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PODBLIGHT OF THE LIMA BEAN CAUSED BY
DIAPORTHE PHASEOLORUM

By L. L. Harter,

Pathologist, Cotion, Truck, and Forage Crop Disease Investigations, Bureau of Plant
Industry, United States Department of Agriculture

INTRODUCTION

The Lima bean (Phaseolus lunatus L,.), compared with some of the other
truck crops, is relatively unimportant from a commercial standpoint. It
is grown in a small way in the garden by nearly every farmer, and on a
commercial scale in a few sections of the United States, particularly
along the Atlantic seaboard and along the Pacific coast. Like many
other crops grown only for home use or on a commercial scale in a limited
area, its several pests and diseases have either been overlooked or ignored.
The Lima bean, however, has its share of diseases, some of which have
been fairly well studied, while little is known about others. To the
latter class belongs the podblight, a disease of considerable economic
importance some seasons in commercial fields. It was first discovered
by Halsted (76)' in 1891, in New Jersey. Further than this nothing is
known as to the time and place of the origin of the disease.

There is no evidence from the search of the literature that the disease
occurs outside of the United States, although it is impossible to state
definitely that it does not, in view of the great number of species of
Phoma, Phyllosticta, and closely related genera under which the casual
fungus may have been described. A careful search through the descrip-
tions of species of the genera Phoma, Phyllosticta, and Phomopsis on
different species of Phaseolus has revealed none located in foreign coun-
tries identical with the organism causing the podblight. It therefore
appears safe to assume at the present time that the disease is indigenous
to the United States.

To judge from published accounts, Halsted (16) was the first to recog-
nize this disease and found it causing much damage to the pods of pole
Lima beans in New Jersey in the fall of 1891. He attributed it to a
species of Phyllosticta, pointing out at the same time that large blotches
were also produced on the leaves. That he studied the disease with some
care and did not content himself with a few passing observations is
evident from the fact that he found that the seeds from infected pods,
when placed under a suitable environment, such as between moist cloths
or paper, developed the fruiting bodies of the fungus.

1 Reference is made by number (italic) to ‘i‘ Lterature cited,’’ p. 502-504.

Journal of Agricultural Research, Vol. XI, No. 10
Washington, D.C. Dec. 3, 1917
kx Key No. G—128

(473)

474 Journal of Agricultural Research Vol. XI, No. x0

In 1892 Ellis and Everhart (15, p. 158) evidently collected the same
organism on the pods of Phaseolus lunatus at Newfield, N. J., and de-
scribed it as a new species of Phoma, Phoma subcircinata, pointing out
that it differed from Phoma leguminum West. in the subcircinate arrange-
ment of the pycnidia and the rather larger binucleate sporules. Halsted
later apparently concurs with Ellis and Everhart that this is a new
species of Phoma, for in 1901 he (17) refers to the disease again by that
name and enlarges his earlier description. ‘Two illustrations are ap-
pended to his article, one showing the characteristic appearance of the
disease on the pods and the other on the leaves, both typical of the trouble
as we know it at the present time.

DISTRIBUTION, PREVALENCE, AND LOSS

For 14 years after this disease was found in New Jersey it had either
not appeared in any other State or had not been discovered. In 1905,
however, Clinton (5, p. 265) reports a leafspot of Phaseolus lunatus at
New Haven, Conn., apparently the same as that described and illustrated
by Halsted. He states that he did not observe the disease on the pods,
but adds that it had not been looked for there. Although agreeing
macroscopically with Halsted’s description of the disease on the leaves,
Clinton entertained some doubt of its identity with Phoma subcircinata
E. and E. because of the fact that the spores averaged larger (5 to 12
by 2.5 to 3.5 uw) and were occasionally septate. He suggests the pos-
sibility that the fungus may be Ascochyta phaseolorum Sacc.

In 1912 Cook (6, p. 517-518) reports a severe outbreak of a disease of
pole Lima beans in the State of New Jersey caused by Phyllosticta sp.,
which occurs on leaves and to some extent on the podsand stems. The
following year it was less severe (7, p. Sor), but in 1914 he (8, p. 472)
reports the disease again as causing much damage, attributing it to
Phoma subcircinata FE. and E. A leafspot caused by Phyllosticta phaseo-
lina Sacc. was also commonly met with.

In view of the relatively few times this disease has been reported
during a period of 25 years, it is evident that it has generally been either
overlooked or disregarded. ‘This is probably due to the fact that the
crop is relatively a minor one and that the disease appears usually after
the low prices do not justify further pickings. It, however, is much
more widely distributed than the published reports indicate. It was
reported (unpublished) to the Plant Disease Survey from one locality
in North Carolina by R. H. Fulton in 1913. The writer received and
studied specimens of badly infected plants from Maryland in 1914 and
1915, and from Virginia in 1915. During the seasons of 1914 and 1915
the disease also caused heavy loss to the crop in New Jersey. In con-
clusion, it may be stated that the disease is rather widely distributed
along the Atlantic seaboard and causes considerable damage to the crop.

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Dec. 3, 1917 Podblight of Lima Bean A75

DESCRIPTION OF PODBLIGHT

Although the ascogenous stage of this fungus has been connected by the
writer with the pycnidial stage, the latter is alone concerned in the injury
to the crop in the field. A macroscopic description of this disease there-
fore will be confined to the pycnidial or imperfect stage. In the field
under suitable conditions the disease first appears on the leaves of plants
from 1 to 2 feet high. Wet weather is conducive to the spread and warm
weather promotes the development of this as well as many other dis-
eases of this type. Serious outbreaks have often occurred immediately
following a few days of rainy, warm weather in fields where previously
the disease was scarcely noticeable. In such a case the loss is often se-
rious; but if dry weather follows new growth of leaves and fruit will be
produced comparatively free of the disease. If, however, the weather
continues wet, a new outbreak ‘is liable to occur.

The leaves function as host for the fungus during the earlier part of the
season, from which it spreads later to the pods. Large, subcircular,
brown, often bordered patches are produced on the leaves (Pl. 42, A).
These patches, varying in size naturally with age, often attain a diameter
of 1 to 3 cm. Infection is not restricted to any portion of the leaflets
but the regions bordering the midrib are most frequently attacked. The
fungus spreads more or less in all directions from the point of infection,
but may often be delimited by a large vein or midrib. In this moribund
tissue, or in tissue soon after it is killed, the fruiting bodies (pycnidia) of
the fungus break through the epidermis. They are arranged more or
less concentrically, and appear first as gray or grayish raised pimples,
which later darken and become nearly black. They therefore may be
seen as conspicuous, minute black specks on the upper surface of the
leaves standing apart, sometimes confluent, on a brown background..
The dead tissues finally become dry and fall out, leaving a ragged hole.

This disease must not be confused with a leafspot of Lima beans and
other varieties of beans, as well as of cowpeas, caused by Phyllosticta
phaseolina Sacc., the spots of which are smaller, more nearly round, and
the pycnidia smaller and fewer in number.

The disease appears on the pods usually the latter part of July or early
in August, at a time when the vines have nearly reached their full devel-
opment. Under field conditions it is not certain at what stage in the de-
velopment of the pod infection takes place. Young pods are rarely
found diseased, and inoculation experiments have shown that they are
infected less easily than ones nearly matured. The disease progresses
rather slowly, requiring a week or ro days after inoculation to produce a
spot of any size or for the production of pycnidia. The growth of a bean
pod is comparatively rapid, and it is likely that infection may take place
when the pods are young, the fungus becoming visible as the pod ap-
proaches maturity. Infection occurs at any point on the surface of the

476 Journal of Agricultural Research Vol. XI, No. ro

pod, but more frequently at or near the ventral suture (Pl. 42, B). If
infection occurs near or at the dorsal or ventral suture, the fungus spreads
in all directions in a circular or semicircular manner, the hyphe growing
among the cells of the pod below the epidermis. The extent of the hyphal
growth of the fungus among the cells is indicated by the darkening of the
pod above the invaded area, evidently the first signs of the exhaustion of
the food supply and approaching death of that part of the pod. This is
soon followed by numerous, minute elevations over the surface, the points
where the pycnidia are forming. These lifted points are at first gray, but
as soon as the pycnidia break through the epidermis they become nearly
black and protrude in a domelike manner from the surface. They are
often, though not always, arranged concentrically and stand apart; or two
or more may be confluent in a cluster or chainlike manner. Upon the
death of the pod the fungus grows rapidly through the tissues, the
pycnidia breaking out more or less over the entire surface (Pl. 42, C).
The fruiting bodies which are formed subsequent to the death of the pod
are usually not formed concentrically.

Under field conditions the fruiting bodies of the fungus are often found
on the dead and more rarely on moribund stems. It is exceedingly
uncommon to find typical infections of living stems, and the writer has
never been able to produce infection of living stems artificially. The
pycnidia, however, appear abundantly after the death of the vine on
stems that have been sprayed with spores of the fungus.

The disease is much more common and destructive on the pole Lima
beans than on the bush Limas.

ETIOLOGY

The podblight of Phaseolus lunatus is caused by a fungus pathogen to
which various names have been given at different times. The use of
different names is largely due to the lack of a knowledge of the life his-
tory of the fungus and to the practice of many mycologists in separating
the genera Phyllosticta and Phoma, respectively, according to whether
the fungus occurs on the leaf, or on the stem, or on other parts of the
host.

Halstead (16) first reported this disease in 1891 as causing considerable
damage to the pods and leaves, and attributed it to a species of Phyllo-
sticta. He was evidently in doubt as to the species, for he does not
refer it to Phyllosticta phaseolina Sacc. (30, p. 149), which had been
described some years earlier and which occurs commonly on Lima beans,
on Phaseolus diversifolius, Kansas (Kellerman), New Jersey (Ellis),
Canada (Dearness) on Phaseolus perennis, Missouri (Galloway) and on
cowpea, Kansas (D. B. Swingle). In 1905 Smith (33) proved the patho-
genicity of this organism on the leaves of Lima beans and other varieties,
such as kidney and wax, and on cowpeas. He found that the pods
were not affected.

