THE MORPHOLOGY AND LIFE HISTORY

OF THE

CHESTNUT BLIGHT FUNGUS

A THESIS

! OF CORNELL UNIVERSITY FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

BY

PAUL JOHNSON ANDERSON

PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL

THE MORPHOLOGY AND LIFE HISTORY

OF THE

CHESTNUT BLIGHT FUNGUS

A THESIS

PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL
OF CORNELL UNIVERSITY FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

BY

PAUL JOHNSON ANDERSON

[Reprint of Pennsylvania Chestnut Blight Commission Bulletin 7, December, 1913]

eW EXCHANGE

JUN 25 "14

TABLE OF CONTENTS

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SUMMARY

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37

43

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(4)

The Morphology and Life History of the
Chestnut Blight Fungus

By PAUL J. ANDERSON, Field Pathologist

Penn’a. Chestnut Tree Blight Commission

INTRODUCTION.

Considering that it has been only seven years since the first article
on chestnut blight was published, the amount of literature on the
subject is becoming extensive. Eighty-five of the piincipal contiibu-
tions are given in the bibliography at the close of this bulletin, but
none of these give us more than the briefest facts concerning the de-
velopment and morphology of the producing organism, Hndothia
parasitica (Murr) And. ‘To be sure, various authors have given such
superficial facts as the size, shape, and color of the spores, asci and
perithecia, the general times of years at which they occur, the macro-
scopic appearance of the stromata, spore horns and “fans;” the be-
havior of the organism in culture has been pretty well covered by
Murrill (2, 3, 4), Pantanelli (34,89) and Clinton (83); inoculation
experiments are recorded by Murrill (2, 3), Clinton (83), Rankin
(101) and the writer (81). Interesting facts and observations have
been added by many others but we know of no one who has made
a detailed study of the life history and morphology. The necessity
of this study is readily apparent; until such study is made we are
dealing with an unknown enemy, our control measures are guess
work and their success a matter of chance. The writer has not ex-
hausted the subject by any means in the work which is recorded in
the following pages. He presents the facts discovered with the hope
that they may be of assistance to others who are working on this
phase. The matter is presented under the heads of Spores, Mycelium,
Pyenidia, Stromata and Perithecia, not because these all represent
distinct stages and because they do not overlap, but because he finds it
more convenient to group the facts about these heads.

The writer is under great obligations to Professors Whetzel and
Reddick of Cornell University, Messrs. Detwiler, Carleton and Heald,
officers of the Pennsylvania Chestnut Blight Commission, Messrs.
Babcock, Kirk, Gates and Keefer, who have assisted him especially in
the laboratory, and to a host of others who have sent specimens and
given valuable aid and suggestions.

(5)

SPORES.

Like most other Ascomycetes, this fungus produces two kinds of
spores: (1) pycnospores, otherwise known as conidia, conidiospores,
asexual spores or Summer spores and (2) ascospores, which are also
called the winter spores or perfect or sexual spores. These will
be treated below in the order named.

PYCNOSPORES.

On active young cankers during the spring, summer and autumn,
slender, curling, yellow tendrils are especially abundant shortly after
rain periods. If one of these “spore-horns” is put in water, it swells
up and then apparently dissolves, but if a drop of this water is placed
under the microscope, it will be found to contain millions of minute,
hyaline bodies—the pycnospores.

Morphology. Murrill (4) who first described the species, gives
their size as 1 x 2-8 microns, Clinton (92:367) as .75 x 2.5-4 microns,
Pantanelli (89) as 1.7 x 3.8 microns. The writer made two hun-
dred measurements of pycnospores from spore horns and got an
average of 1.28 x 3.56 microns. An equal number of measurements
was made of pycnospores produced in pure culture on oat
agar and also of pycnospores from superficial pycnidia on wood,
but the difference in size was found to be negligible. Their
shape is shown in figure 52, being oblong of cylindrical with
rounded ends, or slightly oval. As a rule they are straight,
although occasionally slightly curved. Dr. Mickleborough’s
curved figures (19) are evidently exaggerated; they remind us more
of the spores of a species of Naemospora which grows on the chestnut
and the spore horns of which cannot always be distinguished macros-
copically from those of Endothia parasitica. Although the tendrils
of the latter species are bright yellow, the spores themselves, as seen
under the microscope, are quite hyaline. This color is due to a pig-
ment which is evenly diffused in the spore, or more likely the spore
wall, and can be noticed only when there is a mass of them together.
The pigment is the same as is found in the hyphae and will be dis-
cussed under the head of mycelium.

The wall of the resting spore is extremely thin and is not readily
differentiated by staining. No markings, germ pores or layers can
be detected. The spore is densely filled with protoplasm which is
homogeneous; only occasionally are oil globules or vacuoles seen in
the resting spore. By staining it can be determined that each spore
contains a single small nucleus, which is elongated in the direction
of the long axis of the spore. It usually lies close to the wall, about
equi-distant from the ends, but may be almost in the end. It is shown

ier!

at the center of figure 14. With carbol fuchsin, and various other
stains, a single body in each end of the spore stains very deeply.
The significance of these polar bodies is uncertain. They cannot be
located after germination and it is conceivable that they are used
up in the enormous growth of the spore during that process. The
outside of the spore is covered with a mucilaginous, sticky coat which
is hard when dry and holds the spores together in the characteristic
brittle “horns,” but, on wetting with water, it first swells and then
apparently dissolves and the spores float away free from each other.
The mucilage of the spore horns is however, insoluble in alcohol.

Germination. Unlike the ascospores, the pycnospores do not ger-
minate in cultures in water. Tap water, rain water, spring water
and distilled water have been tried without success except that a
slight and uncertain germination was secured in rain water. A small
percentage of the spores germinated in water made slightly acid with
sulphuric acid. A large number of media have been tried but mostly
with disappointing results. Entirely successful germination was se-
cured, however, in a decoction made by boiling chestnut bark in
water, filtering and then sterilizing the filtrate in the autoclave.
With this solution, a percentage of over eighty has been uniformly
secured, and it has therefore been used almost exclusively in tests
for longevity, vitality, etc. This suggests that there is some soluble
substance in the bark of the chestnut tree that is necessary for their
germination. In order to see if this substance is peculiar to the
chestnut, sterilized twigs of the chestnut, red oak, white oak, black
oak, sour gum, sumach, hickory, walnut, red maple and yellow poplar
were sterilized in test tubes, and then washed with a suspension of
pycnospores. From the fact that they germinated and produced the
characteristic mycelium on all of these species, it is certain that the
substance needed for germination is not peculiar to the chestnut tree,
and that a spore would germinate just as readily if it fell into a
wound of a sour gum or any of the other trees as it would on a chest-
nut. It is also significant that they will germinate perfectly in potato
agar and most any of the ordinary nutrient agars. To determine
whether they would germinate in the humus about the base of the trees
if washed down into it by the rain, twelve petri dishes of sterilized
humus were inoculated by spraying pycnospores over them. Not only
did they germinate, but the mycelium grew and produced typical
pycnidia on this medium. Tannin also is apparently not essential
to germination because they germinate readily in media which are
free from this substance.

