out under
conditions of pure culture.

Obviously, the preferable method would be to dry the spores on _
the seed itself. In order to do this, sterile seeds were necessary. The
difficulty of obtaining such seeds which were certainly free from any
residual effect of the sterilizing agent on the seed coat was so great
that it was finally abandoned.

Preliminary tests with plaster of Paris blocks as the conveyor for
the spores during desiccation were so satisfactory that they were used
exclusively. Thin blocks small enough to slip into a test tube easily,
were sterilized by heat and placed beneath cultures which were dis-
charging spores actively. After a period of 8 to 12 hours the blocks
were placed in sterilized test tubes. These were stored, some in a
glass case in the laboratory and some outside a north window.

From time to time one or more of these blocks were placed on
water agar to which a small amount of alfalfa-leaf decoction had
been added. The amount of the decoction appeared to make no ma-
terial difference up to any amount that could be added to 3 per cent
agar without causing it to lose its ability to solidify upon cooling.
Other culture media were tried, but none gave as prompt a result

LEAF-SPOT OF ALFALFA AND RED CLOVER. 19

as this. The red-brown color of the Pseudopeziza stroma produced
by this medium on these white blocks (Pl. I, B) could be recognized
at an earlier date than the pale-colored growth which developed
when other media were tried.

Spores of Pseudopeziza trifolii were tested in the same way with

those of P. medicaginis, though not as extensively. The results are
given in Table VI.

Taste VI.—Viability test of ascospores of Pseudopeziza medicaginis and Pseu-
dopeziza trifolii viable when dried on plaster of Paris blocks in the laboratory
and out of doors.

Place and date of ending Time Place and date of ending Time
desiccation. (days). a desiccation. (days). Results.
PSEUDOPEZIZA MEDICAGINIS. PSEUDOPEZIZA MEDICAGINIS—
continued.
Tn laboratory:

Hebel agIb- 5. os. eee SAS 32 + Out of doors—Continued.
Jans23 Glo cece ss- cee en 36 + May ZO LOG Les. sev ee oe 120 +
DEC rs NOLO se acca. coe e 37 a Feb. 15, OL GR Ses cee 108 +
Hebi ils 191bees i ceases. 38 + Apr. 7 AQIS Seo ae fibres. 159 +
Mpeg I lO G acct saeco cre i) ee May 20019152 oie s! seco. 212] 0
WDECHIG TOL oon nee 48 ao Sept. 1, LOGE eset she 303 ae
eb 20 1OTS sa kee eens cae 49 ce ie Af ig am a] DX YER 5 RRS pe 329 0
Mars 260 1915 us6 see ke 63 ae
WAG O plOl Ome seem seeemmee 66 0 PSEUDOPEZIZA TRIFOLI,
panel 2 1916 si 52 sees ss o- < 72 +
Say 17 MON aa oe ee 76 a In laboratory:
an 231915: boc = scqsee 76 + Tal OLGA ee yee aes 36 +
Damo Wolo e doreen eee 79 + Mar. 7, aIOICC ee ae 63 +
ami MOIS eee hoses eke 85 0 Feb. 15, MOUSE AE py 2 OSES 64 +
Aug. 26, NOTE ie Pee ess coas ac 93 0 Apr. 6, GIGS eres 8 Mates 112 0

Se CE aCaas aoe anes 94 0 May, lol9l6!e. ees As. See 113 ar
Apr. i, WOLDS es es cases « 100 0 Apr. 20, NQUG IE Rr Seec cpa 120 0
Feb. 12) TO1G 3:5 22S. 92s ware 104 + ert L TOM ses ee aes 258 0
Sepiilgioee eet en 210 OP il ven OSs Meret SMe Dat antes 256 0
aM l5 LIGe sy. .Sehead ae 357 0 Out of aca:

Out of doors: Var 191G 2: mecasemanice 28 ae
Dec. 9, 1915......- 27 + Do a ope 25 +
Dee. 16, 1915.. 34 4x May 4, 1916. 104 a
Dec. 22, 1915. . 52 0 Apr. 7, 1916. Bolt 107 0
Jan. 17, 1916.. 77 a“ Banine 1O1Gs he 265 0
Jan. 13, 1916 78 + Sept. 1, 1916... 265 0
iGo), 1s TONGS aac ob aso 106 4

From these results it appears that drying alone can not be de-
pended upon to kill all the spores of either Pseudopeziza in less than
one year. Severe freezing during drying had no apparent effect.
If conditions for survival are as favorable on the seed as on plaster of
Paris blocks, the spores should be able to live from one season to the
next on the seed. But unless conditions on the seed are more favor-
able than on the block, they should not be able to survive and
germinate during a second year.

PATHOGENICITY OF THE FUNGI.
METHOD OF MAKING INOCULATIONS,

Tn all inoculations that have been made, ascospores alone have been
used as the inoculum. The conidiumlike structures which have
been described are produced almost wholly in the substrate, and
since only rarely will one of them separate from the mycelium, no

&

20 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE.

practicable method for using them has been developed. Since no
evidence has been found indicating that they occur in nature or that
they germinate, no great importance attaches to them.

In the first inoculations an attempt was made to obtain the asco-
spores in water suspension, but when the apothecia were crushed in
water usually only a few spores became separated from the ascus.
The few attempts to make inoculations with such meager spore sus-
pensions failed. The method finally adopted and used with minor
modifications in all inoculations reported takes advantage of the
natural discharge of spores from cultures. If a Pseudopeziza cul-
ture is removed from the test tube carefully it may be cut into frag-
ments and placed on a support, where it will continue to discharge
spores for several days provided it is not exposed to direct sunlight
or high temperatures. A culture may thus be removed from a test
tube and placed over a plant upon which the spores will fall. If the
whole plant is to be inoculated, the culture may be placed in the top
of a bell jar which is set over the plant and turned from time to
time to insure a uniform distribution of the spores.