Dec. 3, 1927 Podblight of Lima Bean 477

That the writer has been working with the same fungus which Ellis
and Everhart described as Phoma subciycinata there is little doubt.
Material, collected by Dr. J. B. Ellis at Newfield, N. J., and identi-
fied by him as Phoma subcircinata, has been examined and found
to be identical with material collected at Vineland, N. J., only 4
miles distant from Newfield. It should be mentioned in this connec-
tion that the material from which Ellis and Everhart made their de-
scription was likewise collected at Newfield, in 1892. In 1890, three
years before Ellis and Everhart’s description of Phoma subcircinata
appeared (75, p. 158), Ellis collected and identified as Phyllosticta
phaséolina some material from Newfield which upon examination the
writer has found to be identical with material later identified by him
(Ellis) as Phoma subcircinata and also identical with the material the
writer has been studying.

Phoma subcircinata EF. and E. was described by Ellis and Everhart
(15, p. 158) as follows:

On pods of Lima Beans, Newfield, New Jersey, October, 1892.

Perithecia subcuticular, 70-90 » diam., sublenticular, subconfluent pierced above,
membranous, black, subcircinately arranged in large (1 cm.), round, faintly zonate
spots, finally spreading and occupying the entire surface of the pods. Sporules
oblong-elliptical, hyaline, 2-nucleate, 5-6 by 2-2.5 u, on simple basidia rather larger
than the sporules.

This description fits perfectly the fungus the writer has been studying,
with the exception of the size of spores and pycnidia. An examination
of the material collected and identified by Ellis as Phoma subcircinata
shows the spores to be somewhat larger (6.4 to 8.0 by 2.4 to 3.6 w, average
7.4 by 2.95 uw), and the pycnidia considerably larger (142 to 276 w, average
185.8 »). Material collected and identified by Ellis three years earlier as
Phyllosticta phaseolina and which he probably later took for Phoma sub-
cwcinata, bore spores (6.0 to 8.0 by 2.8 to 3.2 w, average 7.2 by 2.88 yp)
and pycnidia (197.5 to 260.0 p, average 219.0 pw) of about the same size.
On material which the writer has collected and from which isolations
have been made for inoculation work the spores varied from 6.0 to 8.6
by 2.4 to 4.1 w, average 7.50 by 3.23 mw, and the pycnidia from 158.0 to
475.0 m, average 245.86 yw. From the data at hand it appears evident
that the fungus described by Ellis and Everhart as Phoma subcircinata
is the same as the one the writer has been studying.

In view of the fact that the fungus causing podblight has been con-
nected with a perfect stage, the genus to which the imperfect form should
properly belong is of no great consequence, but it may be of interest to
know that this fungus, like a number of others which have been classed
as Phoma, belongs to the form genus Phomopsis. In morphological
structures the podblight fungus is identical with similar structures of the
genus Phomopsis as laid down in Diedicke’s (z3) revision of the group—

478 Journal of Agricultural Research Vol. XI, No. 10

that is, chambering of the pycnidia, formation of sclerotial stroma,
presence of pycnospores and stylospores, and structure of the pycnidial
wall. No stroma is formed by this fungus on the leaves. If this fact
should be disregarded and Saccardo’s classification followed, it should be
classed as a Dothiorella ora Fusicoccum. Stylospores have been found to
be abundant in most of the material collected and studied, but none were
found in the herbarium material collected and identified by Ellis as
Phoma subcircinata. However, isolations have been made from material
which did not bear stylospores but developed them in cultures later,
which shows that, although not always present, the fungus may pro-
duce them. On the other hand, the organism isolated from specimens
bearing stylospores do not always produce them in culture. This fun-
gus is very similar to a large group of other organisms which have long
been classed as Phoma. In a taxonomic study of the group Diedicke
(73) transferred a number of species of Phoma to the genus Phomopsis.
The podblight fungus does not differ essentially morphologically from
the conidial stage of Diaporthe batatatis (EK. and H.) Harter and Field,
which was first described as Phoma batatae but found later by Harter and
Field (2r) to be aspecies of Phomopsis. It is similar also to the organism
causing fruitrot, leafspot, and stemblight of eggplant, which was known as
Phoma solani on the stem and fruit and as Phyllosticta hortorum on
the leaves, both of which were found by Harter (20) to be caused
by the same organism and to belong to the form genus Phomopsis.

Some years before Phoma subcircinata was described by Ellis and Ever-
hart, Cooke and Ellis (9, p. 93) described Sphaeria (Diaporthe) phaseo-
larum occurring on bean stalks as follows:

Gregaria, tecta. Peritheciis globosis, immersis, minimis. Ostiolis spinaeformibus,
atris, erumpentibus, Ascis clavatis. Sporidiis lanceolatis, quadrinucleatis. Spo-
ridia 0.016 mm.

In the original description no type location was given; neither is it
apparent in this or subsequent descriptions whether the fungus was found
on Lima beans or other varieties. In the absence of these facts, together
with the imperfect description, it would be difficult to identify correctly
the fungus if it had not been more fully described later by Ellis and
Everhart (14, p. 460) as follows:

Diaporthe phaseolorum (C. & E.).
Sphaeria phaseolorum, C. & E. Grev. VI, p. 93.
Diaporthe phaseolorum, Sacc. Syll. 2635, Cke. Syn. 2423.

Exsicc. Ell. N. A. F. 188.

Perithecia gregarious, buried, very small. Ostiola spine-like, slender, projecting
for 4-4 mm. Asci clavate, 30-35 by 6-7 u. Sporidia biseriate, oblong-lanceolate,
4-nucleate, scarcely or only slightly constricted, 10-12 by 3 mu (16 uw long, Cke.).

On decaying bean vines left exposed through the winter, Newfield, N. J. Mostly

around the nodes of the stem, the surface mostly blackened and the stroma limited
within by a black line.

Dec. 3, r9r7 Podblight of Lima Bean 479

The writer collected a quantity of Lima beans (pods and stems) at
Vineland, N. J., in October, 1914, and January, 1915, which bore an
abundance of pycnidia of the podblight fungus, generally known as
Phoma subcircinata E. and E. After isolating the causal fungus the ma-
terial was wintered out at Washington, D. C., and covered with leaves and
dirt. On June 2 perithecia of the genus Diaporthe were found to be
abundant on stems and pods of this material. ‘The perithecia were
scattered and buried in the tissue of the host except the beaks which
were long and projecting and somewhat bent as shown by Plate 43, D.
A pycnidial fungus was isolated from single ascospores which was iden-
tical with the one isolated from the material before it was wintered out
Subcultures of this strain were used in inoculation experiments and pro-
duced infections identical with that produced by subcultures from iso-
lations of single pycnospores. ‘This strain (known as 598), as well as
others isolated directly from pycnospores, produced stylospores. ‘The
perithecial stage has never developed in culture.

The perithecia produced on the wintered-out material varied in size
from 158.0 to 355.5 mu, average 251.9 w; the asci 28.0 to 46.2 uw by 5.2 to
8.0 pw, average 37.4 by 6.73 uw; the ascospores 6.4 to 12.0 u by 2.3 to 4.0 y,
average 9.5 by 2.93 u. ‘The perithecia of type material varied in size
from 158.0 to 237.0 uw, average 215.6 w; the asci 28.0 to 44.0 pw by 4.8 to
8.0 mw, average 33.6 by 7.0 uw; the ascospores 8.0 to 12.0 by 2.4 to 4.0 yn,
average 10.0 by 3.3 u. From the fact that the ascospores, asci, and peri-
thecia together with other morphological characters of the two organisms
are practically identical, it is concluded that they are the same fungus and
should be known as Diaporthe phaseolorum (C. and E.) Sace.

MORPHOLOGY

PYCNIDIAL STAGE

From the point of infection the hyphe grow in all directions, invading
practically all classes of cells except the bast, a layer of which (fig. 1,
A, B, d) is found just below the epidermis. Figure 1, A and B, c, shows
parenchyma cells traversed by the hyphz. Soon after the death of the
cells the pycnidia begin to form, and develop very rapidly thereafter.
The hyphal growth increases in the cavities below the stomata or in the
intercellular spaces. From here it passes up between the bast cells which
are often somewhat separated beneath the stomata. As growth increases,
gnarls of hyphez accumulate under the stomata and between the
epidermis and the bast cells (fig. 1, A, B, b). From this point the fungus
spreads out just beneath the epidermis and develops a more or less
circular plate, the base of which rests on the bast cells. Figure 1, A, shows
an early stage in the development of the pycnidial plate. As the fungus
growth continues, the epidermis is gradually elevated (fig. 1, B, a) as the

480 Journal of Agricultural Research Vol. XI, No. 10

result of the pressure below. Definite cell layers of the pycnidium are
laid down about this time or a little earlier. The pycnidium completes
its full development soon thereafter (Pl. 43, B).

The inner layer of the pycnidium is composed of hyalin cells from
which the conidiophores arise. A slight cavity is formed at the center
concomitant with the release of pressure by the rupture of the epidermis.
Inclosed in this cavity numerous conidiophores are developed, extending
toward the center from which the spores are cut off. As the spores
increase in quantity, the cavity enlarges by the pushing back of the cells
lining the cavity and probably by further lifting of the upper part of the
pycnidium. It has not been possible to determine definitely when the
opening from the pycnidial cavity to the outside through’ the beak is
formed, but it probably takes place only after the pressure within has been
greatly increased by the continued production of spores. It is believed,

Fic. 1.—Diaporthe phaseolorum: A, Au early stage in the formation of a pycnidium. ‘he epidermis (a)
is not yet disturbed but the hyphe (6) have accumulated directly beneath it. Note the coursing of
the hyphe through the parenchyma cells (c). 130. B, A more advanced stage of figure A. Note
the bulging of the epidermis (a). X130.

however, that their expulsion is largely brought about through the ab-
sorption of water by the gelatinous mass in which the spores are em-
bedded, a condition which has been found by Stewart (34) to occur for
Guignardia aesculi (Pk.) Stewart.

The writer does not wish to leave the impression that the pycnidia
develop only in the stomatal chambers, but that this is a common and
natural place for their formation there can be no doubt. Even in young
pycnidia it is not always easy, and sometimes is impossible, to tell
whether or not they were being developed just beneath a stoma. The
bast cells of the pods of the Lima bean are very thick walled and are
generally pressed so close together that it would be difficult, if not impos-
sible, for the fungus to pass between them. These cells, however, are
separated at certain places, and often just beneath a stoma, so it would
be logical that they should form in such a cavity. From these facts it is
evident that, in many cases at least, the pycnidium is developed beneath
a stoma, and also at such other places where the bast cells are separated.