Two methods of artificial germination have been used. In the
first, a slide is supported on two glass rods in a petri dish as a moist
chamber, and a drop of the bark decoction containing a suspension
of the spores placed on the center of the slide. In the second method

8

a film of pycnospores in water is spread on a sterile cover glass and
permitted to air dry. It is then covered with a drop of potato agar
or some other nutrient agar and inverted over a Van Tieghem cell.
This second method was used when it was desired to study the pro-
cess of germination because it offered the advantages of keeping the
spores stationary, and at the same time they could be put under the
immersion lens.

The time required for germination varies widely with the tempera-
ture. Fulton (48:52) says: “Conidia germinate best at a temperature
of 60 degrees F. and distinctly less rapidly at temperatures 10 de-
grees below or above that point.” The writer, on the other hand,
secured the most rapid germination at 89 degrees F., the shortest
time secured for the appearance of germ tubes being twelve hours.
At temperatures ranging from 60 to 75 degrees F., germination oc-
curs in from 18 to 36 hours. At lower temperatures it often re-
quires four or five days. No effort was made to find the exact maxi-
mum and minimum temperatures. Some experiments by D. C. Bab-
cock in our laboratories indicate also that light hinde:s germination.
From the data given, it appears that the very warm periods of the
summer are most favorable for infection by pycnospores. That
winter conditions are not favorable is indicated by the following
experiment: At the beginning of every month during the last year,
twenty-five or more inoculations in healthy chestnut trees have been
made with conidia. At the present time, (June 15, 1913), none of
those made after September or before April show any signs of pro-
ducing cankers. Cankers are appearing about the inoculations made
in April. Apparently then, infection will not necessarily result even
if conidia do gain access to wounds during the winter.

The process of germination is preceded by an enormous swelling
of the spores. This swelling begins in fifteen to twenty hours after
they are placed in neat bouillon agar, and is then very rapid until
the germ tube is pushed out. As previously stated, a mature spore
measures about 1.28 x 3.56 microns. At the end of 18 hours,
50 spores which were just on the point of pushing out germ
tubes, gave an average of 6.86 x 10.53 microns. The largest
one observed was 9.05 x 14.48 microns. The volume of the
spore just before germination is thus more than _ eighty-five
times that of the resting spore. This increase in size is shown
in figure 38, at the center of which are a number of resting
spores. The various shapes assumed by the germinating spores will
also be observed here. They may become cylindrical, oblong, ellipti-
cal, isodiametric, ovate, pyriform, reniform or dumb-bell shaped. in
which latter case they resemble ascospores. The contents become
coarsely granular, and often large vacuoles are seen, due to the rapid
swelling. The first indication of a germ tube is a small protrusion or

9

pimple at one end which rapidly increases in length. So far as ob-
served, the tubes are always at the ends of the spores. <A few hours
after the beginning of the first tube, another one starts at the other
end of the spore. Only very rarely do both start at once. The rate
of growth, size of the tubes and order of the laying down of the septa
are brought out by the series of camera lucida drawings of single
spores at short intervals reproduced in figures 39 and 40. This is an
average growth in potato agar in Van Tieghem cells at 21-26 degrees
C. The pycnospores generally produce two germ tubes. Very
rarely a third one comes out laterally. From three to six hours after
germination starts, the first septum appears in the tube and other
septa are laid down often enough after that to make the cells of the
mycelium 4-10 times as long as broad. As the germ tube lengthens,
the cells composing it increase in diameter but the septa, being solid
plates, do not increase in size correspondingly; hence the constric-
tions at the septa which become more marked as the mycelium -be-
comes older (Fig. 8). Sometimes a septum divides the spore dur-
ing this process. After a time it is difficult to locate the old spore
since the first cells of the germ tube become exactly like it, and it is
now merely one of the cells of the hypha. The branching of the germ
tube is shown in the figure just referred to.

The swelling of the spores is due not merely to aemechanical imbibi-
tion of water; it is really a process of growth. To be sure, dead
spores will swell, but only to about half the size acquired by living
spores. Pycnospores, stained just before the germ tube is pushed
out, show that the increase in size is accompanied by active nuclear
division. Even at this time, two to six nuclei, rather larger than the
original nucleus, may be made out. Also the polar bodies have dis-
appeared and the protoplasm is not dense. The nuclei push out into
the germ tube almost as soon as it starts. The wall in the meantime
has increased in thickness until it almost equals the diameter of the
resting spore and is quite distinct in stained sections. A germinating
spore is shown in optical section in figure 13.

Vitality. All experiments up to- the present indicate a remark-
able vitality of the summer spores. Reasoning from analogy to what
is known or believed of the imperfect spores of most fungi, one would
not expect them to survive winter conditions. But the case is quite
the contrary. During every month of the past winter pycnospores
were taken from the woods, (a) from spore horns, (b) from pycnidia
imbedded in the stromata and (c) from superficial pycnidia on bare
wood and tested for germination in bark decoction. The percentage
of spores which germinated ranged between 54 and 71 per cent.,
being only slightly lower than that of fresh conidia in culture, and
showing only slight variation for the months. Apparently, then,
weather conditions such as we have had in Pennsylvania during the

10

past winter, have very little if any effect on their vitality. Heald and
Gardner (938) also found that freezing does not affect the vitality
of the pycnospores. Tests made at various times during the summer
of 1912 show also that the hot and dry weather of summer does not
affect their vitality. Three series of tests were conducted to deter-
mine their longevity. In the first series, spore horns were detached
from the bark and stored in open vials in the laboratory. At the
end of each month, sterile twigs have been inoculated with the spore
horns. Every test has been successful, including the last, which was
at the end of one year. In the second series the spore horns were
left attached to the bark, which was kept dry in the laboratory,
and germination tests made in decoction as given above. The last
test—at the end of 11 months and 15 days—gave a germination of
65 per cent. In the third series, pycnidia in the bark were stored.
This series has been running only eight months; the last test gave a
germination of 40 per cent. All these series are being continued
and there is little doubt that they will retain their vitality much
longer than a year since very little decrease in the percentage has
been noticed. On the other hand, if the conidia are separated by dis-
solving the spore horn in water and then dried, they do not retain
their vitality very long. The writer has not seen them germinate
when kept in this eondition longer than one month, but more experi-
ments are necessary.

Inoculation experiments with conidia are, described in detail by
the writer and Babcock in Bulletin 3 of the Pennsylvania Chestnut
Tree Blight Commission. In general it has been proved that almost
any kind of a wound in the bark may be infected with pyecnospores,
whether they are introduced dry or suspended in water.

ASCOSPORES.

On older cankers, as shown in figure 46, the mature stromata
are beset with projecting papillae. The black speck at the apex of
each papilla is the opening of a little flask in which the winter spores
are produced.