If single leaves are to be inoculated, the culture or fragments of the
culture may be placed for a short time over these leaves in succession.
A more uniform discharge of spores for long periods is obtained in a
dark room at 16° to 20° C. If inoculations are made in the field, they
should be made at night or on a cloudy day. The plants may be wet
with a fine spray before the spores are discharged, or if time permits
this may be deferred until the spores are on the leaf. In the latter
case, a larger number of infections are usually secured, due appar-
ently to the fact that spores falling on large drops of water are held
from sinking by surface tension and germinate too far from the leaf
surface to effect penetration. After the inoculated plants are sprayed,
they should be kept in a moist chamber for at least 12 hours.

This method has been employed in all inoculations made, unless
otherwise stated.

CONDITIONS UNDER WHICH INOCULATIONS WERE MADE,

If inoculations are to be entirely conclusive in result, the control
plants must remain free from the disease. It has been found impos-
sible to keep plants free from leaf-spot for infection experiments
during the summer at Madison, Wis., where the work was done. This
has been due to the fact that alfalfa fields are located so close to the
greenhouse that spores are easily blown in through the ventilators.
But it has been found that if all diseased alfalfa foliage was removed
from the greenhouses in the autumn after the ground outside froze, it
was possible to keep alfalfa plants free from infection with leaf-spot
during the winter and spring. Therefore all inoculations have been
made or at least repeated during the winter months. This precaution

LEAF-SPOT OF ALFALFA AND RED CLOVER. a}

has not been necessary in the case of inoculations with Pseudopeziza
trifolti, since the Pseudopeziza leaf-spot has not occurred on any spe-
cies of Trifolium about Madison during the time this work was in
progress. The following host plants have been inoculated with pure
cultures of both species of Pseudopeziza.

HOST PLANTS INOCULATED.

Medicago sativa with Pseudopeziza medicaginis.—In seven or eight days,
under greenhouse conditions, infections begin to show as minute brown spots
scattered over the foliage. If infection is very abundant these leaves quickly
die. If only four or five infections are scattered over each leaflet, apothecia
begin to appear in about two weeks. For instance, one inoculation made on
December 2, 1915, produced abundant apothecia on the spots by December 15.
On December 20 spores discharged from apothecia on one of the leaves of this
plant were cultured, and the fungus was recovered. An inoculation made on
April 17, 1915, on plants in the field before the natural infection developed
showed abundant spotting 11 days later. The weather turned cold after this
date and no fruiting was observed.

Leaves of all ages are attacked. Leaves which have grown to full size
appear to develop more abundant infections than leaves which are not full
grown, but leaves which are yellow and weak do not seem to become infected
as easily as those which are more vigorous. ;

None of the infection experiments performed during three winters has failed
to develop a greater or less amount of typical leaf-spot.

Medicago sativa with Pseudopeziza trifoliimInoculation experiments have
been repeatedly conducted parallel with those already cited on alfalfa, using
plants of different ages. No infections visible to the naked eye have been
produced.

Medicago lupulina with Pseudopeziza medicaginis.—Plants of this host have
never been very thrifty under greenhouse conditions, and therefore not a large
number have beeen available for inoculation. In no case has any infection been
obtained. Attempts to secure infection by setting Medicago lupulina plants
in the garden among alfalfa plants which were heavily infected with Pseu-
dopeziza medicaginis also failed to produce infection.

Medicago lupulina with Pseudopeziza trifolii—Only two inoculations have
been tried. No infections resulted.

Melilotus alba with Pseudopeziza medicaginis—On March 28, 1915, several
leaves of a vigorous sweet-clover plant were placed beneath fragments of a
culture of Pseudopeziza medicaginis which was discharging spores. At the end
of 24 hours the culture was placed over the entire plant, which was kept in a
moist chamber 48 hours longer. After four days the leaves first inoculated
showed minute brownish spots. These did not increase in size. After two
weeks the portions of the leaves bearing the minute spots were embedded in
paraffin and sectioned. In these sections the brown spots were found to
eonsist of dead shrunken cells in which traces of mycelium could be found.
But this mycelium appeared to be shrunken and dead and not advancing into
the living cells of the host. From this, it appears possible that under favorable
conditions P. medicaginis may be able to cause a very slight spotting of sweet-
clover leaves.

Trifolium pratense with Pseudopeziza medicaginis.—Inoculations of red-
clover plants in the greenhouse with pure cultures failed to produce any infec-
tion. Red-clover plants grown in the garden in close proximity to badly dis-

22, BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE.

eased alfalfa plants have never shown a trace of this leaf-spot during the two
years that these diseases have been under observation.

Trifolium pratense with Pseudopeziza trifolti.—tiInfection from inoculation
with spores from pure cultures has been at all times easy under greenhouse
conditions. Frequently such abundant infection has been obtained that the
leaves are killed before the spots develop to a fruiting stage. The incuba-
tion period of the disease appears to be slightly longer than that of Pseudope-
ziza medicaginis, varying somewhat with the abundance of the infection. Ifa
leaf is heavily infected, the individual infections appear to develop more
rapidly and produce small characteristic killed areas earlier than if infections
are few. Numerous infections appear in 11 or 12 days, and in the greenhouse the
leaf frequently dies a few days later. Fruiting bodies have not been observed
on such leaves, but a reisolation has been made from infections two weeks after
inoculation.