Dec. 3, 1917

Podblight of Lima Bean 481

The pycnidia vary greatly in size (158.0 to 475 mw, average 245.86 p

from host).

They are irregular in shape, chambered with an inner

hyalin layer from which the conidiophores arise, and a dark outer layer
much thickened and darkened above (PI. 43, B, C), all characters typical

-of the genus Phomopsis. Sometimes the contiguous sidewalls of two or
more pycnidia are ruptured, forming a single cavity (Pl. 43, E). On the
other hand, they may be pressed together but separated by a single dark
wall and their respective hyalin inner layers, thereby

forming a chain of pycnidia (Pl. 43, C). The pye-
nidia can hardly be said to be embedded in a true

stroma.

The pycnidia on the leaves and stems are lenticu-
lar in shape, rarely, or not at all chambered (PI. 43,
A), fewer in number, and smaller than on the pods

Fic. 2.—Diaporthe phas-

and usually isolated. They varyinsizefrom 197.5 to — ectorwm: Long, slender.

260.0 mM, average 219.0 ym. fragile sporophores with
Th b 1 d hi li a two pycnospores at-
€ pycnospores are bome on slender, nyalin, sim- tached. (2) Sporo-

ple conidiophores (fig. 2,a), 134 to 3 times the length —phores; (6) pycno-

of the pycnospore. They are mostly oblong to fusoid

spores. Xs5oo.

(fig. 2, b; 3), hyalin, 1-celled, usually straight, seldom slightly curved,
with two large oil droplets. These droplets, sometimes pressed to-
gether in the center of the spore, give the appearance of a septum, a
characteristic common to the genus Phomopsis and so deceptive that
at a casual glance one might believe that a true septum was laid down.
That the spore is but 1-celled can readily be demonstrated by clearing

QO
EN

Fic. 3.—Diapor-
the phaseolo-
rum: A group of
py cnospores.
Note the oil
droplets and
variations in
size and shape.
X 500.

it with a little salicylic acid. The variation in the size of
the pycnospores from various sources is seen from the
following measurements:

From host.—6.0 to 8.6 by 2.4 to 4.1 w, average 7.5 by
BB 3 pe,

Stems of Medlilotus alba.—5.6 to 10.0 by 2.4 to 4.0 p,
average 7.82 by 3.11 mu.

Corn meal.—5.4 to 8.2 by 2.4 to 3.4 w, average 6.49 by
2.79 M.

Irish potato cylinders.—5.4 to 8.5 by 1.7 to 3.0 y, aver-
age 6.36 by 2.59 yw.

Corn-meal agar.—5.1 to 8.3 by 2.4 to 3.4 mw, average 6.78by 2.85 p.

Rice.—5.4 to 8.5 by 2.0 to 3.4 mw, average 6.45 by 2.80 yu.

It will be noted that the spores are relatively large in artificial cul-
tures on stems of Melilotus alba, and small on cooked Irish potato cyl-
inders. This difference has been found rather constant, and is prob-
ably associated with vigor of growth. Of all media tried stems of
M. alba were found exceedingly favorable for spore formation, while
cooked Irish potato cylinders were rather unfavorable.

482 Journal of Agricultural Research Vol. XI, No. ro

Under favorable conditions, such as in a nutrient broth or beef agar,
the pycnospores germinate in about six to eight hours at room temper-
ature. Previous to germination the spores
become almost spherical and slightly en-
larged. Usually but one germ tube develops, _
but occasionally two (fig. 4). One or both
of the oil glcbules may disappear at this
stage or remain until the germ tube is several
times the length of the spore. Growth is
rather slow at first, the hyphal growth attain-
ing a length compared witn the diameter
of the spores as shown in figure 4 in 48
hours. During this time branching may oc-
cur, and septa rarely are formed till later.

The bodies called stylospores are frequently
found either alone or accompanying the
Fic. 4.—Diaporthe phaseolorum: Ger- pycnospores in pycnidia on the pods. They

minating pycnospores. X50. have never been found on the leaves.
They are long, slender, hyalin, nonseptate, straight or curved bodies
(fig. 5) borne on short, rather stout awl-

shaped simple hyalin stylosporophores (fig.
6). Figure 5 shows some __ stylosporo-
phores with stylospores attached. They
are produced but sparingly the early part

of the season or when the pycnospores
are most abundant. As the season advances
the stylospores increase in number and
appear to be associated, though not always,
with a saprophytic existence. Following estes
the death of the host and the gradual _ phoreis attached to the stylospore.
disappearance of the pycnospores the stylo- eee ue oe: gents
spores increase and finally become very
numerous. Specimens which bore practically no stylospores in the fall
bore them in increasing numbers throughout
the winter. There is a possibility from the
data at hand that the stvlospores may be
associated with a lack of food supply. They
are produced in some culture media freely,
but not until after the food supply becomes
more or less exhausted. Although in cultures
Fic. 6.—Diaporthe phaseolorum: A the pycnospores are usually exuded from the
group ofstylosporophores. soo.

beak in seven days, the stylospores have never
appeared sooner than 11 and generally not before 15 to 30 days after inocu-
lation. The average size of the winter crop of stylospores developed

Dec. 3, 1917 Podblight of Lima Bean 483
eee SI al lec cela AER kc SS
under natural conditions is considerably larger than those produced in the
fall or in culture. Measurements mdde of the fall stylospores from the
host vary from 11.7 to 31.0 uw by 1.4 to 2.0 p, average 22.83 by 1.73 un,
while those collected on February 7 varied from 20.6 to 54.4 wm by 1.38
to 2.4 wu, average 32.44 by 2.04. They are produced in culture on rice,
corn meal, Irish potato cylinders, and stems of Melilotus alba and vary
in length from 11.7 to 54.4 u
and in width from 1.38 to
2.4 p.

The function of these bodies
is not known. Repeated at-
tempts have been made to
germinate them but without
success. Reddick (29) has
suggested the possibility that
they were paraphyses and
Saccardo (31, p. 264) regards
them as conidiophores. Von
Ho6hnel (23, p. 526), described
a new genus, Myxolibertiella, .
to include those species hav-
ing stylospores and pycno-
spores. He first placed the
genus in the order Melanco-
niales but later in the order
Phomatales. Bubék (2) and
Diedicke (13): both regard
them as spores. The latter
investigator for convenience
designated the Phoma-like
spores by the Greek letter alpha (@) and the stylospores by beta (@).
Shear (32), like Bubak, Diedicke, and others, regards these bodies as
spores and designates them as scolecospores.

There is nothing peculiarly distinctive about the mycelium. It is
hyalin, frequently branched, and septate. It varies considerably in
width and is often supplied with enlargements which bear a slight re-
semblance to chlamydospores. When young the cells are supplied with
small protoplasmic granules. When the hyphe are old, the cells are
filled more or less with oil globules or droplets (fig. 7).

Fic. 7.—Diaporthe phaseolorum: Mature hyphe. Note the
septations, oil droplets, and granules. soo.

ASCOGENOUS STAGE

Specimens of Lima beans consisting of stems and pods collected in
October, 1914, and January 1, 1915, at Vineland, N. J., were wintered
out at Washington, D. C., and covered over with dirt and leaves. These

484 Journal of Agricultural Research Vol. XI, No. 10

specimens were kept under observation through the remainder of the
winter and spring months. Pycnidia and pycnospores were found from
time to time, as well as an increasing number of stylospores. On June 2
pods brought into the laboratory bore a considerable number of mature
perithecia of an ascomycete, which was identified as Diaporthe phaseo-
lorum (C. and E.) Sace. The writer was away for five weeks previous to
June 2 so it is not certain just when the development of perithecia began,
and for that reason it was not possible to follow the complete course of
development. The perithecia are scattered and buried beneath the
surface, most of the beak protruding. Plate 43, D and F, shows dia-
grammatically the arrangement of the perithecia and relatively the
depth they are sunken below the surface. Whether or not the perithecia
are all formed in the cavities of old pycnidia has not been definitely de-
termined. The pycnidia are rather numerous
and often coalesce, and as a matter of chance a
perithecium might preempt a pycnidium and be
formed therein. It is believed, however, that
thisisnot therule. The perithecia are not irregu-
lar in shape, as might be expected if they were
formed in old pycnidia; but they usually bear a
rather definite and constant form (Pl. 43, F).
They also have separate and distinct walls which
Fic. 8—Diaporthe phaseolo differ from the layers of the pycnidium. In no
rum: (a) Asci and (6) asco-

Spores uo<noa. case have the spores or stylospores ever been
found associated with a perithecium, although

these bodies were present on the host at the time.

The perithecia are slightly flattened, 158 to 355.5 mu, average 251.9 mu
in width and from 110.6 to 205.4 wu, average 166.5 win depth. The beaks,
many times longer than wide, are usually curved or hooked (PI. 43, D),
rarely straight, tapering gradually tothe end. The perithecium, circum-
scribed by a dark layer of cells, is lined within by a thin, hyalin layer
from which the asci arise. The perithecium is evidently almost or
completely matured before the beak is formed. Plate 43, D, shows
beaks just appearing through the epidermis. Sections through peri-
thecia with beaks in the incipient stage show the wall layers to be per-
fectly formed and many mature asci present. The asci (fig. 8, a) are
sessile, clavate or fusoid, 28.0 to 46.2 yw by 5.2 to 8.0 w, numerous,
and contain eight spores arranged mostly biserially. The ascospores
are apparently bound together or embedded in a gelatinous mass, since
they are firmly held together even after the apparent disappearance of
the ascus wall. The ascospores, (fig. 8, b) measuring 6.4 to 12.0 by 2.3
to 4.0 u, are spindle-shaped, oblong-lanceolate, 1-septate at or near the
center, slightly or not at all constricted, and contain two to four oil
dropiets.

Dec. 3, 1917 Podblight of Lima Bean 485

The ascospores germinate readily in most any nutrient medium in
about six to eight hours by the development of a single germ tube from
one or both cells (fig. 9). The hyphe branch is 12 to 24 hours, and septa
are laid down at about the same time. Growth is very rapid. At ordi-
nary room temperature a colony from a single spore becomes visible
to the naked eye in about five days. The ascogenous stage has not been
produced in culture. The transfer of a colony from a single ascospore
resulted after 7 to 10 days in the production of the pycnidial stage on
the same culture media on which pycnidia were developed by a transfer
of pycnospores in the same length of time. In other words, there is
no difference between the growth of the ascogenous strains and pycnidial
strains on the same culture media.