Morphology. The shape of the spores is shown in figure 37, being
oblong to oval with rounded or more or less blunt pointed ends, 2-
celled and constricted at the septum when mature. Clinton (92:368)
in Connecticut, evidently does not consider the constriction as con-
stant. His photomicrographs however—as they have been reproduced
in his plate XX VIII—show beautifully constricted spores. They are
quite hyaline both as seen under the microscope and when seen in
mass. Murrill (4) gives their size as 9-10 x 4-5 microns, Pantanelli
(89-73) the same as Murrill, Clinton (92:368), says they vary from
6-10 x 2.75-5 microns and average (92:427) 7.45 3.2 microns, based
on the measurement of one hundred spores. His measurements

11

are the smallest of any we have seen. ‘The average of one hundred
and forty measurements made by H. W. Anderson and reported
in Bulletin 4 of the Pennsylvania Chestnut Blight Commission,
was 8.53 x 4.49 microns. These were from points in Pennsylvania.
In the same bulletin seventy-five measurements of ascospores made
by Rankin in New York are reported and give an average of 8.8
x. 4.4 microns. One hundred measurements of spores from points
in Pennsylvania and Maryland more recently made by the writer
gave 8.68 x 4.51 microns as the average.

The walls are thicker than those of the pycnospores and are also
more resistant to chemicals. With strong sulphuric acid they may be
made to swell until their thickness often equals the diameter of the
contents but they do not dissolve. This treatment shows no strati-
fication of the walls and no germ pores or markings of any kind.
The septum is also swollen greatly by this reagent; in fact, in none
of its reactions does it seem to differ from the wall, and it is evidently
of the same composition. It is a true septum and not merely a di-
viding line between the protoplasts. This fact was particularly no-
ticed because Saccardo in his description of the genus Endothia in-
timates that it is a false septum, and also because it differs in this
respect from the long-spored southern Endothia, as reported by H.
W. Anderson before the American Phytopathological Society in Janu-
ary, 1913.

The spore is densely filled with homogeneous protoplasm. Only
occasionally have anything like oil globules or vacuoles been seen.
The writer has not found the large globules (or vacuoles), repre-
sented in Murrill’s figures (4), to be common. Chemical tests have
shown no glycogen or other storage products except proteids. As
shown in figure 37, each cell of the spore contains two or four nuclei;
occasionally there is one or three, and in some cases the number is not
the same in both ends of the spore; more than four in one cell have
not been found. The nuclei are best brought out by staining with
iron-alum haematoxylin. The ascospores, like the pycnospores are
sticky and adhere with great tenacity to any object with which they
come in contact. The nature of the sticky covering has not been
exactly determined, but it is conceivable that it is due to the matrix
of epiplasm in which the spores lie while in the ascus.

Germination. They readily germinate in tap water, spring water,
rain water or any of the ordinary media used for this purpose. A
higher percentage was secured in chestnut bark decoction, however,
than in pure water but as a rule more than ninety percent germinate
even in water. They germinate as soon as mature without a period
of rest. Spores were produced in September from inoculations made
the previous June, and as soon as mature, were tested and gave a good

12

percentage of germination. The same methods for artificial germina-
tion were used as were described in treating of the pycnospores.

The time required is much shorter than for pycnospores. At
room temperatures they push out a tube in from six to twelve hours.
The shortest time secured was one hour and twenty-five minutes after
ejection from the perithecium. As for the effect of temperature on
germination, Fulton (48:52) says: “Ascospores germinate best at a
temperature of about 70 degrees F., but a good percentage of germina-
tion occurs at 85 degrees and 45 degrees F. Even at 38 degrees F.
the germination of ascospores was 25 per cent in 24 hours and
reached 70 per cent in three days.”

Like the pycnospores they swell before germination, but not to
such an extent. The resting ascospore measures approximately 4.5
x 8.5 microns. Fifty spores measured after ten hours in nutrient agar
averaged 7.27 x 13.84 microns—representing an increase of about
four times the volume of the resting spore. The largest one was 17.2
x 9.05 microns. During the swelling the shape remains practically
the same except that the sinus becomes deeper. The first germ tube
usually appears at the end, but this is not always the case—some-
times it is lateral. The second tube to appear is in the other cell;
this is generally followed by a second one from each of the cells,
making a total of four germ tubes, which is the rule for the asco-
spores of this species. Their order of appearance, size, manner of
septation and branching is best explained by reference to the suc-
cessive camera lucida drawings of single spores in figures 41 and 42.
The germ tubes from the ascospores grow much more vigorously than
those from the pycnospores. By sowing ascospores on chestnut bark
agar, in summer weather, mature pycnidia have been produced in five
days. The early and rapid development of the mycelium from
the ascospores is probably due to the larger amount of food material
available in the spores.

During germination the contents of the spore becomes granular
and vacuoles often appear. The nuclear behavior is the same as
that of the conidia described above.

Vitality. So far as has been determined, weather conditions have
no effect whatever, on the vitality of the ascospores. During every
month for the last year they have been collected from the woods and.
tested, but the differences in the percentage of germination for the
months have been entirely negligible. Their longevity is indicated
by the following two series of experiments: In the first series, ascos-
pores ejected from the perithecia were caught on glass slides and then
stored and tested every two weeks for germination by covering them
with a drop of water. They continued to germinate for five months
and six days. After that they would not germinate. In the second
series, bark containing mature perithecia was stored in the labora-

13

tory and tested every month. The last test—at the end of approxi-
mately twelve months—gave a germination of above 90 per cent.
There is no doubt that this experiment will give a much longer
record, since they germinate almost as well now as they did a year
ago. These experiments also show that the spores will live much
longer when they remain in the perithecium than if they are ejected
and free from each other. 'Phese tests of course, indicate only the
time they would retain their vitality if they were kept.dry. If, on
the other hand, they were in a moist place, they would germinate
at once and unless they gained entrance to their proper host or
possibly, some suitable substratum for a saprophytic existence, they
would die without causing any damage.

The results of a large number of inoculation experiments are given
by the writer and Babcock in Bulletin 3 of the Pennsylvania Chestnut
Tree Blight Commission. In general, the same thing may be said
of them as was said of the pycnospores; any kind of a wound in the
bark deeper than the cork layer may be readily infected either by dry
ascospores or with ascospores in suspension in water. In fact, there
seems to be very little difference in the ability of the ascospores and
pycnospores to produce the disease on the trees.

MYCELIUM.

This is the absorbing system of the fungus. It consists of millions
of fine branching threads—the hyphae—which grow into the living
tissues of the bark and sap wood, killing and digesting them in its
progress round the tree. It is thus the immediate agent in producing
the canker and ultimately killing the tree.

In culture. The beginning of the mycelium is the germ tube; the
mature mycelium with its millions of hyphae is produced simply by
the continued elongation and branching of the germ tube. In all es-
sential points it is alike, whether produced from an ascospore or a
pycnospore. A few hours after the germ tube starts it begins to di-
vide into cells by laying down septa. (See figures 38-42.) Shortly
afterwards, branches are pushed out from these cells and these in turn
become septate and give off branches until a thick tangle of filaments
is produced. These processes, so readily followed in the simple germ
tube, are in all essentials the same in the later growth of the mycelium.
Branching is nearly always preceded by septation; it is always mon-
opodial and it is very rarely that more than one branch is produced
from a single cell. The sinus at the septum, seen in the younger
mycelium, is less distinct in older hyphae. The manner of branching
is shown in figure 8. The individual hyphal cell is best studied in
agar culture although it shows some slight differences from the cell
in the bark, as will be explained later. The diameter of the hypha in
agar culture varies from 2 to 12 microns, and the length of the cells

14

from 20 to 50 microns. The apical cells have very dense protoplasm,
but, further back in the hyphae, large vacuoles appear, as shown in
figure 8. The protoplasm is not homogeneous but shows larg gran-
ules and certain refractive bodies. The wall is very thin and easily
collapses when dried. Each cell contains several small nuclei as
shown in the figure.