Less numerous infections may not show for two weeks or even longer, but
at the end of three weeks or more they may produce typical apothecia if the
air of the greenhouse is sufficiently moist. These apothecia have been found
on leaves nearly or quite dead. In this respect the fungus behaves differently
than does Pseudopeziza medicaginis. Apothecia on dead leaves are very diffi-
cult to discover unless the leaf has been in a moist atmosphere for some time,
when they appear as dark-amber gelatinous masses on the leaf surface. The
exposure of these gelatinous masses of asci to dry air for even a few minutes
causes them to discharge the larger part of the spores present and to shrink
to a minute mass only a little darker in color than the leaf and therefore
difficult to identify. The best development of apothecia has been obtained by
placing infected plants outside the greenhouse during protracted periods of rainy
weather.

Trifolium hybridum with Pseudopeziza medicaginis.—No success in obtaining
visible infections has been attained.

Trifolium hybridum with Pseudopeziza trifolii.—Of the eee plants inecu-
lated only one survived in a vigorous condition for a sufficient period to show
infection. This showed an abundant spotting, which was in every way charac-
teristic of the Pseudopeziza spot on red clover except that the spot appeared to
be somewhat limited in development by the veins, thus showing a slight ten-
dency to become angular. No fruiting bodies were produced. Apparently
infection takes place only under the most favorable circumstances. This plant
appears to be a much less congenial host than red clover.

GERMINATION OF THE SPORES ON THE LEAF,

While study was being made of the leaf-spot fungus in the host
tissue it was found that the mode of penetration could be observed
very readily by decolorizing the leaf soon after inoculation was
made. This method of study was used, not only to determine the nor-
mal penetration of these fungi into their own hosts, but to determine
the relation of these parasites to other closely related plants reputed
to be hosts of these fungi but upon which infection had not been
obtained. In case preliminary inoculations failed to give visible
results it was more simple and rapid to determine whether the spores
of that fungus could penetrate the host in question and develop after
penetration had taken place than to conduct other extensive inocula-
tions. Thus, a study of penetration has formed a part of all inocu-

LEAF-SPOT OF ALFALFA AND RED CLOVER. 23

lation trials. The results of the two methods of attacking host rela-
tionships should be considered together.

METHOD OF STUDY.

The most of the data given here have been obtained by the follow-
ing simple procedure: Leaves which have just reached full develop-
ment are selected for inoculation. A culture of Pseudopeziza known
to be discharging spores abundantly is supported over the leaf or one
of the leaflets so that the spores as they are discharged will all fall
upon it. The leaf may be removed from the plant for studies which
do not involve a period of more than two days, since results obtained
from such leaves have always been found by comparison to agree
with results obtained from leaves attached to the plant. The leaf
may be sprayed with very fine spray before the spores are discharged
upon it, but more abundant penetrations are usually obtained if the
spores are allowed to stick to the leaf before it is wet. When the
leaf has been kept moistened for at least 12 hours, usually longer,
it is removed, dropped into a mixture of equal parts of acetic acid
and alcohol and promptly heated to the boiling point. Leaves which
are killed promptly in this fashion decolorize in better condition than
when slower killing takes place in cold acetic alcohol. The acetic
alcohol is changed until all color has been removed from the leaf.

The leaf may then be mounted in this liquid on a slide under a
cover glass and examined under the microscope. The epidermal cells
should be perfectly clear, and the entire structure of the leaf to its
very center should be visible. The spores remain attached to the leaf
during the treatment, and the method of entry and the mycelium
within the leaf can be clearly seen. No method of staining has been
found to improve the visibility of the fungus.

Although this method works best in the case of alfalfa leaves, it
works well enough with the various clovers to give entirely satis-
factory results.

METHOD OF PENETRATION.

In all of the hundreds of penetrations observed the method of
entry has invariably been as here stated. The spore is found stuck
fast to the leaf. The germ tube emerges from the spore either within
or at the margin of the area of contact of the spore with the leaf and
passes directly through the cuticle into the epidermal cell. Occa-
sionally a spore sends out its germ tube along the surface of the
leaf, but such a germ tube has never been observed to enter the leaf.
Apparently the germ tube must enter the leaf at the moment of
emergence from the spore, if at all. Ordinarily there is no per-
ceptible thickening or alteration of the wall in consequence of this
penetration. The actual opening appears to be extremely minute.

24 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE.

After passing through the wall, the germ tube quickly expands to
normal size. When it reaches the center of the cell it usually divides
(fig. 5) into two or three branches, which pass into the adjoining
epidermal cells or down into the palisade layer. Cell walls do not
appear to offer any obstruction to advancing hyphe. No marked
disorganization of the cell contents appears to result. from this in-
vasion until hyphz become very numerous.

The actual time required for a spore to germinate and transfer its
contents to the germ tube inside the leaf has not been determined
accurately, but in most cases it must be less than 12 hours at 18° to
29°C.

This description applies to the method of penetration of Pseudo-
peziza medicaginis and P. trifolii in their respective hosts. The
following notes have been made of the penetration of germinating
spores of these
fungi in other
reported hosts
that have been
available:

Trifolium pra-
tense by Pseudo-
peztza medica-
ginis. — Inoculated
leaves were de-
colorized in two,
three and five
days after inocu-
lation. After
three days’ the
host cells beneath
many of the germinated spores had yellow granular contents. The yellow
color made the exact relation of the germ tube to the cell impossible to deter-
mine. In five days it could be observed that in the case of at least a part of
these yellowed cells the germ tube had passed through the epidermal cell wall,
but had not advanced far into the cell.

Medicago sativa by Pseudopeziza trifoliimA leaf inoculated on the plant on
March 28, 1915, appeared to show penetration in 60 hours. Five leaves inocu-
lated on December 4, 1916, and decolorized four days later showed many yel-
lowed cells beneath germinated spores. Suitable fragments were embedded
and penetrations found in sections. The germ tube had not advanced beyond
the first cell which it entered.