INOCULATION EXPERIMENTS

The inoculation experiments on which the conclusions of the parasitism
of the organism have been drawn were made in the greenhouse. It was
found that Lima beans grow normally and produce fruit as well under
greenhouse conditions as in the open field. Opportunities
were therefore afforded to study the parasitism of this dis-
ease under controllable conditions and at any time of the
year most suited to the needs of the work.

Controls were held with each experiment, the number
and results of which are shown in Table I following a dis- We. 9.—Diapor-
cussion of the inoculation experiments. The pods unless — M?,,, Phgseole-
otherwise stated were inoculated by inserting spores and mating asco-
hyphe into a wound made by a sterile needle. While “°" “°°
later evidence will show that under suitable conditions infection may
take place without wounding, the relative humidity of a greenhouse is
usually so low that spores smeared on the surface often fail to germi-
nate. It was necessary, therefore, to protect the spores from drying out
by inserting them under the epidermis or by other means. In most
cases it was impossible to protect the spores against drying, owing to
the fact that when a Lima bean plant is fruiting abundantly it is gener-
ally too large to confine in an infection cage, though this was done with
some smaller plants (see p. 486).

All inoculations were made from cultures grown on stems of Melilotus
alba. The age of the culture was not considered or found important
further than that the culture should be vigorous and fruiting abundantly.
Spores were exuded from the pycnidium in about seven days after the
culture was inoculated and continued to do so for 30 to 4o days there-
after or until the culture was dried. Spores were well suited for inocu-
lation purposes until the culture medium was dried out.

Inoculations were made with six different strains. By a strain is here
meant a difference in the source and not a difference in morphology or

486 Journal of Agricultural Research Vol. XI, No. ro

parasitology, since all were found to be identical organisms. For the
sake of convenience in presentation each of the strains was given a sepa-
rate number as follows: Strain 286 was isolated on October 26, 1914,
from a Lima bean pod collected from a small patch at Vineland, N. J.
Stylospores and pycnospores were abundant. Strain 420 was isolated
from a Lima bean pod collected during the fall of 1914 by Mr. M. B.
Waite in Maryland. Stylospores, as well as pycnospores, were present
in abundance. Strain 447 was isolated from material collected on
January 1, 1915, from the same field as No. 286. Strain 567 is a reisola-
tion made on April 12, 1915, from a pod that had been inoculated in the
greenhouse on March 13, 1915, with strain 447. Pycnospores were
abundant, but there were no stylospores. Strain 598, as previously
stated, resulted from the isolation on June 4, 1915, of a single ascospore
from a pod that had been wintered out at Washington, D. C. Strain
6o1 was isolated on August 7, 1915, from leaves of Lima beans collected
at Cape May, N. J., on August 5, 1915.

Other isolations of the same organism not used for inoculation pur-
poses have also been made for the purpose of identification and com-
parison. Frequent reisolations were made from inoculated pods, one
strain (567) of which was used to make inoculations. From specimens
inoculated with this strain the causal fungus was again recovered, thus
fulfilling the requirement of Koch’s postulates, claimed necessary to
prove parasitism.

The first infection experiments were made on January 15, 1915, when
20 nearly mature pods on six small plants were inoculated, ro with
organism 286 and ro with organism 420. After the pods were inoculated
by inserting spores and hyphe, the plants were sprayed with spores
suspended in water. They were then kept in a paper-covered infection
cage for 24 hours. On February 20, pycnidia were abundant on the
stems and on six pods inoculated with No. 286. Of those inoculated
with No. 420, five pods were dead, and pycnidia were already on four
and developed on one more after 10 days in a moist chamber. In this
case there were no leaf infections, though the stems, particularly the
lower parts, were covered with pycnidia, the pycnidia developing most
abundantly on the moribund stems and pods.

On March 13, 21 small plants were sprayed with organism 447. There
were only three small, immature pods in all and they were inoculated by
inserting spores and hyphe. These plants were inclosed for 48 hours
in paper-covered infection cages. Twelve days later the three pods were
drying up, and pycnidia were present on one and developed on the two
others after 7 days in a moist chamber. Here again no leaves were
infected, and the pods did not show typical infections. These results
were disappointing at the time, but it was discovered later that young
leaves and pods were difficult to infect, mature parts being much more
susceptible to the disease.

Dec. 3, 1917 Podblight of Lima Bean 487

On April 9, 1915, twelve almost mature pods were inoculated by
inserting spores and hyphe in the usual way with organism 447. The
plants were too large to place in the infection cage. Seven days later
the tissue for half an inch or so around the point of infection was visibly
darker. Three days later some of the pods were withering, and pycnidia
began breaking through the epidermis 4 to 34 inch from the point of
inoculation. As long as the pods remain firm, the pycnidia form in
more or less concentric rings; but concomitant with the withering of the
pods the fungus rapidly invades all the tissue, and pycnidia break through
the epidermis indiscriminately ina manner shown by Plate 42,C. Eight
of these pods finally became infected.

April 20, nine half-grown pods were sprayed with a suspension of
organism 447 in sterile water and kept in an infection cage for 24 hours.
These specimens were kept under observation until June 2, but no
infection had taken place. It is believed that the failure here is again
due to the relative immaturity of the pods. These results agree with
those of Pool and McKay (27), who were able to infect only mature
leaves of sugar beets with Phoma betae (Oud.) Fr. Apparently the writer
has succeeded in infecting young pods by wounding, but there is reason
for doubt, owing to the fact that a small wound frequently results in
serious injury and death of the pod, in which case the fungus quickly
invades it as a saprophyte. On April 23, spores of organism 447 were
inserted in five pods, and by June 2 pycnidia and spores were abundant
on three and a few days later on one more, one remaining sound.

On June 2, pods bearing perithecia which contained mature asci were
brought into the laboratory and thoroughly washed and disinfected in
mercuric chlorid. The perithecia were carefully picked out and macer-
ated in sterile water in a watch glass. Twenty-one nearly full-grown
pods were inoculated by inserting ascospores and bits of broken tissue
into wounds. ‘Twelve days later thirteen pods showed unmistakable
evidence of the disease, and by June 21 all but four of the pods were well
covered with the fruiting bodies. The writer does not claim any proof
of the connection of the conidial and perfect stage from this experiment.
It is included here because it forms a link in the chain of experiments
performed with this organism. ‘The results are justly open to the criti-
cism that spores and hyphe of the imperfect stage may have been
inserted, and it is not unlikely that such was the case.

On June 3, 1915, fifteen pods were inoculated in the usual way with
organism 567. By June 14, pycnidia were abundantly produced over
an area an inch or more in diameter on ten pods, and on four others
by June 21. One pod alone remained sound.

On June 14, seventeen pods were inoculated with organism 598
(ascospore strain). In four days the tissue around the wound on some
pods began to darken, showing unmistakable and characteristic symptoms
of the disease. As the pods were killed by the fungus, they were gathered

488 Journal of Agricultural Research _ Vol. XI, No. 10

and all but one bore numerous pycnidia by August 9. Cultures from some
of these infected pods produced a growth characteristic of the pycnidial
stage. On the same date spores of organism 598 were smeared on the
surface of five nearly full-grown pods. There were no signs of infection
on June 21, but by July 7 three showed evidence of infection, two remained
sound. Seven pods of various ages were sprayed with organism 598 and
confined in an infection cage for 30 hours. Pycnidia appeared on one of
these pods on June 21 and on two others on August 9 as well as on the
stems and some of the dead branches. ‘The writer was away for sometime
previous to August 2, and it is not definitely known just how the disease
developed. It is probable, in view of the fact that the pods were dead
and studded all over with pycnidia, that they succumbed sometime
earlier.

On June 21 twenty-one pods were inoculated with organism 598 in the
usual way. One diseased pod was removed on June 29, eighteen on July
7,and one on August 17. One of the inoculated pods could not be found,
and was therefore regarded as not infected. There were ten checks held
for this experiment, one of which became infected and the only one of all
the checks (66) held at various times.

On August 17, ten pods were inoculated with organism 601, and by
September 3 six were well covered with pycnidia. Four remained sound.
Of 19 pods inoculated with 601 on September 7, ten showed typical in-
fection on September 25. Nine remained sound.

A summary of the results is given in Table I.

TABLE I.—Summary of inoculation experiments of Lima beans

Num-
: Date of in- : umber in- | Num- | Num. | ber of
Organism No. erative How inoculated. yemuberse pa as ee bir eo
fected.
IQI5
BOOM cme dcteriacins te Jan. 15 | By insertion of | 10 pods.... 6 5 oO
spores in a wound.
OOM ce eaten eta ess dow ae (6 Co renee asics merce latcac (loan 5 5 °
AA ere eee oneal pea” LE Sill PLAYER: oss casectens nl ed Panes... ° 6 °
Wounding......... Supodsics <i 3
AAG area elastase Sys sya eis Apr. 9 | By wounding......| 12 pods.... 8 5 °
BAG a aie arses wallets Apr. 20. SpLayimg. ..'5....-\0e | iPOUS. . 2. ° 5 °
GAP sir tose 's Sas} Apes a> By wounding.) 2 Bepadsmc as 4 5 °
Fragmentsof peri-| June 2 |..... MO heii ior 2IAMOAS «15150 gz 8 °
thecia contain-
ing asci from
pods.
567 [iite esas. soe GOs nsec mer oe 15 pods 14 6 °
GCOS e coisas toate ehes iS era Pee doe aaa T7podSe we 16 6 °
BO Salaa sea we wWatertellons Cole wane Spores smeared on | 5 pods..... 3
pods.
BOSretactectncier wales downs Sprayedsisn.. heal 7epOGSeaeee 3
BOScina sas. oe - «af ee. ers eb yp WONG ine sey a. 21 pods.... 20 bie) I
601 i Mb emits erin Se ro hoe No Menas ae topods 6
601 Nepte7hlaen ee do fea Ee 19 pods ae) 5 °

Dec. 3, 1917 Podblight of Lima Bean | 489

PERPETUATION OF THE FUNGUS

The finding of mature asci of this fungus on June 2, 1915, and on June
II, 1916, and later on the dead vines and pods wintered out at Washing-
ton, D. C., and on similar material under field conditions at Vineland, N. J.,
at about the same season of the year shows one way the fungus may live
through the winter. This method of perpetuation is, however, not the
only one. At intervals from October until June pycnospores were fre-
quently found, and the fungus was isolated at will from field material.
It is interesting to note in this connection that the pycnospores were
produced periodically during the winter season, depending upon weather
conditions. Following a period of a few days of relatively warm, rainy
weather, they could nearly always be found in more or less abundance.
For example, pycnospores were found on material examined on October
24, January 2, 14, and 21, February 7, March 6, April 14, and June 2.
Isolations were made on October 24, January 2, February 7, May 31, and
June 2. Similar results were obtained with Phomopsis vexans (Sacc. and
Syd.) Harter, the pycnospores of which were found in May and June on
wintered-out material. However, the same material examined in Decem-
ber, January, February, and April bore no pycnospores but the stylospores
appeared to be more numerous during the spring months. The appear-
ance of pycnospores was roughly coordinated with rainfall and tempera-
ture, since on May 22, following a period of rainy weather and relatively
high temperatures, they were found in great abundance.