The yellow pigment. The mycelium grows luxuriantly on a large
number of artificial media. Cultural studies have been reported in
detail by Murrill (2) and Clinton (83). Results secured by the writer
largely duplicate theirs, and will not be recorded here. For ordinary
purposes the writer has used potato agar. On this medium, at the
end of from four to six days the mycelium begins to turn yellow, due
to the production of a pigment in the cells. The same pigment gives
the characteristic color to the spore horns and the stromata on the
bark. It is apparently evenly diffused in the cells or cell walls. The —
writer has noticed that old agar cultures of the fungus often become
purple or wine colored. Other experimenters have told him they have
had the same experience and were at a loss to explain it. The con-
nection between the purple color and the yellow pigment, as worked
out by H. W. Anderson, is this: The pigment is yellow and insoluble
when in an acid or neutral medium, but in an allsali medium is readily
soluble and takes on a purple color. This can readily be demon-
strated by pouring a solution of sodium hydroxide or any other alkali
over the yellow mycelium. The fungus, in its growth on the agar,
gradually causes it to become alkaline in character, and the pigment
goes into solution and colors the medium purple. Pantanelli (34)
says that the pigment is a lipochrome. Quite recently it was isdlated
and its chemical reactions determined in some detail by Cecil Thomas
of Wabash College.* In this excellent piece of research, he shows
that it does not resemble a lipochrome in any way except in color
and solubility but that it is one of the colored compounds known
chemically as the aurines. It is best isolated by extracting with
alcohol and then precipitating with hydrochloric acid.

The fans. In order for the germ tube to gain access to the host
tissue the spore must germinate in a wound. As reported in Bulletin
3 of the Pennsylvania Chestnut Tree Blight Commission, all attempts
to produce infection without a wound have failed. The germ tube is
not able to bore through the cork layer nor to enter through lenticels.
Even if one secured an occasional infection without making a wound,
it would be difficult to prove that the bark was free from small abra-
sions which had escaped the notice of the experimenter. But if ger-
mination takes place in fresh wounds, the germ tube will thrive on the
injured and dead cells until it has produced a mass of mycelium.
Then, gradually accumulating strength as it increases in size, the
mycelium en masse pushes out through the living tissues of the bark.

*Master’s Thesis. Publication of the Botanical Department of Wabash College.

15

Single threads do not seem to possess the power to penetrate alone
among the living cells. Starting from a narrow point, the hyphae
erow out in ray-like bundles, completely destroying the parenchyma
and collenchyma and cambium cells as they go. All the rays start-
ing from a single point are contiguous and they form a fan-like mat of
mycelium as shown in figure 50. These fans are flat because they
are not able to destroy the segmental bast zones but must squeeze
between them. The edge of the fan is quite regular and is surrounded
by a darker gelatinous band of the disintegrating host cells.
Whether the cells are killed by a toxin secreted by the parasite
or whether they are killed by the mechanical action of the mass of
hyphae was not determined. The fans vary in length from one-eighth
to three-quarters of an inch. The young ones, on the advancing edge,
are pure white but as they become older they become light yellow or
buff in color. This color, however, is not due to a development of
pigment, since the pigment is never found in the fans; it is probably
due to a decomposition product of the disintegrating host cells, which
stains the mycelium. Each ray consists of a loose bundle of hyphae
running almost parallel and branching only sparsely. They are much
more uniform in diameter than the hyphae in agar culture. They are
about 7 microns in diameter and are divided into cells about 30
microns long. They are not anastomosed in any way; a section of a
ray showing their relation is represented in figure 9. The individ-
ual cells of the hyphae are densely filled with rather coarsely gran-
ular protoplasm. As the fans become older, however, the cells be-
come vacuolate. Like most of the other cells of this fungus, they
are multinucleate. The fans are produced only in the growing sea-
son. Although the canker spreads slowly in the winter, no white
fresh fans are found in that season.

Rate of growth. The rate of growth of the mycelium under natural
conditions on the tree can be measured by the increase in the size of
the cankers. During the last twelve months, a large number of
cankers have been outlined at the end of each month as shown in
figure 49, and the averages computed for the months. Table I gives
the increase in diameter during the last year. The increase in
length—up and down the tree—is greater but not so important since
it is not the growth in this direction that kills the tree. The table
shows the effect of winter temperatures on the growth. The last
winter in Pennsylvania, however, was exceptionally mild, especially
the months of December and January.

Even the most rapid growth in the summer time—as indicated by
the table—is less than one millimeter per day. But on artificial
media, such as chestnut bark agar, the writer has often seen a growth
of three millimeters per day. Also, in the dying bark after the tree is
cut, the mycelium will spread at a much more rapid rate than when

16

it is invading the bark of a healthy tree. In the latter case, it does
not advance by producing fans but by individual strands.
TABLE 1.

Showing the monthly rate of growth of cankers. Transverse diame-
ters of the cankers.

&

=|

~

a

S)

=|

‘ 7

g z

Month. a a

o ~

3) Cae

on

ca me

= eo

=o re!

eB oe

a <

|

Afri & Wes Skeet, dookbcanubpodaodous os node Se F550 Bac 31 1.88
DU eL Olas. ae 200 2.78
August, 1912, ... 186 2.83
September, 1912, pee 140 1.85
OCTOBER eel Ge ear yaa stetei crorstelelelclolecterstebetevefehe SENET ee aie ee ere MIR ter Ste ieie fats ath aie cles accie’ aievarslevevels 53 1.92
November, 1912, 27 0.00
WECOMDE Le OL rc trsteictercletere siele eles etic ieenia era raiatelavaretsoye eicieleistatelstelsteletaretsia ata: s c1oyelp ic clelv\e’e!'s\elv/eisle 27 *130
RU SANRYT ELT op eS saeete = evade olatasel=) Neleve mista cfoteteiatate eraiaiala/aieiciaraie cdetoua Sta}athictaisie'etata\s (ctr! e\e(e_« »'sir\ sis, diwie/sialsin\n 89 81
DEVAN yueel Glare: tctetetersie cielotereretalele\ sie oteterete teaver ale aieyetoferale phot lorelate ele eteleleltersts%s ois =(0\/lainis\elo\sie\e\eisia 89 0.0
AURA CH yy god Spd Sones sie erases caters lols taco miata eravernteietac aloe ateins meio teiste iicintals Inieietetatetaioietaieter eis e.elslsve,e]0\s\s/n.dielow's\s 84 An
PAS TNT MOTs tee eteeaysyere cles storota tele rovore eretoteratetcrm ie lokam etoteatetetaya clekatoletere res efevel ofelalareinia(e cia)afcisla(elsielateis/ejoterr PAl 1}
WER Be ICES SS aaa aoe gages aan nonbOd bebouyodh ado topbaSudbeCOnnune canobadodnocecducsosanedeg 41 2.4

*Doubtful record. No growth at all on a large number of other trees examined.