Medicago lupulina by Pseudopeziza medicaginis—Four series of leaves re-
moved from the plant were tried. Penetrations were noted in 23 hours at 22° C.
Penetrated cells always show yellow granular contents. Even after 75 hours
it was doubtful whether the germ tube had advanced beyond the first cell
penetrated.

Medicago lupulina by Pseudopeziza trifolii—One series of leaves was removed
from the plant. Penetrations were abundant and the penetrated cells yellowed,

(A

Fic. 5.—Penetration of the epidermis of an alfalfa leaf by the
germinating ascospores of Pseudopeziza medicaginis. X 800.

LEAF-SPOT OF ALFALFA AND RED CLOVER. 25

but even after 52 hours the germ tubes had not advanced beyond the ¢ell first
penetrated.

Melilotus alba by Pseudopeziza trifolii—Three days from inoculation a few
germ tubes were found distinctly inside epidermal cells, but they had advanced
but slightly. Penetrated cells were not yellowed.

Melilotus alba by Pseudopeziza medicaginis—The cultures used for inocula-
tion in this series did not produce many spores. Nevertheless, after four days
two penerations were found. The penetrated cells were very slightly yellowed.

Trifolium hybridum by Pseudopeziza trifolii—mNo examination was made
until two days after inoculation. At this time penetrations were abundant
and. easily seen. In a few cases germ tubes had advanced into cells adjoining
those first penetrated. No yellowing of penetrated cells was observed.

Trifolium hybridum by Pseudopeziza medicaginis.—In 24 hours penetrations
were abundant, but the germ tubes had not advanced far into penetrated cells.
Penetrated cells showed no yellowing.

It appears that the results which are shown above are exactly
parallel to the results obtained from inoculations. In every case
where infection in varying degree has been attained, penetration has
occurred abundantly and the growth of the hyphe within the host
cells has been rapid without causing discoloration of the cell con-
tents. In cases where visible infection has not been obtained, the
relative number of penetrations is usually reduced, but in any case
growth of the germ tube ceases promptly upon entering the epidermal
cell. Thus, the resistance which the plants that can not be infected
offer appears to be due not to any mechanical obstruction to entry,
but to something within the epidermal cell which prohibits growth.

These infection experiments supported by penetration studies have
failed to produce a completely successful infection of any of the
hosts tried except those from which the fungi were isolated. This
result is very different from that which was anticipated from a
consideration of the host lists. Have we here a group of closely
similar fungi highly specialized in their host relationships?

It is noteworthy that in both host lists only two or three of the
species of Trifolium are native to America. All the rest have been
introduced if they occur here at all. Moreover, some of these intro-
duced hosts which are widely distributed do not appear to be
attacked by this parasite except in certain limited areas. For ex-
ample, Pseudopeziza medicaginis has been found on Medicago lupu-
lina only in New York; P. trifolii has been reported on 7rifoliwm
repens only by McClatchie (1895) on the Pacific coast? and not at
all on 7. hybridum. It is not likely that these fungi have been over-
looked on these hosts in other localities, and therefore the conclusion
that they do not always pass to these hosts can hardly be escaped.
Thus, there is reason to suspect that these two species, as now re-

1 An excellent collection of Pseudopeziza on Trifolium repens now in the possession of
the writer was made by Mr. C. W. Hungerford at Olga, Wash., on Sept. 3, 1916.

26 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE.

garded, are made up either of several closely similar species or of a
group of specialized races.

Especially in the case of the Pseudopeziza on Medicago lupulina
the fragmentary evidence indicates a species distinct from that on
alfalfa. In addition to the evidence already given, the measurements
of 113 ascospores collected under standard conditions is adduced.

(Table VII.)

Taste VII.—Lengths of 113 ascospores of Pseudopeziza medicaginis on Medi-
cago lupulina, measured to the nearest micron.

Length (microns),

Ascospores of —
9 10 11 12 13

Pseudopeziza medicapimisse=-- 2. -4ses-- eee ae = Brae sc number. | 1 14 59 31 8

When Table VII is compared with Table II, it will be noted that
these spores are even larger than those of Pseudopeziza trifolii. Un-
fortunately, the cultures which were made of this fungus were de-
stroyed, and complete evidence of the relationship of these fungi was
not obtained.t Of the other hosts of P. medicaginis, less can be said.
Field observation indicates that the fungus in Medicago falcata and
M. hispida denticulata occurs by infection from Pseudopeziza in al-
falfa. A collection of the fungus has been found in only one in-
stance each on Melilotus alba and Vicia villosa. An examination of
a portion of the collection on Melilotus alba reveals a lesion that is in
all respects similar to that which the fungus causes on other hosts,
but no asci and spores by: which the fungus could be identified were
found. Certainly the occurrence of the fungus on these hosts is not
common or of economic importance. No material of Pseudopeziza
on Onobrychis sativa or any species of Trigonella has been available
for study. |

Of the host list of Pseudopeziza trifolii only a few species have
been available for study. The fungus from 7’rifolium pratense has
not infected any other species to produce fruiting of the fungus on
that host. Furthermore, it has not been found fruiting on any other
host in America except in the one instance already mentioned. Yet
reports well supported by herbarium specimens indicate that 7'ri-
folium repens and T. hybridum are abundantly infected in Italy.
Whether this infection is by the same species of the fungus or not
can not be determined at present.

1[It is of interest to note that Pseudopeziza medicaginis was first described on what is
now known as the variety wildenowii of Medicago lupuiina. If it should be shown that
Pseudopeziza on Medicago lupulina wildenowii is a distinct species from that on Medicago
sativa the name Pseudopeziza medicaginis will be restricted to the fungus on the original
host, and a new species name will be required for the fungus on alfalfa.