It is likely also that the organism may live for a considerable time in
the seed. The writer has isolated it in pure culture from the interior of
both seeds and pods after surface disinfection in mercuric chlorid. From
these results it is apparent that the fungus invades all parts and it is not
unlikely that it is carried fron one season to the next on the seed. That
the fungus is not readily killed by drying is evident from the fact that it
was isolated from dried specimens kept in the laboratory for nine months.
It would probably survive such conditions even longer. These results
are supported by the earlier work of Halstead (17), who in 1901 called
attention to the fact that the seeds were invaded by the fungus and sug-
gested the possibility that it was carried through the season by that
means.

MODE OF INFECTION

It is not always easy to explain how a parasite gains entrance into its
host. It is safe to conclude that, if a fungus is parasitic, it may enter
through wounds, it being merely necessary to show that the host is
subject to wounding under natural conditions. Other types of penetra-
tion as, for example, through the unbroken epidermis or by way of the
stomata are not always easy to demonstrate. There is no doubt that
physical factors such as rainfall, temperature, humidity, and stomatal
movement all play an important rdle in the penetration of the epidermis

490 Journal of Agricultural Research Vol. XI, No. ro

or stomata by certain parasites. In this connection it is interesting to
note the results of Pool and McKay (28), who have studied in some
detail the relation of stomatal movement to infection with Cercospora
beticola Sacc. These investigators showed that the germ tubes entered
only when the stomata were open, the movement of which was closely
associated with a relatively high humidity, warm temperature, and
light. Their results further showed that mature leaves were more readily
infected and possessed a pore opening almost twice as large as young
immature ones. The writer obtained similar results with the Lima bean
fungus. It was found, as already pointed out, that young immature
pods were not infected without wounding while mature pods could
easily be. This difference may be due at least in part to the difference
in the size of the stomatal aperture but more likely to the fact that im-
mature stomata do not open, thus preventing the entrance of the hyphe.
Under humid conditions the spores germinate and produce a germ tube

Fic. 10.—Diaporthe phaseolorum: Some of the germ tubes of the pycnospores penetrating the stomata two
days after the pods were sprayed, and other germinated spores lying alongside the stomata and growing
toward or away from the aperture. X 260.

20 or 30 times the length of the spore in 24 to 48 hours. This is rather
a feeble growth but the spores do not germinate readily in water, and
then only a small percentage at that. On pods that had been sprayed
with a spore suspension in water the germ tubes were found penetrating
the stomata as shown by figure ro.

It is evidently a matter of chance when a germ tube finds its way into a
stoma, since, if there was any attractive force, the germ tubes might be
expected to head that way, which is apparently not the case. The germ
tubes, as often observed by the writer and illustrated by figure 10, grow
just as frequently alongside or away from the stomata as toward them,
showing that there is no appreciable chemotactic influence. Entrance of
the stoma would be easy so far as relative size of germ tube and stom-
atal aperature are concerned. In fact, it would not be impossible and
it may actually happen that the spores fall directly through the pore
into the intercellular space beneath. Measurements of the pores showed
them to vary in length from 22 to 28 wand in width from 5 to 7 uw when
open, the germ tube of the spore in width from 2 to 3.5 uw, and the spore
itself from 6 to 8.6 uw by 2.4 to 4.1 w. Considering the relative size of

Dec. 3, 1917 Podblight of Luma Bean 491
peewee ee re
the stomatal pore and the spore of the fungus, it is quite reasonable to
conceive of an infection even during dry times, an event which actually
does sometimes take place. It must of course be admitted, and the ob-
served fact supports such a conclusion, that the number of infections by
a fungus of this type during dry weather is relatively small compared
with those during a period of rainfall or damp weather. Jones, Giddings,
and Lutman (25) pointed out that moist, damp weather was conducive
to the germination of the spores of Phytophthora infestans and made the
interesting observation that the germ tube penetrated the leaf of an
Irish potato through the epidermis as well as through the stomata.
Similar observations were made by Jones (24), who found that the germ
tube of Macrosporium solani EK. and M. may penetrate the epidermis, as

well as the stomata of Irish potatoes. ‘The writer found no instance of
penetration of Lima bean pods except through the stomata, and Pool

and McKay (28) make a similar statement for Cercospora beticola.

The inoculation experiments already described have shown that
infection takes place readily through wounds, perhaps made chiefly
by wind, and by means of pickers. In ordinary farm practice the pole
Lima bean is usually trained up several feet above the ground. Exposed
thus to the wind, the vines are whipped around and the pods wounded
by rubbing against the stems, poles, or wires on which they are trained.
A careful examination will readily show the bruised spots on pods ex-
posed to such conditions. Pickers also, in searching through the foliage,
may likewise brush or press the immature pods against stems or poles,
bruising them in a similar manner. The reddish, streaked appearance
in localized spots often observed on bean pods is characteristic of wounds
made by constant brushing or rubbing of the pod against a hard surface.
It is in such areas that infections are frequently found.

The relation of insects to infection has not been studied.

DISSEMINATION

The dissemination of the podblight of Lima beans must be considered
from two standpoints: (1) Over short distances, and (2) over long dis-
tances, as from one district or State to another. As to the first of these,
there are several ways in which the spores may be carried from plant
to plant or from one field to another. It is not unlikely that insects,
whether they feed on the pods or not, play an important part in the
distribution of the spores. During wet periods the spores are exuded
from the pycnidium, and any insect frequenting the diseased pods is
likely to carry the spores about on its body. Pickers, too, in searching
through the foliage for mature fruit, are likely also to distribute the
spores to unpicked pods, where, under favorable conditions, new infec-
tions will start. It is interesting to note in this connection that this
and certain diseases of other crops seldom become serious until about
picking time. There are two possible explanations for this. One is

492 Journal of Agricultural Research Vol. XI, No. 10

that the host may be more susceptible at this age; and the other that
picking of the fruit, especially if the foliage is wet with dew or from
rain, results in a liberal distribution of the spores.

That strong winds may be responsible for severe outbreaks of this
and other diseases is evident from the fact that Cook (6, p. 517-518)
found a serious outbreak in a certain section of New Jersey previously
visited by a severe windstorm. It is, of course, natural to conclude,
as Cook assumed and the writer has frequently observed, that injury
to pods and foliage during such a storm would add to the chances of
infection. It is believed that the part played by the wind in dissemi-
nating fungus spores has been very greatly underestimated. It is a
well-known fact that the pollen of pine trees is carried long distances.
Dr. Albert Mann, of the Bureaw of Plant Industry, found?! a large
variety of fungus spores collected on a gelatin plate exposed for about
15 minutes at a distance of 1 mile above the surface of the earth. This
readily shows that spores are freely air-borne over long distances, and
this fact alone may account for the serious outbreak of a disease in
isolated sections. :

Owing to the fact that the causal organism can readily be isolated
from the seed, it is likely that this affords the principal means of dis-
tributing it over long distances. Fruiting bodies have also been found
on infected seed. This shows the great necessity of saving seed only
from fields where this disease does not occur.

CULTURAL CHARACTERISTICS OF THE FUNGUS

The fungus produces hyphz on nearly all of the culture media in
ordinary use. The pycnidia, on the other hand, are rarely produced
on agars, but freely on media containing starch, such as rice or corn
meal. A study of the growth and production of fruiting bodies was
made on the following media: String-bean agar, Irish potato agar, beef
agar, corn-meal agar, cooked rice, cooked corn meal, potato cylinders,
and stems of Melilotus alba. ‘These media were selected because they
are in common use in the laboratory of Cotton, Truck, and Forage Crop
disease investigations, and because some (corn meal and rice) were
known to be particularly favorable for the growth of other very similar
organisms, thus affording an opportunity for comparison. Other media
might have served the same purpose.

The experiment was carried out by inoculating five tubes (100 c. c.
flasks, in case of corn meal) with spores from a 1o-day-old culture on
stems of Melilotus alba. After inoculation the tubes were kept in the
light and exposed to the temperature of the laboratory room (21°-25°C.).

STRING-BEAN AGAR.—This on the whole has been a poor medium for this fungus.
Growth of hyphe started promptly and was visible in two days in slanted tubes of

1 Unpublished data.

Dec. 3, 1917 Podblight of Lima Bean 493

the medium. At the end of 9 days three dark bodies, which were at that time thought
to be pycnidia, and which probably were sterile ones, were developed. ‘These cul-
tures were kept under observation for 72 days, and no more pycnidia and no spores
were found.

IRISH POTATO AGAR.—There was a visible vegetative growth in two days which
continued to increase for about one week. A few scattered pycnidia appeared in all
tubes but one. These were kept under observation for 72 days, and no spores or
stylospores were ever produced.

BEEF AGAR.—A rapid vegetative growth during the first seven days of growth.
Thereafter the hyphe became fluffy and cottony. No pycnidia were formed in any
of the tubes during the whole course of the experiment.