Vitality. The mycelium, like the spores has a remarkable vitality.
That it is not injured in the least by low temperatures in winter is
proved by the fact that successful isolations were made from under
the bark during every month of the last winter, and also by the vigor
with which the-canker resumes growth in the spring. To see if freez-
ing would affect it when exposed while growing under artificial con-
ditions, colonies were started on agar plates which when they were
about one inch in diameter, were put out of doors and kept frozen up
during the whole month of February which was the coldest month
of the winter. When brought back into the laboratory, they resumed
growth as vigorously as fresh colonies. Desiccation also has no
detrimental effect, as shown by the following experiments: In the
first one, bark was removed from a canker and stored under perfectly
dry conditions in the laboratory. Isolations have been made each
month and at present—at the end of ten months—the isolations are
just as successful as when the experiment was started. The second
was like the first except that diseased wood was stored instead of
diseased bark. This has been in progress only six months, but the iso-
lations are still successful. That a pile of bark or chips may be a
source of infection for a long time on account of the mycelium is

17

indicated by the following experiment: One year ago, some diseased
logs were peeled and the bark thrown into piles. Isolations have been
made from these heaps at the end of every month—being careful to
avoid contaminations from spores of the fungus—and up to the pres-
ent have been entirely successful. The writer has been unable to find
any especially resistant cells in the hyphae which tide it over.

The mycelium also invades the sap-wood to a depth of about four
or five rings. The hyphae are not different here except that they
are smaller than in the bark and do not enter the wood as fans. They
grow through and destroy the cells of the medullary rays and wood
parenchyma to some extent, and are found in the vessels in abundance,
but the walls of the latter are not affected by them.

PYCNIDIA.

The summer spores in all cases are produced in pycnidia. The
stages in the development of this organ are most readily observed on
artificial media, such as potato agar or chestnut bark agar. The
process is the same whether it takes place on agar or under the cork
layer of the tree or superficially on the exposed wood. But on agar it
is more simple and more easily followed. It will therefore be taken
up in detail as it occurs on artifical media, and then more briefly on
the bark and on the wood, noting particularly the points in which
they differ.

Development on artificial media. The first stages can be watched
directly under the microscope in Van Tieghem cells. Cultures of
pycnospores are made just as stated previously in describing the
methods of artificial germination of these spores. At the end of
twenty-four hours they are germinating, and in about four or five
days, at summer temperatures, the beginnings of the pycnidia can
be seen. They appear first where the weft of mycelium is the thickest
but they are more easily followed if one finds them on more
isolated branches. At certain points short ceils are developed in
the hyphae by laying down of new walls, thus dividing the old cells.
The cells also increase in diameter and in the amount of cell con-
tents. Each of these short cells now sends out stubby, septate
branches, the cells of which in turn send out other branches. Such
a stage is shown in figures 1 and 2. By the continued branching—
or budding—of these cells, a tuft of hyphae is formed which re-
minds one of a witches’ broom. This tuft seems also to exert an
influence on the neighboring hyphae and the more distant branches
of the same hypha, because they now grow toward it and mingle
with its branches so that in another day or two, the mass of hyphae
becomes so dense that a surface view no longer shows what is oc-
curring. The little blocks of agar are then fixed in fixing solution,

2

18

sectioned and stained to be studied in cross section. Figure 3 shows a
cross section of a pycnidium grown in this way. It is merely a solid
ball of hyphae densely intertwined but not grown together in any way
by their lateral walls. The hyphae appear to be all alike in every
particular, that is, there is no differentiation of wall cells and core
cells.

The succeeding stages are best studied by the following method:
A single culture is made at the center of an agar plate and permitted
to grow until it has almost reached the edge of the plate. Beginning
at the center, concentric rings of pycnidia are formed as shown in
figure 51. Starting from the outermost, the pycnidia of each ring
are one day younger than those of the next succeeding ring. This
gives a perfect series of successive stages, from those which are so
small that they can barely be seen with the naked eye to fully mature
ones pushing out spore horns at the center of the plate. A perfectly
flat cross section of one on the outer ring is given in figure 4 and
shows that it corresponds to the stage observed in Van Tieghem
cells and represented in figure 3. It is merely a solid tangle of un-
differentiated hyphae. There is, as yet, no evidence of a cavity at
the center. In the next older stage, figure 5, the hyphae begin to
pull apart slightly and become loose at the center but are not other-
wise differentiated. Those branches which extend into this loose area
begin to lay down cross walls at regular intervals and as the cells,
thus formed, become mature they are cut off successively from the
ends of the hyphae and lie free in the cavity (Figure 57). These short
cells are the first pyenospores. As all the branches projecting into
the central area are cut up to make spores, the cavity is naturally
enlarged. But other branches now push in from the surrounding
hyphae and more spores are cut off from their apices until the cavity
becomes densely filled with them. The size of the cavity increases
then, first, by the constant cutting off of the branches and, second,
on account of the increased pressure from within caused by the pack-
ing of the spores. Also the crowding for space by the new conidio-
phores would tend to distend the walls. This pressure from within
causes the hyphae which are on the periphery to be crowded together
and to form a sort of a wall. This wall Jayer is not so distinet in
the pycnidia on agar because there is nothing on the outside to re-
sist the pressure but in the pycnidia on the bark it is quite distinct.
Also, the membranes of the wall cells become somewhat thicker at
this time. “A section from the wall in this stage, showing the rela-
tion of the conidiophores, is shown in figure 6. There is no ostiole
whatever at this time but a little later the hyphae become loose at
a point on the upper wall of the pyenidium and the spores are forced
out through this by the pressure from within. The ostiole is thus
formed by the same process as the cavity itself. It is very indefinite

19

at first but as it becomes older and wider, it becomes surrounded by
a more definite wall just like that of the cavity.

When fully mature, the cavity may be as much as a fourth of a
millimeter in diameter. It is usually almost circular in cross section,
but sometimes shows the convoluted form which will be described
later as occurring in mature stromata on the bark. The conidio-
phores form a dense, brush-like fringe and extend directly out into
the cavity from every point of the wall. They are of uneven lengths,
the majority being 20-40 microns long and about 1.5 microns in dia-
meter. Four of them are shown highly magnified in figure 7. In
an unstained section, the septa of the conidiophores cannot be made
out but, when properly stained with iron-alum haematoxylin and
erythrosin, the septa show up very plainly as unstained lines across
the sporophore. It will be seen that almost the whole length of the
conidiophores is divided into regular cells, each of which contains a
single nucleus. As the cells become mature, they break off success-
ively as conidia. Just how many break off from a single conidio-
phores was not determined. The majority of them are simple, but
branched conidiophores, as shown in figure 7, are not uncommon.
But they are never so frequent or so much branched in this type of
pycnidium as in the types to be discussed later. In the older
pycnidia they are longer than in the young ones. Among the con-
idiophores are certain longer branches which project further into the
cavity. These are evidently the structures which Pantanelli (89)
calis paraphyses. ‘Yet he seems to have some hesitation in designat-
ing them by that name, because in a footnote at the bottom of the
page he adds; “Non tutte si possono considerare come parafisi o
pseudoparafisi, perche talvolta formano conidii alla loro estremita.”
The writer also found pycnospores on the tips of them and. they are
also divided into the same regular uninucleate cells as the conidio-
phores. They branch like the conidiophores and, as for their length,
all lengths can be found from 75 microns down to 10 microns. One
would be excusable for wondering on what basis they would be
distinguished from the conidiophores.