LEAF-SPOT OF ALFALFA AND RED CLOVER. Pad

LIFE HISTORY OF THE CAUSAL ORGANISM IN RELATION TO
THE HOST PLANTS.

AMERICAN STUDIES BEARING ON LIFE HISTORY.

The great economic importance of the alfalfa crop in America
has inspired the small amount of work which has been done upon
this disease with a point of view quite different from that shown in
the European studies already reviewed. Life history has been studied
with a view to the possible control of the disease.

The first work was done by Chester (1891) at the Delaware
Agricultural Experiment Station. In 1889 twenty plats of alfalfa
were seeded in different parts of Delaware, with seed from the
same source. The disease appeared on all of these plats at about the
sixth week after planting. The plat under closest observation at
Newark showed a yellowing of the leaves accompanied by black spots
before Pseudopeziza was found fruiting on some of the dark spots.
Evidently some other disease was associated with Pseudopeziza.
Chester concludes from this experiment that the disease is carried
by the seed and next tries a method of disinfecting the seed in order
to prevent such conveyance. Seed was treated with copper sulphate
and planted in heat-sterilized soil in cans. The diseases appeared
on all the plants from these cans. Therefore, Chester concludes that
the source of the disease must be a general atmospheric infection.
Unfortunately, in none of his experiments does he give any details
regarding the proximity of his plats or plants in cans of sterile soil
to alfalfa which was infected with the leaf-spot and which might
have been a source of wind-borne spores.

The only other attempt to study the disease which has been re-
ported was made by Coombs (1897a) in Iowa. On August 20 alfalfa
seedlings 3 weeks old grown under bell jars were treated as follows:
One was left as control, one sprayed with germinating Pseudopeziza
spores, and one sprinkled with powdered diseased leaves showing
disease. Next, plants growing in the field were cut back, and after
the débris was removed, the roots were protected by large bell jars.
In the next six weeks the plants outside the bell jars became diseased,
while those inside were healthy. However, when the jars were re-
moved the plants immediately became diseased. As a result of this
work Coombs concludes that two things are established: (1) That
plants are infected by spores from the air and (2) that the disease is
strictly local and not systemic.

It will be seen at once that these conclusions are based on a very
small amount of experimental evidence. Such important factors as
the high temperature and absence of dew or rainfall inside the bell
jars do not appear to have been considered as possible conditions

28 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE.

which might have prevented infection even had the spores been
present.

At this point, it is convenient to mention an English discussion of
this disease by Ivy Massee (1914) in which an effort is made to
throw light on the method by which the fungus is conveyed from one
locality to another. In this article Miss Massee states that so far as
England is concerned the dissemination of the disease is due to
diseased seed which is badly cleaned. She says, “I have recently
examined a sample of commercial seed and found the fungus present
in abundance on minute fragments of leaves and calyces, and rarely
on the seed itself.” Unfortunately, Miss Massee does not state the
methods by which she was able to make the identification of the
fungus with such certainty on this single sample of commercial seed.
Even if she was correct in this observation, it still remains to be
proved that the fungus which she found was alive and capable of
infecting the plants grown from this seed.

The many scattered observations of the disease merely contribute
data regarding distribution, seasonal occurrence, and environmental
factors. Most of these reports indicate that the disease is worse on
plants during the first summer of their growth than later, but
Coombs (1897) speaks of attacks as being worse after the first year.
Most reports indicate that the disease usually gains headway slowly
in the spring and becomes worse later in the season, but this is not
always the case. Stewart, French, and Wilson (1908, p. 384-387)
speak of the disease as being worse in dry years while most reports,
especially from drier regions, indicate that the disease is worse in
wet seasons.

The facts bearing upon the overwintering of the fungus in the
field are surprisingly meager. The only definite bit of observational
evidence is that of Chester (1891), who states that he found live
asci on leaves in midwinter. Voges (1909) suggests in addition that
the fungus survives the winter in living leaves.

After surveying these scattered references in American literature
we find that there is a general belief that Pseudopeziza medicaginis
is distributed with the seed and survives the winter on dead leaves.
This opinion has been reached, not so much as the result of careful
experimental evidence, which in fact ‘is meager, but more as the
cumulative effect of the expressed opinions of competent observers
who have watched its development during a series of years.

METHOD OF OVERWINTERING.

The first evidence of the method of overwintering of the leaf-spot
fungus in the field was obtained in the spring of 1916. On April 11
overwintered alfalfa leaves showing abundant Pseudopeziza spotting
were brought into the laboratory and placed in a moist chamber.

LEAF-SPOT OF ALFALFA AND RED CLOVER. 29

Two days later, when one of the apothecia was crushed out in water,
asci containing spores apparently mature were found. The leaf was
then supported over an agar surface. In two hours a large number
of spores were discharged, which germinated promptly. At this
time the young alfalfa shoots had hardly emerged from the mulch
of the débris of the previous season’s growth.

On May 6 spots began to appear on the alfalfa foliage of some of
the plats under observation. When the spotted leaves were decol-
orized, characteristic Pseudopeziza spores with germ tubes penetrat-
ing the epidermal cells were found in nearly all the spots.

A search of the overwintered foliage discovered a large number of
fresh-appearing apothecia developed on leaf areas that had been
diseased the previous year. When the overwintered leaves were
placed over agar plates, a large number of viable ascospores were
caught.

Fragments of overwintered leaves bearing apothecia were placed
over ten marked leaves of a healthy alfalfa plant in the greenhouse,
and the plant was kept in a moist chamber for 24 hours. On May 17
seven of the ten marked leaves showed more or less of the charac-
teristic Pseudopeziza spotting.