CORN-MEAL AGAR.—Growth was not visible in two days, but in four days the
hyphe had spread over an area 4 inch in diameter. In six days dark specks appeared
which later developed into mature pycnidia. At the end of 13 days the slimy white,
creamy masses began to exude from the pycnidia. These cultures were frequently
examined thereafter for stylospores, but none were found until after 34 days, when
they appeared in great abundance. When stylospores and pycnospores are both
produced, the former always appear after, and never before, the production of the
latter.

COOKED RICE.—Rice has proved to be a good medium for the growth of this organ-
ism. Hyphez were conspicuous and pycnidia began to form in 4 days. From that
time the pycnidia increased in number and spores began to exude in 7 days. A
heavy, black stromatic mass, in which the pycnidia were embedded, was formed.
As the medium dried out, these stromatic masses were left as conspicuous domelike
projections over the surface of the medium. The pycnospores were exuded in slimy,
yellowish, viscid masses in such quantities as to flow over the top of the stroma and
mostly to cover the surface of the medium. Stylospores were found sparingly in 22
days and abundantly in 34 days.

COOKED CORN MEAL.—Corn meal was found to be one of the best media for this and
other closely related fungi, such as Phomopsis vexans, Plenodomus destruens, and
Diaporthe batatatis. In 5 days the hyphe covered most of the surface of the corn
meal in roo-c. c. flasks, and pycnidia began to form. The pycnidia were produced
in great numbers in a more or less well-developed stroma which bore characteristics
similar to those on rice. The small slimy droplets containing pycnospores were
first noticed at the end of 13 days. The slimy exudate increased in amount until
the droplets became quite large and flowed over the sides of the stroma, eventually
covering nearly the whole surface of the medium. The stroma and pycnidia were
dark on top and somewhat raised above the surface of the medium. As the corn meal
dried out, the surface became more or less crusty and rugose. Stylospores were found
for the first time at the end of 22 days.

POTATO CYLINDERS.—On this medium abundant mycelium was produced. A
cottony, white growth covered one-third of the potato slant in two days and com-
pletely infourdays. In five days incipient pycnidia were seen which later developed
into a more or less stromatic mass. These masses were at first whitish on top, but
later turned darker. In 22 days pycnospores were exuding slightly, and stylospores
were present. At the end of 34 days the stylospores had become more abundant than
on any other medium; the pycnospores, on the other hand, were relatively few.

STEMS OF MELILOTUS ALBA.—This medium, everything considered, is the best
ofanysofarused. Itgivesa maximum amountof spores with a minimum of mycelium.
There were no signs of growth in two days, but in five days numerous pycnidia began
to form, and these soon became conspicuous as black specks studding the surface of
the stems. The beaks were comparatively long and conspicuous. In one week
under optimum conditions the viscid, creamy droplets in which the spores are em-

494 Journal of A gricultural Research Vol. XI, No. r0

bedded were noticeable on the end of the beak. These droplets gradually enlarged
and finally overflowed the pycnidium, coalescing with similar droplets from other
pycnidia and thereby forming an almost continuous liquid covering of the substratum.
A stroma was not formed on stems of Melilotus alba. In this particular experiment
no stylospores were formed. In other cultures, however, of the same organism on
M. alba stems a few stylospores were found.

EFFECT OF LIGHT ON GROWTH

The literature is full of the records of experiments on the effect of light
on growth and development of chlorophyll-bearing plants, as well as
fungi. It would be useless to review all or any considerable part of these
records here. It is evident, however, from a perusal of some of these
publications that no general, sweeping conclusions can be drawn that
will apply to all fungi alike. Recent results, on the other hand, show that
under similar conditions individual fungi may be expected to respond
differently. In support of this last statement, reference may be made
to the results of Coons (ro), who found that light was a determining factor
in the production of pycnidia with Plenodomus fuscomaculans (Sacc.)
Coons. The writer (19), on the other hand, working with a different
species of Plenodomus, P. destruens Harter, found that the absence of
light did not inhibit the production of pycnidia. That the absence of
light very greatly inhibited, but did not entirely prevent, the production
of pycnidia by Diaporthe batatatis, was shown by Harter and Field (21)
in another experiment. The writer has obtained similar results, as yet
unpublished, with other fungi.

The influence of light on growth and production of fruiting bodies of
the pycnidial stage of Diaporthe phaseolorum was determined by inocu-
lating stems of Melilotus alba in test tubes and exposing one-half of the
set to light and excluding the light from the other tubes by wrapping
with black paper, which was found by previous test to prqhibit the
passage of light. ‘The tubes were all tightly plugged with cotton. The
cotton plugs alone were left exposed, so as to permit aeration. The two
series were then placed on a table in the center of the laboratory room
and kept under observation for 25 days. The cultures began to dry up
about that time and development was accordingly arrested. The tubes
kept in the dark were examined for a few minutes every few days for
the purpose of taking notes and were for that length of time exposed
to the light. While this brief period can not be regarded as having
no influence on the results, exposure to the light for such a short time
would certainly have no marked effect. The growths of mycelium in
the cultures in the light and those in the dark were about the same at
the end of three days, but from that time the mycelial growth in
the dark was relatively more abundant and more cottony. In tubes
in the light, on the other hand, there was, as has always been, a minimum
of mycelium on stems of Melilotus alba. At the end of six days pycnidia

Dec. 3, 1947 Podblight of Lima Bean 495

were forming in large numbers in all tubes in the light and in smaller
numbers in all those in the dark. The ratio between the number of
pycnidia formed in the light and in the dark remained nearly constant,
the fruiting bodies forming in all tubes, but they were better developed
in the light. In one week spores began to exude from the pycnidia
both in the light and in the dark. From this time spores continued
to be exuded, but here again the amount of the exudate was relatively
less in the dark. ‘Toward the close of the experiment, however, there
appeared to be a relative increase in the exudate from the pycnidia
formed in tubes in the dark. But one conclusion can be drawn from
these results, that while the dark retards the production of fruiting
bodies and accelerates vegetative growth, it is not as marked as the
results obtained with other fungi by the writer and others.

INFLUENCE OF TEMPERATURE ON GROWTH

To determine the influence of temperature on growth 12 (No. 2, 3, 5,
G8 NdwLOy 15, PZ. 18, 19, and 20) of the 20 chambers in an Altmann
incubator were used. Tubes of cooked rice were inoculated with spores
of the fungus and immediately placed in the chambers, where they were
kept exposed to the respective temperatures for a period of 27 days, at
the end of which time growth had practically stopped. No growth took
place in chambers 2 (1° G),.3 (627 C.), 19 (35-4° C.), and 20 (38.8° C.),
and but a scant growth in 18 (32.9° C.). The optimum growth was
found in chambers 15 (26.5° C.) and 17 (30.5°C.). The growth of hyphz
decreased relatively with the increase and decrease of the temperature
from these limits of the optimum. In chambers 9 (17.4° C.), 10 (19.4°
ys '(26-5°° C.) 747 (30.5° C.), and 18 (22.99) Cx) stromatic masses of
psetidoparenchymous tissue were produced which macroscopically looked
like pyenidia. A cateful examination of these bodies, however, showed
that, while they bore some resemblance to pycnidia, no cavity had been
produced within them and no pycnospores were found. ‘The cause for
this was later found to be due to a lack of sufficient aeration, resulting
probably in a reduced oxygen supply. A subsequent experiment in
which cultures of the organism on cooked rice were exposed in a chamber
in which most of the oxygen had been removed by a mixture of pyrogallic
acid and sodium hydroxid showed that while mycelial growth continued
under such conditions the production of fruiting bodies was inhibited.
In addition to the fact that the cultures in the Altmann chambers were
kept in the dark, the absence of oxygen, which in itself was found, as
already shown, to reduce somewhat the production of fruiting bodies,
evidently accounted for the failure to produce pycnidia in the cited cases.

The growth of the podblight fungus in artificial cultures is schemati-
cally shown in figure IT.

496 Journal of Agricultural Research Vol. XI, No. x0

INFLUENCE OF TOXIC AGENTS ON GERMINATION OF SPORES

Perhaps no chemical substance has been more generally studied in its
relation to the growth of plants than copper salts. This is perhaps due
to its well-known efficiency as a fungicide, following the discovery of which
a large amount of work was undertaken to determine the concentration
of copper salts necessary to prevent germination or bring about the death
of the spores of a great variety of fungi. Much of this work was done
with saprophytes, and consequently has little more than a scientific
interest, while the practical side of the problem was evident from the fact
that the toxicity to germinating spores of many well-known parasites was
established. Because of the various uses that chemical substances may
be put to, such as antiseptics, disinfectants, etc., the toxic action of many

salts, metals, and acids,
go alone and in combina-
tion, on _ flowering
plants and fungus
spores have been ex-
tensively studied, and

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Fic. 11.—Diaporthe phaseolorum: Graphicrepresentation of growth on contradictory. In gen-
cooked rice at different temperatures. - -
eral, however, all in-

vestigations have shown that copper salts, among other chemical
substances, are exceedingly toxic to all germinating spores and to the
seedlings of flowering plants. Coupin (zz) has shown by experiments
that 1 part of copper sulphate to 700,000,000 parts of water is suffi-
cient to retard the root growth of wheat seedlings, and the writer has
proved (z8) that a small amount of a single alkali salt, such as
sodium chlorid, sodium carbonate, and magnesium sulphate in water
cultures, inhibits the growth of the same plant. Heald (22) found
1/51,200 and 1/25,600 gram-molecule per liter of copper sulphate to be
toxic to Pisum sativum and Zea mays, respectively. According to Moore
and Kellerman (26) 1 part of copper sulphate to 500,000 parts of water
killed Closterium moniliferum in 4 days and 1 part of copper sulphate to
3,000,000 parts of water killed Anabaena flos-aque in three days.
Uroglena americana was even more sensitive and was killed in 16 hours
in 1 part of copper sulphate to 10,000,000 parts of water.