Factors influencing production. As indicated above, the time
required for the production of pycnidia on artificial media is very
short. Whén ascospores, naturally ejected from the perithecia, are
caught on plates of sterile chestnut bark agar, they germinate in a
few hours and at the end of from five to seven days—where they
fall thickly on the agar—a pycnidium containing mature spores
will be formed at every point where a spore or group of spores fell.
These pycnidia differ in no way from those described above. When
cultures are made from pycnospores by making streaks on potato
agar, pycnidia containing mature spores are usually developed with-
in eight days at ordinary summer temperatures. At lower tempera-

20

tures, the time required is much longer. As previously mentioned,
plates. of the fungus exposed to out-of-doors temperatures during
the last winter showed considerable growth of the mycelium but in
no case were pycnidia produced on these plates. Also on the trees,
where the spread of the cankers was measured each month by a
painted outline, it was observed that no pycnidia or even “blisters”
were developed on the diseased areas that were added during the
winter. These experiments indicate that the fungus will grow at a
lower temperature than that at which it will produce pyenidia.

Another factor which influences the production of pyenidia is
light. When plate cultures are grown in total darkness on chestnut
bark agar, no pycnidia are developed, while on plates made at the
same time and grown in the light, the usual rings of pycnidia ap-
pear (Figure 57). Experiments were also tried in which the plate was
left in darkness until about hailf-covered with mycelium and then
brought into the light. Circles of pycnidia were developed, beginning
with the ring which marked the outermost limit of the colony when
removed from the dark chamber. The concentric rings which always
appear on agar cultures are due to the alternation of night and day.

When young trees in the woods are inoculated, the pycnidia do
not become evident as soon as on artificial media. But, even here,
the spore-horns have been observed in three weeks on inoculations
made with pycnospores. ‘Blisters,’ indicating the development of
the pycnidia under the cork layer, have been observed in eighteen
days. é

Development of pycnidia on the young canker. The first outward
indication of the pycnidia is the appearance of numerous little raised
“blisters” just back of the advancing edge of the canker (Figure
45). They are perfectly smooth littke mounds and, under the hand
lens, appear slick and somewhat translucent. Contrary to published
statements of investigators of this disease (e. g. 4: 187), they bear
no relation whatever to the lenticels. They seem rather to avoid the
lenticels. On account of their smooth, unbroken surface they can-
not be confused with the latter at this stage, but at later stages,
when they are broken open at the apices, they often give the er-
roneous appearance of having been formed in the lenticels. They are
much more numerous than the lenticels, often being so thick as to
be in contact with each other. If the cork layer is carefully re-
moved, the beginning of a single pycnidium will be found under each
of these raised places. At this stage they are hyaline, more or less
globose or biscuit-shaped cushions with a moist gelatinous appear-
ance, about half imbedded in the disintegrating collenchyma tissue,
the other half projecting upward and raising the cork layer to form
- the pimple. In size, they vary from those on the outermost edge
which are almost microscopic to those a millimeter in diameter just

21

before the breaking of the phelloderm. There is no stroma at this
time, but each one is very early surrounded with a fringe of loose
mycelium which is the forerunner of the stroma. It is at first white
but begins to turn yellow even before the cork layer is broken.
When a cross section is made of this moist-looking cushion, it is
found to be a closely wound ball of hyphae corresponding to figure
4, as decribed under the development of the pycnidium in culture.
There are no pycnospores and as yet no indication of a cavity. Frem
the periphery toward the center of the canker the cushions are suc-
cessively larger and more of the developing stroma about them until
the cushions are entirely covered by the mycelial weft, which is
now bright yellow. The cavity, sporophores and pycnosperes are
developed from this cushion in exactly the same way as described
above on agar plates and will not be again described. Where the
pycnidia originate very closely together, the stromata often come
into contact and coalesce so that we now have a compound stroma—
to all appearances, a single stroma containing several pycnidia. This
condition has been found by the writer in mature stromata several
times but seems to be rather the exception—a single much con-
veluted or labyrinthiform pycnidium in each stroma being the rule.
Apparently, even when by coalescence several pycnidia are thrown
into one stroma, the receding walls of the chambers soon come into
contact and portions of them are broken down so that there is now
one large, irregular cavity. So far as observed, the stroma never
precedes the pycnidium. A pycnidium first starts and later the
stroma forms about it. There is no rind layer on the stroma previous
to the breaking of the cork layer. This latter process is brought
about through pressure exerted by the growing pycnidium beneath.
By this time the spores have developed and soon push out in curling
tendrils through the rent in the cork layer.

Spore Horns. They are light yellow in color at first and have a
waxy appearance. As they become older they take on a reddish east.
They vary in size from the diameter of a hair to a half-millimeter and
in length from a millimeter to more than 2.5 em. The writer and
J. R. Guyer measured an exceptionally long one that was two and
one-half inches in length. On young cankers on smooth-barked trees,
they are usually small in diameter, single and twisted into several
coils, but on the bark of old trees, where they come from the lines
of stromata in the crevices, they are large, stout and irregular and
often a whole line of them are united comb-like. Figure 48 shows
this condition in which they are coming out from rough, burnt-over
bark. In cross section, the horns are usually flat or irregular in
shape, and only rarely circular. This accounts largely for the way
they curl. The irregular twisting is shown in figure 47. When dry,
they are hard and brittle, and it takes some little effort to break