Five or six of these overwintered leaves bearing apothecia were
placed on the ground under a rank growth of alfalfa plants in the
greenhouse. The plants were then sprayed, but not covered to pre-
vent evaporation of water from the foliage. On May 21 the alfalfa
foliage in the vicinity of these overwintered leaves was found in-
fected with leaf-spot.

On March 31, 1917, overwintered alfalfa leaves bearing leaf-spot
lesions were collected in an alfalfa plat. At this time no spores
could be found in the apothecia. These leaves were kept in a moist
chamber for a week, care being taken to soak them in water twice
a day to remove the products of decomposition. At the end of the
week asci with mature spores had developed in the old apothecia.

Thus, it appears evident that Pseudopeziza medicaginis survives
the winter on diseased foliage which escapes decay. When the
weather becomes sufficiently warm in the spring and moisture is
provided by protracted rains or the shelter offered by the young
growing foliage, new asci develop apparently in the old apothecia
and, in addition, new apothecia are produced around the old one
(Pl. Il, A). The spores thus produced furnish the primary infec-
tion in the spring.

Apothecia producing spores indistinguishable from those of Pseu-
dopeziza trifolti were found on overwintered clover leaves in the
spring of 1916 in northern Wisconsin, but since no inoculations were
made with these spores, tlieir identity was not determined. However,

30 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE.

it does not seem unlikely that this fungus winters in the same way
that has been demonstrated for its close relative on alfalfa.t

METHOD OF DISTRIBUTION.

SUGGESTIONS FOUND IN THE LITERATURE.

Since the method of overwintering of these fungi has been traced,
it is now possible to see their entire annual cycle in a field in which
they have once been introduced. But thus far no information has
been gained which serves to indicate how they are conveyed into
new localities in which the host plants are grown for the first time.
This phase of the problem is of special importance in connection
with the alfalfa leaf-spot. A large amount of recorded experience
indicates that this disease appears wherever alfalfa is grown, re-
gardless of environment. A knowledge of the source of infection in
these new localities might suggest feasible control measures.

Scattered through the various discussions of the alfalfa leaf-spot
are found four suggestions that have been put forth to explain the
constant appearance of Pseudopeziza on alfalfa in newly seeded
fields: It is suggested (1) that the fungus is carried with the seed,
(2) that it is conveyed in soil that is used to inoculate the new field
with the bacteria producing nodules on the roots, (3) that the fungus
spores are generally distributed in the air, and (4) that other host
plants near by furnish the source of infection. Since none of these
suggestions are supported by carefully controlled experimental evi-
dence, they must be subjected to examination before they are used as
working hypotheses in experimental work.

The first suggestion, that the fungus is carried with the seed, de-
serves careful attention. This might happen in three ways. Spores
might adhere to the seed coat, spores or fragments of the fungus
might accompany the seed, and living mycelium of the fungus might
oecur within the seed. When the conditions under which seed is
produced are examined it is found that the fungus spores are prac-
tically all discharged and blown away before the seed is thrashed,
thus making it highly improbable that spores are attached to the seed
except as a rare occurrence. Commercial seed is so well cleaned that
there appears to be small chance that fragments of the fungus are
often conveyed with the seed.

The possibility that fragments of the fungus as well as the spores
may be carried with the seed appears unlikely in the case of most
commercial seed. Débris consisting of plant parts is so hght in com-
parison with the seed that it is easily removed. Nevertheless, Ivy
Massee (1914) states that she has examined commercial seed and

1On April 6, 1919, apothecia of Pseudopeziza trifolii were found abundantly on living
overwintered clover leaves at Madison, Wis., showing clearly that young leaves infected
late in the autumn under favorable conditions may carry the fungus over winter.

LEAF-SPOT OF ALFALFA AND RED CLOVER. 31

found the fungus present. This can hardly be a common occurrence
in America.

The possibility that the fungus may be present in the seed as
mycelium is open to the objection that this involves a larger or
smaller amount of systemic infection, both of the plant producing
the seed and of the seedling. No evidence of such a relation of these
fungi to their host plants has been found, and therefore this method
appears highly improbable.

The second suggestion, that the fungus is conveyed with plant
débris that accompanies soil which is transported to new fields, may
and probably does account for a small amount of the distribution of
the fungus. But owing to the restricted extent of this practice, this
method must be of minor importance.

The third hypothesis, that spores of Pseudopeziza are generally
distributed in the air, has been advanced several times in a vague
way either in a discussion of conditions where large areas of dis-
eased alfalfa were growing at no great distance or with an implied
belief that some other host plant in the vicinity was the source from
which this general infection arose. In the vicinity of areas of dis-
eased alfalfa it is highly probable that spores are borne to a consid-
erable distance by wind, but it is not often that the spores are pro-
duced so abundantly that they are likely to be conveyed great
distances in large numbers.

The final hypothesis, that other hosts provide the source of infec-
tion, has been rendered less probable by results already presented.
The only common hosts that can be considered are red clover and yel-
low trefoil (ledicago lupulina). Pseudopeziza on red clover appears
to be a distinct species from that on alfalfa, and no evidence has
been obtained indicating that the fungus on yellow trefoil can cross
to that host. ,

Thus, a summary of the available evidence does not point clearly
to any of these suggestions as the one most likely to contain the
truth. However, the suggestion that the fungus is carried with the
seed affords most opportunity for experimental study and, if found
true, affords the greatest opportunity for the application of control
measures. The following experiments in seed sterilization were car-

ried out.
EXPERIMENTAL METHODS AND RESULTS.

Laboratory experiments.—If the fungus spores are carried adherent
to the seed they must inevitably germinate upon the seed coat and
produce apothecia there, if at all. In order to determine to what
extent the fungus is capable of developing upon the seed coat, spores
were discharged upon seeds sterilized with formaldehyde. The seeds
were then germinated upon agar in test tubes. After a time minute

32 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE.

fungous colonies were found developing upon the seed coats, whether -
they remained attached to the cotyledon leaves or fell to the agar.