These few references show the extreme sensitiveness of some of the
flowering plants and some alge to copper salts, and at the same time the
variability in their resistance. Although not strictly comparable with

Dec. 3, 1917 Podblight of Lima Bean 497

flowering plants, because of the different methods or experimentation
and standards of measurement, the spores of fungi show equally great
variability and sensibility to copper salts and other toxic substances.
Clark (3) found that Penicilliwm glaucum was injured by copper sulphate
at a concentration somewhere between N/2,048 and N/1,024, while
Aspergillus flavus would endure a concentration of N/512 to N/256, two
to four times as great. Crandall (12), on the other hand, showed that
the apple scab fungus is quite resistant to copper sulphate, the spores
being only slightly retarded in germination in a solution of 1 to 100,000,
while a concentration of about 1 to 10,000 was necessary to entirely
prevent germination. At this latter concentration some of the common
molds, however, grew quite well. Temperature was found by Brooks
(r) to influence the percentage of germination of the spores of certain
fungi in the same concentration of a toxic substance. By an extensive
series of experiments he found that Penicillium glaucum gave medium
growth in N/128 copper sulphate at 20° C., Monilia fructigena did not
germinate in N/r6 copper sulphate at 25° to 30° C., but at 15° C. 12 per
cent, at 10° C. 30 per cent, and at 5° C. 49 per cent.

No attempt has been made to review all the extensive literature on the
effect of toxic substances on plant growth. ‘The results of the work of
the different investigators are very conflicting, due, perhaps in part at
least, to the different methods of experimentation employed and the
different standards of measurement.

During the time the podblight fungus of Lima bean has been under
investigation many interesting observations have been made trom time
to time on the effect of certain chemical substances commonly used in the
laboratory on the germination of the spores. The use of a solution of
mercuric chlorid for disinfecting field specimens preparatory to isoiating
the fungus led to a study of the strength of this solution necessary to kill
the spores ma giventime. A similar study was made with formaldehyde
because of its possible use in disinfecting infected seed, and with copper
sulphate, a well-known constituent of Bordeaux mixture, sometime rec-
ommended as a spray in controlling the disease in the field.

METHOD OF EXPERIMENTATION

Preliminary experiments showed that the use of Van Tieghem cells
to determine the toxic-limit for the germination of the spores was ex-
ceedingly unreliable and was consequently abandoned. Although this
method has been generally used, it is likely that many of the conflicting
results may be attributed to it. In fact, the writer found that each
test in Van Tieghem cells might be expected to give different results,
probably owing to the variation in vapor pressure or to some slight
accident overlooked, unexpected, and unaccounted for. After con-
sidering the different methods that might be used, the writer concluded

498 Journal of Agriculiuval Research Vol. XI, No. ro

to employ the method frequently used in medical bacteriology to de-
termine the germicidal value of certain chemicals. This takes more
into account the actual death of the organism rather than the strength
that inhibits growth. It seems that the same method should apply
equaily well to fungi and is easier of determination and more reliable,
since the length of time the organism is exposed plays an important
part also, and can be more accurately gauged. It is a well-known fact
that a fungicide may merely inhibit growth when the spores are exposed
to it for a certain length of time but may kill them on prolonged ex-
posure. In these experiments the time the spores were exposed to the
fungicide was relatively short, the concentration of the solution being
proportionately strong to produce death in a comparatively short time.
This method eliminated the necessity of taking into account, among
other things, the temperature which Brooks (rz) found to play such an
important part in germination of the spores of some fungi.

The pycnospores of Diaporthe phaseolorwm were found to grow well in
beef agar +15 on Fuller’s scale; and therefore this medium was used in
which to test germination. The spores from a culture on Melilotus alba
were transferred by a small platinum loop to a solution of the fungicide
of the desired concentration and mixed by a wide circularly swinging
motion of the arm with the plugged end up, or by vigorous shaking. At
the end of one minute, two minutes, and every two minutes thereafter
up to the suspected limit of endurance, a loopful of the mixture was
transferred to a tube of liquefied agar and, after mixing by shaking, a plate
was poured. Control plates were made by transferring a loopful of
spores from the same test culture to a sterile water tube and a loopful
from this tube to a tube of liquefied agar. This method was followed
with each of the fungicides and repeated as often as necessary to establish
the toxic limit. The plates were kept at room temperature in the
laboratory. ‘The colonies in the control plates appeared in about two
days. Notes were taken from time to time for seven days, after which
they were thrown out, for, if growth took place at all, it would do so
before that time.

RESULTS OF EXPERIMENTS

The results of these experiments are given in Table II.

The mercuric-chlorid and copper-sulphate solutions were made in
advance and kept corked in the ice box until needed. Because of the
instability of the formaldehyde, the solutions were prepared just before
using and in such a way that the fractional parts given in Table II are
based on a hypothetical formaldehyde solution of a strength of 100 per
cent.

Dec. 3, 1917 Podblight of Lima Bean 499

TABLE II.—The results of exposing pycnospores of Diaporthe phaseolorum to different
strengths of mercuric chlorid, copper sulphate, and formaldehyde for different lengths of
time ;

Copper sulphate. Formaldehyde. Mercuric chlorid.
Time of
exposure. ;
N/12.5 | N/31.25 | N/62.5 I:I00 1:250 t:500 | N/135 | N/1,350 | N/2,700 | N/4,050
Minutes
I | @100 100 100 75 85 100 ° ° 50 100
2 35 Os abinti es fo) 30 Boje) ° ° ° 100
4 & 55 QOH Risse ste se ° CoG fal ogaackeee neice Latta ee 75
OS eercrer IGS Fede t torte) lial |(stane) oteta ail lon otetteeatchs TO) liererpsiotelleets, «tetas okey aeehcnsys 50
(St AA oes 15 iis Aldi Coocal oreo Ol | srayeteell lenge rete fee teres canes °
POM enaretsteverts OM ifefreiiettelaillerrelielelele)iai|isivayoe.e afislltetic lel'ei ia ctfell ete cttete/teiltete onaltanel el |teten etietretene) |emerctettate
ED || eater os 5 Aireilecevsioporen a iortretens aiailleie o1S ciate lies see atelee rars eyerel | eter tered a Pet ORs
TA |Pctte sone Dai oteraaree Mell cat Konan couse avian eevee os co/G Roree [fas lalevcti lta te-ateor rate acon he bedeicall eens
NOM |p pies llovemer ote ZO ietarectons lite sea eves crevarmie ali Sgie eas [letatere atone te orate sie Itramna aes
Total pears estes ai epee reciept ek teh aicarerteil era tode det ancl l Geaio So enehe [laevane: chaall susns coh euaslap sacred Seelllcaetonchel o Mlleieeuereke
ZAO} HAs oasoy] econ ole oD Ria Nevaeh alts ohn |latalie cle shasreil\alis e, wivin!(aille,fohayeivoleil/e'cciel, nice) aire IMO at eels: cell chen eneita

@ In this and corresponding columns are given the percentage of germination as estimated at the close of
the experiment. Water controls were made in every casein which the germination was arbitrarily placed
at roo percent. ‘These plates, together with those poured from a 1 minute exposure, served as controls for
comparison in estimating percentages of germination.

Reference to the table will show that the spores were nearly all killed
in four minutes in a N/12.5 copper-sulphate solution, and in 14 minutes
in a N/31.25, and in 20 minutes in a N/62.5 solution. It is interesting
to compare these results with those of Clark (3), who found that the
spores of several fungi exposed in a copper-sulphate solution were killed
or injured as follows: Aspergillus flavus (?) Link N/4 and greatly injured
in N/256, Sterigmatocystis niger Von Tieghem N/& and greatly injured in
N/128, Oedocephalum albidum Sacc. N/64 and greatly injured at N/256,
Botrytis vulgaris Fr. greatly injured at N/r28, and Pemiciliwm glaucum
Link N/z and greatly injured at N/256. As will be seen, there is a wide
difference between the strength of the solution necessary to kill and that
required to injure greatly the spores. The writer found that none of the
spores exposed for one minute to a solution of mercuric chlorid at a con-
centration of N/135 and N/1,350 would germinate, and that only 50 per
cent germinated when exposed for the same length of time in a concen-
tration of N/3,700. At the greater dilution, N/4,050, 50 per cent germi-
nated after an exposure for six minutes. Clark (3) found that spores of
A. flavus, S. niger, P. glaucum were killed in a N/4,096 mercuric chlorid,
O. albidum in a N/16,384, and B. vulgaris in a N/65,536. The spores of
these fungi were killed in the solutions of the toxic substance more dilute
than that required to kill the pycnospores of Diaporthe phaseolorum, but
they were likewise subjected to the solution for a longer time. In
another work Clark (4) has shown that Rhizopus nigricans was killed in
75 seconds in a solution of mercuric chlorid 1 to 3,700.

Formaldehyde was found to be very toxic to the spores, only 10 per
cent germinating after a six-minute exposure in a 1-to-500 solution, and

500 Journal of Agricultural Research Vol. XI, No. ro

30 per cent after a two-minute exposure in a 1-to-250 solution, and
only 75 per cent in a 1-to-100 solution after one minute. Clark (3)
found that 1 part by weight in 273,066 parts of water permitted the
germination of but 10 per cent of the spores of Oedocephalum albidum
after 11 hours’ exposure, as compared with 95 per cent after four hours
in the controls, and greatly injured the mycelial development.

As regards the results, it should be stated that in all cases where the
spores were not killed the germination was very much retarded. The
colonies in the controls became visible to the naked eye in about 48
hours. A long exposure in a weak solution retarded germination to
about the same degree that a short exposure in a correspondingly more
concentrated solution. That the germination of Lima bean seeds is not
injured by exposure for 10 minutes in the strongest solutions of mercuric
chlorid, formaldehyde, and copper sulphate to which the spores were
subjected is shown by a series of experiments discussed under ‘‘Control
measures.”

CONTROL MEASURES

Halsted (77) was the first, and probably the only one, who has ever
done any experimental work on the control of the podblight of Lima
beans. In these experiments spraying the dwarf Limas with Bordeaux
mixture or soda-Bordeaux mixture yielded very favorable results.
Although he evidently did not spray the pole Limas, he suggests that the
disease could be checked by so doing. It is now known that the fungus
attacks the leaves before the pods are set, and it is likely the pods become
infectedfromthem. In view of this fact, the first spraying should be done
when the plants are 1 or 2 feet high, and should be repeated often enough
to keep the foliage covered.

From badly infected pods the fungus invades the seed, where it may
live for a considerable time. Infected seed are usually dark, shriveled,
and immature. They probably would not grow, and, if they did, would
give weak, sickly plants. Only plump, bright seed should be planted.
Sound seed may be disinfected to kill the adhering spores by submerging
for 5 or 10 minutes in a solution of mercuric chlorid (1:1,000), formalde-
hyde (1:100), or copper sulphate (1:100), after which they should be
rinsed in water.