22

them loose. It is doubtful if a wind is ever strong enough to break
them off and carry them away when dry. But when they become
wet, they swell and the spores—of which they are entirely com-
posed—separate and wash down the tree, but as soon as the rain is
over, new spore-horns appear with surprising rapidity. Just how
long a pyenidium will continue to produce spores has not been deter-
mined. During the last season, on young cankers produced by in-
oculation in the spring, the horns were abundant after each rain
until the latter part of the summer, when the pushing out of the
stromata indicated the beginning of the perithecial stage. After
that, very few spore-horns were found on these cankers. Heald and
Gardner (93) have shown that the pycnospores are produced in the
winter. Except in cases where they were protected and kept dry,
so that tendrils produced in the sumraer were not washed away,
the writer has not seen spore-horns in the winter, but this is’ preb-
ably due to the fact that they are produced at such a slow rate that
they are washed away before their size makes them noticeable. They
first began to appear this season, (1913), about the middle of April.
Pycnidia in the older stromata. About the middle of the summer,
on cankers produced by inoculations in the spring, there is an active
increase in the amount of stromatic tissue, and the pycnidia in the
top of this new stroma are pushed out through the cork layer. Mean-
while they continue to increase in size. During this increase, the
cavity does not remain round but becomes intricately labyrinthi-
form, as shown in figures 11 and 55. This shape is easily explained
when one considers the method by which the pycnidium increases
in size. As previously indicated, the walls are constantly receding
in all directions. The new stromatic tissue is mingled with portions
of the disintegrating host tissue, and when the receding wall comes
in contact with this tissue, it continues to recede on both sides of
it, but the part around the obstruction remains as a process jutting
out into the cavity. This is repeated many times until often the
entire stroma will be found honeycombed with numerous but com-
municating irregular chambers. A simple case is shown in figure
55. This explanation accounts for the shape of the pyenidium only
in part because this type is sometimes found on agar cultures where
there are evidently no such obstructions. When cross sections of
the stromata are cut, a single section usually shows a number of
cavities which do not appear to be connected, but if the entire stroma
is cut into serial sections, it will usually be found to contain but
a single many-chambered pycnidium. Occasionally however, the
writer has found stromata which contained three or four distinct
pycnidia.
The pyenidial form of this fungus has often been referred to th
genus Cytospora, based on the idea that the stroma typically con-

23

tains a number of pycnidia. Evidently this is a mistake. If there
is need of a distinct generic name for this stage, it should be referred
to Endothiella, a genus erected by Saccardo, (Ann. Myc. 4:73),
based on the imperfect form of Endothia gyrosa. Sacecardo did not
apply this name merely to the superficial type on wood, but under
this word he included all forms of the pyenidial stage. The laby-
rinthiform pycnidium in the mature stroma becomes larger than
the forms developed on agar and on wood. Cavities more than a
millimeter in diameter have been found by the writer. Besides
differing somewhat in shape and size, this type also differs from the
type on agar in that the wall layer is more distinct, and the conidio-
phores are more branched and longer.

Superficial pycnidia. Another form of the pycnidium is found on
the cut ends of stumps and logs and both on the wood and the inside
of the bark where the latter has broken loose and an air space is
left between it and the wood. These are superficial, single pycnidia.
A group of them is shown in figure 12. <A favorite place for them
is on the inside of the bark where it has drawn away from the
stump around the top, after the tree is cut. Also after a log or
stump on which there was a canker is peeled, the pycnidia will de-
velop on the surface very quickly if it does not dry out too soon.
Their production is largely dependent on the water supply. This
is illustrated by the fact that in dry weather they will develop on
the lower side of a log lying on the ground, but not on the upper
side. Their shape also varies with the amount of moisture. In the
more moist, Shaded situations, they are long pear-shaped or conical,
as shown in figure 12, or the base may be flattened out slightly on the
substratum. But on tops of stumps—where they occur abundantly
on the outermost four or five annual rings, and where the supply
of moisture is not constant—they are flattened out on the substratum
and do not stand out free as shown in the figure. Also they have
more of a tendency to run together here. In color they are deeper
red than the stromata, but have light yellow conspicuous ostioles
which project upward in a sort of neck or beak.’ They are surround-
ed by no stroma whatever, and stand out free so that they can easily
be picked off with a dissecting needle. They measure about a quarter
of a millimeter in diameter and the same in height. The outer wall
is perfectly smooth as seen under the hand lens. Often several of
them grow together, but their ostioles remain distinct and we have
the appearance of a single pycnidium with several ostioles.

The writer has not seen all the developmental stages of this type,
but theze is no reason to believe that they differ essentially from those
on agar or under the cork layer. A cross-section of one when ma-
ture, (fig. 54), shows no differences in the configuration of the cavity,
the character of the conidiophores, etc. The walls are thicker and

24

much more dense, however, and the ostiole is more perfectly formed
than in the others previously observed.

Usually, this type of pycnidium is not followed by the perithecia,
but in two cases, where they were between the bark and the wood,
the writer has found perithecia developing among them.

STROMATA.

The stromata are more often seen and better known than any
other stage of this fungus. They are the reddish brown cushions
mentioned in the introduction, which are scattered thickly over the
canker and make it so conspicuous and easy of diagnosis. A canker
thickly beset with them is shown in figure 44. The beginning of the
stroma has been mentioned in treating of the pycnidium. As stated
there, it always starts as a loose growth of hyphae around the
pycnidium. It does not precede, but follows the first stages in the
development of that organ. This stage of the stroma may often be
observed on agar cultures where the pycnidia are rather far apart.
A fluffy growth of light yellow mycelium surrounds the pycnidium,
and covers it over until often nothing can be seen but a mass of
spores oozing from the top of a loose ball of hyphae. If these are
imbedded and sectioned, they will be found to contain a loose tangle
of undifferentiated hyphae surrounding a central pycnidium. No
rind layer is produced under these conditions. This corresponds
to the stage on the bark which precedes the rupturing of the cork
layer. But as soon as the cork layer is broken, the stroma under-
goes a change. There is a rapid increase in size, and at the same
time, a differentiation of the cells at the tips of those branches which
reach the exposed surface. These cells now become shorter and
thicker, acquire heavier walls, and are densely crowded together,
so that in cross section they appear as a pseudoparenchymatous tis-
sue (Fig. 10). The rind thus formed covers all of the exposed sur-
face of the stroma, and also grows up around the necks of the
perithecia (Fig. 11). The cells are pretty well filled with protoplasm
and stain deeply. They also contain more pigment than the other
cells. The interior or medulla of the stroma remains the same. As
shown in the base of figure 10, it is merely a loose tangle of hyphae
which are much branched and more often septate, but in all other ~
respects, like the usual vegetative hyphae. The cell contents, nuclei,
vacuoles, walls, ete., are just the same. They also contain a large
amount of pigment. Stone cells, bast fibres and remnants of the
walls of the collenchyma cells are scattered through the basal parts.
A diagrammatic drawing of a stroma showing the location of the
pycnidium, perithecia and rind layer is given in figure 11. When
they first come through the cork layer, they are lemon yellow in color
but with age the color deepens to orange, reddish brown and finally

25

cinnamon brown, But when cut into, they are found to be lighter
colored on the inside than on the surface. Fully mature, they aver-
age about 2.4 x 1.2 millimeters in size, being usually elongated hori-
zontally as shown in figure 44. They average about 1.3 millimeters
in depth. The size however, depends largely on the location and
the season. If they grow in a moist situation they are much larger
than where they are exposed to desiccation. On old rough bark,
they do not occur as shown in figure 44, but come out only in the
crevices of the bark, often united in a solid line for several inches
so that they apparently form one long stroma. Otherwise they do
not differ from those described above.

PERITHECIA.