In no case were the cotyledon leaves attacked. Subsequent attempts

to infect cotyledon leaves of seedlings did not produce macroscopic

lesions. The seed coats bearing the minute fungous colonies were

subsequently transferred to fresh agar slopes to keep them moist, and

finally they developed minute apothecia. This development was so

slow, however, that it is doubtful whether it could proceed so far

under field conditions, where periods of drying would occur and com-

petition with other fungi would be encountered.

Efforts to grow the fungus on soil sterilized or unsterilized have
been entirely unsuccessful. |

If the fungus occurs on the outside of the seed or in débris it can
easily be destroyed by the surface sterilization of the seed. If such
treated seed can then be grown under conditions which will exclude
other sources of infection and which will also be favorable for the
development of the fungus, the occurrence of the disease will indi-
cate that the fungus is carried within the seed.

A satisfactory method of seed sterilization has been worked out
by Mr. A. H. Gilbert (in an unpublished manuscript). He found
that treatment with a solution of 1 part of bichlorid of mercury in
1,000 parts of water for five minutes rendered the seed sterile, while
treatment for 10 minutes injured the seed. These treatments were
repeated, and it was found that treatment for eight minutes was
more than sufficient to render the seeds sterile without injury, pro-
vided they were washed promptly after treatment, All sterilized
seed mentioned in the experiments here described were treated in
this way.

Suitable conditions for growing the treated seed were difficult to
obtain. Two places were tried—in the greenhouse during the winter
months and in the open field in localities as remote as possible from
other alfalfa. Experience in the greenhouse in the winter of 1915
showed that unless great care was taken with infected plants, the
fungus was likely to occur occasionally on other alfalfa plants in the
same house. During the following two winters all inoculated plants
were cared for so thoroughly that in not a single instance did the leaf-
spot develop upon any other plant in the houses until the disease
appeared in the fields outside in the spring. Alfalfa plants grown ©
close to red clover infested with Pseudopeziza trifolit remained free
from leaf-spot. The following greenhouse-plat trials were made:

(1) Four grams of sterilized alfalfa seed were exposed to a discharge of
ascospores of Pseudopeziza medicaginis for three days before sowing in the
garden greenhouse on March 38, 1916. Thousands of viable spores must have

been attached to the seeds at the time they were sown. By April 15 the plants
were 6 inches tall and very vigorous. When the experiment was discontinued

LEAF-SPOT OF ALFALFA AND RED CLOVER. 33

on May 10 no trace of Pseudopeziza had appeared. The plants were then 10
to 12 inches tall, very vigorous, and in a dense mat apparently favorable for
the development of the fungus. At the end of this experiment the plat was
inoculated with Pseudopeziza from overwintered leaves and was quickly over-
run with the disease, showing that the greenhouse conditions were favorable
for its development.

(2) On January 27, 1917, a plat about 4 feet square was sown in the garden
greenhouse with unsterilized Kansas-grown alfalfa seed. This plat developed
normally without leaf-spot until May 5, when the disease was present in the
field outside.

(3) On February 16, 1917, a plat 2 by 3 feet was sown in a garden green-
house with sterilized alfalfa seed 3 years old. This plat likewise developed
normally with no leaf-spot until May 10, at which date leaf-spot was abundant
outside the greenhouse.

Several other plats were started and developed in the green-
house without leaf-spot, like those referred to above, but owing to
insect injury the conditions were not as favorable for the develop-
ment of the disease as those described. In fact, all greenhouse plats
started from seed, whether sterilized, unsterilized, or even treated
with spores before sowing, have developed without the appearance
of leaf-spot until the disease occurred abundantly in an alfalfa
field close outside the greenhouse.

Field plats.—In the selection of locations for’ plats three conditions
were sought: (1) Remoteness from large areas of growing alfalfa;
(2) the greatest possible distance from farms where alfalfa has been
grown; and (8) accessibility, so that a visit to the plat would be
possible. The second condition was very difficult to secure. Small
plats of alfalfa are surprisingly abundant even in localities where it is
not grown as a farm crop. In consequence of this fact, only one of
the eight plats started in 1915 was found upon examination to be
sufficiently remote to give results of value.

In 1915 the assistance of the States Relations Service secured the
cooperation of several agricultural county agents whose intimate
knowledge of local conditions made possible the selection of a larger
number of suitable locations. To these men the writer is indebted
for any degree of success that was attained in these experiments.

The seed which had been sterilized superficially was furnished to
the agricultural county agents, who allotted it to the men on whose
farms the plats were to be located. In the autumn all the plats
were visited except the one at Bruce, 8. Dak., which was under the
observation of Dr. A. G. Johnson, and the presence or absence of
leaf-spot was determined. In a number of cases alfalfa was found
growing nearer the plat than was previously supposed. The results
noted on such plats—always an abundance of leaf-spot—are excluded
from the summary in Table VIII. However, if, as sometimes oc-
curred, the near-by plants were very few in number and no other

34 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE.

plants were known to exist within a 5-mile radius the results have
been included.