A series of experiments has shown that germination was not injured by
such a treatment. Healthy Lima bean seeds were sorted out; a part
were treated for 5 minutes and a part for 10 minutes. After rinsing in
sterile water, they were laid between moistened filter paper and ger-
minated. The results showed that seeds exposed for 10 minutes gave
just as good germination as those soaked in sterile water for the same
length of time. It should be mentioned in this connection that those
soaked in sterile water were all more or less covered with saprophytic
molds, while those treated with mercuric chlorid, formaldehyde, and
copper sulphate were comparatively free.

Dec. 3, 1917 Podblight of Lima Bean 501

SUMMARY

(1) The podblight of Lima beans is probably indigenous to the United
States.

(2) It was first recognized by Halsted in New Jersey in 1891, and has
since been found in the States of Virginia, Maryland, Connecticut, and
North Carolina.

(3) The disease forms circular brown spots on the leaves, and large
unsightly spots on the nearly mature pods and on the stems. Numerous
pycnidia are produced in the diseased areas.

(4) The loss, which in some fields is very large, is confined mostly to
the pods.

(5) The Lima bean podblight fungus has been known as Phoma
subcircinata EK. and E. almost since its first discovery, although in a few
cases it has been referred to as a species of Phyllosticta.

(6) The writer isolated single ascospores of a Diaporthe found on
specimens wintered out and made inoculations with pure cultures of
such strains which produced a disease identical with that produced by
inoculations with pure cultures of a pycnospore strain. In 1892 Diaporthe
phaseolorum was completely described, which agrees with the ascogenous
fungus the writer has been studying. The fungus causing the disease
is therefore referred to as Diaporthe phaseolorum (C. and E.) Sace.

(7) The hyphz course through and among the parenchymous cells
of the pod, forming pycnidia just under the epidermis at points where
the bast cells are more or less separated.

(8) Stylospores are abundantly found on field material and readily
produced in culture on some media. The pycnospore stage belongs to
the form genus Phomopsis and not to Phoma.

(9) The disease is readily produced by artificial inoculation with spores
from both the pycnidial and ascogenous strains.

(10) Wounding of the pod is not necessary for infection, as the germ
tubes may enter through the stomata.

(11) The fungus is carried through from one season to the next, either
by periodic production of pycnospores and the persistence of hyphe or
by the production of an ascospore stage.

(12) The disease may be carried on the seed and spread by such
agencies as wind, by the process of picking, and possibly by means of
insects.

(13) The fungus fruits well on stems of Melilotus alba, on rice, corn
meal, and other starch media, and but poorly or not at all on the agars
tried with the exception of corn-meal agar.

(14) Cultures in the dark fruited more slowly than those in the light
and had increased vegetative growth.

(15) No growth takes place at a temperature below 6.1° nor higher
than 35.4° C. during a period of 27 days.

502 Journal of Agricultural Research Vol. XI, No. 10

(16) Dilute solutions of formaldehyde, copper sulphate, and mercuric
chlorid are toxic to the spores. The germination of the spores is greatly
retarded when exposed to a solution too dilute to kill them in a given
time.

(17) To control the podblight it is recommended that clean seed be
selected. They should then be disinfected in mercuric chlorid, formalin,
or copper sulphate. The plants should be sprayed in the field the first
time when they are 1 to 2 feet tall, and often enough thereafter to keep
the foliage covered.

LITERATURE CITED.
(1) Brooks, Charles.
1906. TEMPERATURE AND TOXIC ACTION. In Bot. Gaz., v. 42, nO. 5, Pp. 359-375)
12 fig. Literature cited, p. 375.
(2) BuBAK, Franz, and KaBsart, J. E.
1905. VIERTER BEITRAG ZUR PILZFLORA VON TIROL. In Osterr. Bot. Ztschr.,
Jahrg. 55, no. 2, p. 73-79. (Contiaued article.)
(3) CLarK, J. F.
1899. ON THE TOXIC. EFFECT OF DELETERIOUS AGENTS ON THE GERMINATION
AND DEVELOPMENT OF CERTAIN FILAMENTOUS FUNGI. In Bot. Gaz.,
v. 28, no. 5, p. 289-327; no. 6, p. 378-404, 10 diagr. Bibliography,
P- 402-404.

(4)

Ig0I. ON THE TOXIC VALUE OF MERCURIC CHLORIDE AND ITS DOUBLE SALTS. In
Jour. Phys. Chem., v. 5, no. 5, p. 289-316, 7 fig.
(5) CuinTon, G. P.
1906. REPORT OF THE BOTANIST. NOTES ON FUNGUS DISEASES, ETC., FOR
zgo5. InConn. Agr. Exp. Sta., 29th Ann. Rpt., [1904]/o5, p. 263-277.
(6) Coox, M. T.
1913. REPORT OF THE PLANT PATHOLOGIST. In N. J. Agr. Exp. Sta., 33d
Ann. Rpt., [1911]/12, p. 509-527.

(7)

1914. REPORT OF THE PLANT PATHOLOGIST. In N. J. Agr. Exp. Sta., 34th
Ann. Rpt., [1912]/13, p. 793-817, 13 fig.

(8)
1915. REPORT OF THE PLANT PATHOLOGIST. Ju N. J. Agr. Exp. Sta., 35th
Ann. Rpt., [1913]/14, p. 467-476.
(9) Cooke, M. C., and Euis, J. B.
1878. NEW JERSEY FUNGI. In Grevillea, v. 6, no. 39, p. 81-96. (Continued
article.)
(10) Coons, G. H.
1916. FACTORS INVOLVED IN THE GROWTH AND THE PYCNIDIUM FORMATION OF
PLENODOMUS FUSCOMACULANS. Jn Jour. Agr. Research, v. 5, no. 16,
p- 713-769. Literature cited, p. 766-769.
(11) Couptn, H.
I90I. SUR LA SENSIBIEITE DES VEGETAUX SUPERIEURS A DES DOSES TRES
FAIBLES DE SUBSTANCES TOXIQUES. In Compt. Rend. Acad. Sci.
[Paris], t. 132, no. Io, p. 645-647.
(12) CRANDALL, C. S.
1909. BORDEAUX MIXTURE. Ill. Agr. Exp. Sta. Bul. 135, p. 199-296, 8 fig.
(13) DiEpIcKE, H.
IQII. DIE GATTUNG PHOMoPsIS. Jn Ann. Mycol., Jahrg. 9, no. 1, p. 8-35, 3 pl.

Dec. 3, 1917 » Podblight of Lima Bean 503

(14) Exus, J. B., and Everuart, B. M.
1892. THE NORTH AMERICAN PYRENOMYCETES. 793p.,41 pl. Newfield, N. J.
(15)

1894. NEW SPECIES OF NORTH AMERICAN FUNGI FROM VARIOUS LOCALITIES.
In Proc. Acad. Nat. Sci. Phila., 1893, p. 128-172.
(16) HatstTeED, B. D.
1892. LIMA BEAN DISEASES. InN. J. Agr. Exp. Sta., 12th Ann. Rpt., 1891,
p. 287.

(17)
I90I. BEAN DISEASES AND THEIR REMEDIES. InN. J. Agr. Exp. Sta. Bul. 151,
28 p., 9 fig., 4 pl.
(18) HarTER, L. L.
1905. THE VARIABILITY OF WHEAT VARIETIES IN RESISTANCE TO TOXIC SALTS.
U. S. Dept. Agr. Bur. Plant Indus. Bul. 79, 48 p. Bibliography,

p- 47-48.
(9)
1913. THE FOOT-ROT OF THE SWEET POTATO. Iu Jour. Agr. Research, v. 1,
no. 3, p. 251-274, 1 fig., pl. 23-28.
(20)
1914. FRUIT-ROT, LEAF-SPOT, AND STEM BLIGHT OF THE EGG PLANT CAUSED BY
PHOMOPSIS VEXANS. Jn Jour. Agr. Research, v. 2, no. 5, p. 331-338,
1 fig., pl. 26-30. Literature cited, p. 338.
(21) and FIEeLp, Ethel C.

1913. A DRY ROT OF SWEET POTATOES CAUSED BY DIAPORTHE BATATATIS.
U.S. Dept. Agr. Bur. Plant Indus. Bul. 281, 38 p., 4 fig., 4 pl.
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504 Journal of Agricultural Research Vol. XI, No. 10

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PLATE 42
Diaporthe phaseolorum:

A.—A leaflet of Phaseolus lunatus L., showing a number of ragged holes of various
sizes caused by the Lima bean podblight fungus. This specimen was collected at
Cape May, N. J., August 5, 1915. The disease was produced on pods by inoculation
with the organism (601) isolated from this leaf. Natural size.

B.—A green Lima bean pod photographed ro days after inoculation with the ascos-
pore strain. Pycnidia are abundantly formed over an area about 4 inch in diameter;
outside of this is another somewhat darkened area which has already been invaded
by the fungus, but the pycnidia have not yet broken through the epidermis. Natural
size.

C.—A pod showing the characteristic manner in which the fungus rapidly overruns
it soon after it is killed, pycnidia forming indiscriminately over the entire surface.
Natural size.

Podblight of Lima Bean

PLATE 42

Journal of Agricultural Research Vol. XI, No. 10

PLATE 43

Journal of Agricultural Research

Vol. XI, No. 10

PLATE 43
Diaporthe phaseolorum:

A.—A vertical section through a pycnidium from the leaf. 130.

B.—A vertical section through a pycnidium from a pod showing the beak and the
thickened dark wall surrounding it. Note also conidiophores arising from hyalin inner
ayer. Some spores are attached. 130.

C.—A vertical section through the epidermis and underlying host of a portion of a
diseased pod showing the connection and chambering of the pycnidia. 6s.

D.—A somewhat diagrammatic drawing of a portionof the surface of a Lima bean
pod, showing the distribution of the perithecia, the shape of the beaks and their pro-
jection from the surface. The beaks of two perithecia may be seen just breaking
through the epidermis. X16.

E.—A surface view of a portion of a bean pod showing arrangement of the pycnidia
and fusion of the beaks. X65.

F.—A vertical section through a perithecium from a pod. Note the dark wall
surrounding the perithecium in which the asci are inclosed; also the portion of the
beak buried below the surface of the pod. The outer end of the beak has been cut
away. X130.

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