TABLE VIII.—Summary of data of plat tests to determine whether leaf-spot can
be prevented on alfalfa sown in isolated localities by the superficial steriliza-
tion of alfalfe seed.

she ne res plstenee)
J ate ate Stes oneares
State and town. sown, visited. CEar alfalfa Leaf-spot,
: (miles),
Maine: 1915, 1916,
IN IGY R612) ORS es PE oe ere er Rear oie June 28 | June 14 1 6 | Present,
1916. =
VAN DIONE sae sees costa as eens ae eee eee May 6 | Sept. 12 20 3k Do.
AITTIOLGN S40 eter eis 2 ete esse eee JUNE Sees es donee. 40 5 Do.
Gorham ts oa... -- a deseise teciesieeebie cesar May 20] Sept. 5 1 3 Do,
(Eigrrisons see). 28 te CaS ee ee re Phere June 1 OlzE-= 2 34 | Doubtful.
IMaSSHUDOLO DS sas ah oto cet i cecen tee tonne May...-- Sept. 6 40 5 | Present.
Windsore. Set. Gec sf tecee Pec et Sec oeee June....| Sept. 12 40 5 Do.
South Dakota:
IBTHCeR. So22 SoS eRe ee Oia 2 ba 2 May 20} Aug. 11 40 5 Do.
Wisconsin:
Doering ....... sree ok See oe ck te Se eee eee June....| Oct: 2 40 10 | Doubtful.
IME CONN OLAS sna nase eae Seana eee ae May....- Oct. 4 10 3 | Present,
RadiSsSOnes-fe. -c- fia. 58epe rate tees oe cer June s-|--- Wozee a 40 3 0.
Merrill! 7.82 7). oe nie soe cet yoet ster eee eer ae LO omaee Oct. +2 40 5 Do.
Dorteee sa j3cb =. sess chee eee Beto bo sacres le doees-s 40 Do
SCRE ERCEROESEStSCS: jtaedeeoucnasas --do. Goze. 40 124 Do
Tomahawk. ses see ae eee eee ciiaia-c ase eet Be 0 Ke ae ee dos: 40 10 Do
Ce cece arta cle Sarre es Ac se Obc reel eet Gece 40 10 Do
WGITPOD Ss ccm secs cae e-em asGh aces ciameece dora Oct. 5 40 5 Do

From Table VIII it appears that the one small well-isolated plat
started in 1915 did not develop leaf-spot until the following year.
Of the 16 plats started in 1916 only 2 failed to show an abundance
of leaf-spot in the autumn of the same year. One of these plats,
located at Harrison, Me., was in very poor condition, only a few
scattering spindling plants about 4 inches high being found. How-
ever, other plats in almost as poor condition showed leaf-spot. The
second doubtful plat, at Doering, Wis., was in very vigorous con-
dition, but it had been cut just previous to inspection, leaving very
little foliage. Unfortunately, it was not feasible to .revisit the plat
the following year.

These results are in accord with previous experiments and experi-
ence. Surface sterilization of seed apparently accomplishes nothing
in excluding leaf-spot from alfalfa fields. But these results do point
very clearly to one conclusion, that the leaf-spot fungus is not car-
ried on or in débris mixed with the seed. The greenhouse experi-
ments, which are only suggestive because of their limited extent,
indicate that the fungus is not carried within the seed.

Thus, in conclusion, it is necessary to say that no positive evidence
pointing toward the method by which this disease gains access to
remote alfalfa fields has been found. Evidence has been obtained
which apparently eliminates other plants previously under suspicion
as host plants of the fungus from consideration as sources. A lim-

LEAF-SPOT OF ALFALFA AND RED CLOVER. 35

ited amount of evidence indicates that the fungus is not carried
with the seed. Yet the fact of the almost universal occurrence of the
disease in remote localities is well established. The explanation of
this fact still furnishes a very interesting and apparently very
difficult problem.

SUMMARY.

(1) One of the most important diseases, if not the most important
foliage disease, of alfalfa is the leaf-spot caused by the fungus
Pseudopeziza medicaginis (Lib.) Sace. A similar but less important
leaf-spot of red clover is caused by the fungus Pseudopeziza trifolii
(Biv.-Bern.) Fekl. The morphological differences between these
fungi are so slight that doubt has frequently been expressed whether
they are not identical. Several conflicting opinions as to the life
histories of these fungi are found in mycological literature. This
study attempts to determine the relationship of the two fungi here
mentioned and to trace as far as possible their life histories in rela-
tion to their host plants.

(2) Pseudopeziza medicaginis on alfalfa and Pseudopeziza trifoli
on red clover have been obtained and studied in pure culture. Efforts
to cross these fungi from one host to the other have not been success-
ful. Morphological as well as physiological differences have been
found which in the opinion of the writer justify retaining the fungi
as distinct species.

(3) None of the imperfect fungi which have been regarded as a
stage in the development of these fungi have been found to be re-
lated. Apparently no other spore form than the ascospore occurs
in nature.

(4) Infection is produced by the direct penetration of the germi-
nating ascopores through the cuticle and epidermal cell wall of the
leaf. The mycelium developing into a small stroma about the point
of entry produces in about two weeks an apothecium.

(5) The fungus lives over winter on dead leaves which escape
decay, and ascopores produced in the spring furnish the source of
new infection.

(6) Efforts to exclude the disease from alfalfa fields sown in
localities remote from other alfalfa by the surface sterilization of
the seed have given no degree of success. Evidently, in these experi-
ments at least, the fungus was not carried on the surface of the seed—
probably not with the seed at all. The demonstration of the source
of infection in such fields still furnishes an interesting problem.

LITERATURE CITED.

BERTHAULT, PIERRE,
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BIvONA-BERNARDI, ANTONIO.
1813-16. Stirpium rariorum minusque cognitarum in Sicilia sponte pro-
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p., 14 pl. Panormi. '

BLASDALE, W. C.
1902. Fungous parasites. Jn U. S. Dept. Agr., Bur. Plant Indus. Bul. 12,
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1869. Mémoire sur les Ascoboles. Jn Ann. Sci. Nat. Bot., s. 5, v. 10, p. 191—
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BREFELD, OSCAR.
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1888. Esperienze per combattere la Peronospora della vite eseguite nell’
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