Previous to the beginning of the perithecial stage, the cork layer
has been broken only by the emerging spore-horns. The small
amount of stroma that is developed lies entirely beneath this cork
layer, that is, none of it is erumpent as yet. The change to the peri-
thecial stroma has been observed within eight weeks after inocula-
tion. On trees inoculated in June the stromata have been observed
in August. The stroma increases very rapidly in size and pushes
off more of the cork layer. Not only does it fill up the enlarged rent
in the phelloderm, but it also grows out over the torn edges to some
extent so that they are included in the stroma as shown in figure
11. If one peels off the cork layer now, either the entire stroma, or
at least the top comes off with it. The stroma now has an erumpent
superficial appearance as shown in figures 48 and 44.

Primordia. When we speak of the perithecial stroma, however,
we do not mean that it contains perithecia as yet. Spot infections
have been under observation where the perithecial stromata were
in abundance on all the cankers in the early spring, but there was no
outward appearance of perithecia during the entire summer. On
the other hand pycnospores may be pusked out from these stromata
in numerous spore-horns during the entire season. Cross sections
of these stromata show that the pycnidia are now located in the
periphery, the mass of stroma having been formed beneath them and
pushing them out through the cork layer. Their location is shown
in figure 11.

The most noticeable feature in a cross section at this stage is the
numerous primordia—the earliest stages in the development of the
perithecia. These arise usually in the tissues of the bark below the
base of the original pycnidium and by their growth and the growth
of the new stromatic tissue about them, they push these disorganized
elements upward and apart so that scattered fragments of them are
found included throughout the base of the stroma. The primordia
do not always originate however in the lower layers. At times they

26

may be found well up in the stroma without a trace of the disorgan-
ized bark about them. A stained cress section shows one or two
very prominent large, deeply stained cells at the center of each
primordium, and running around these in close concentric circles
are enlarged strands of mycelium. ‘These latter also stain quite
heavily so that the stain may be taken out of all the rest of the
stroma and still leave the primordia quite prominent.

The number of primordia in a single stroma may be very large—
over one hundred having been counted in one. They fill up most of
the available space in the base of the stroma and are often so close
that they give the appearance of double or triple primordia. AI of
them however, do not develop into mature perithecia on account of
the lack of space and possibly of food supply. When the perithecia
are mature there are usually fifteen to thirty in a stroma. This
means that one out of every four or five primordia reaches maturity.
Their degeneration takes place at all stages almost up to the mature
perithecium, but by far the greater number never get past the as-
cogonial stage. Sections of the stroma at any subsequent stage will
show these starved primordia in the base. Both the ascogonial cells
and the enveloping hyphae lose their contents almost entirely, and ap-
pear as empty cells which no longer take the stain like those of the
healthy primordia and are usually pressed out of shape by the
growth of the latter.

The large central cells are part of the organ which was first known
as. the Woronin Hypha but now more commonly called the ear-
pogonium. The cells of the carpogonium lying within the envelop-
ing hyphae as described above are the ascogonial cells, or simply
the ascogonium. In a thin section usually only one or two of them
is seen, (Figs. 19 and 20), but if serial sections are examined, it
will be found that they number from two to five in each primordium
and are wound into a circle or, more often, a spiral of one or two
coils. Occasionally, the entire structure may be seen in one section
as shown in figure 21. The cells are elongate, oval and slightly
curved to fit into the segment of the spiral of which they are a part.
Fully mature, each measures about 10 x 25 microns. They are deeply
constricted at the septa and apparently are only loosely connected;
in fact in prepared sections they are very frequently not in contact
at all—especially the older ones.

They are very densely filled with protoplasm, and for this reason,
easily brought out by differential staining, retaining the protoplasmic
stains with great tenacity. They are best stained with Heidenhain’s
iron-alum haematoxylin and erythrosin. The nucleoli are especially
tenacious of the haematoxylin, and in a properly differentiated cell,
the writer has counted as high as eighteen nuclei. They may be
quite readily brought out by Flemming’s triple stain. These two

\

27

stains have been used interchangeably, their relative efficiency de-
pending on the points to be brought out and the stage under con-
sideration. Outside the nucleolus, however, the resting nucleus does
not retain the stain when treated with the haematoxylin and a
definite nuclear membrane is made out only in the more favorable
cases. The usual appearance of the nucleus is shown in figure 20,
merely an intensely stained nucleolus surrounded by a circular clear
area. The nuclei are much more numerous in the ascogonial cells
than in the cells of the enveloping hyphae, usually only about two
to five appearing in each of the latter. They are also larger and
more prominent.

The ascogonial spiral does not terminate inside the primordium
but is continued up through the stroma as a large-celled, prominent,
deeply staining thread. The thread can be traced entirely to the
surface of the stroma. The cells are of a less diameter than in the
cells of the ascogonium and not curved and do not show such deep
constrictions at the septa. The cell contents, including the prom-
inent nuclei, are the same as in the ascogonium. Fourteen nuclei

‘have been counted in a single cell. This thread has been called the

trichogyne and the writer will continue to use that term, not im-
plying by so doing that it has the functions of a true trichogyne.
They are often found branching, and in the upper part of the stroma
they may be distinguished in great numbers on account of their

avidity for stains. It is not so easy to trace them through the

pseudoparenchymatous rind because the cells of the latter are_quite
compact and stain deeply. The apical cells usually project slightly
beyond the surface.

So far as could be determined, the trichogyne is a useless organ
in the development of the perithecium. It is probably a remnant
of an ancestry in which a copulation with a free spermatium was
essential to the further development of the carpogonium. Lindau*
has suggested as the function of a similar organ in the lichens the
breaking of a way through the thallus for the emerging apothecium.
A similar function here, that is, making a path for the advancing
neck of the perithecium, is very doubtful. The trichogyne threads
become less distinct as they become older and finally cannot be seen
any more.

The stage containing the mature ascogonia is evidently a resting
stage for it has been found more numerously than any of the other
developmental stages of the perithecium. As a rule, the primordia
of one stroma are all in the same stage. The writer hoped to find
stromata in which the primordia were all in a younger stage, in
which he could determine the exact origin of the ascogonium. Up
to the present however, he has not secured such a stroma, and has

*Lindau, G. ‘‘Uber Anlage und Entwicklung einiger Flechten Apothecien.’’ Flora, 1888.

28

had to depend on a relatively small number of apparently incipient
primordia which were found in older stromata. The earliest stages
found are represented in figures 15, 16 and 17. They show merely
a coiled hyphal branch, somewhat larger than the stromatal hyphae
which surround it and taking the stain very deeply. In figure 15
there is no indication of a differentiation of the surrounding hyphae
to form the envelope. Figures 16 and 17 show the beginning of such
a differentiation. Whether this young ascogonial branch is a new
formation, or whether it is merely a transformed pre-existing branch
of the mycelium, could not be determined with certainty, but the
writer is inclined to the latter view by what evidence he has seen.
The envelope is differentiated from the surrounding hyphae, and is
in no direct connection with the ascogonial branch. As the as-
cogonial cells increase in size, the number and size of the enveloping
cells also increases as indicated by the succession shown by figures
AG LT elS, ete:

Degeneration of the ascogonium and growth of the enveloping
hyphae. Figure 21 shows the highest point of development in what
we have called the ascogonial stage. The entire primordium is now
about 50-75 microns in diameter. The material from which this fig-
ure was drawn was taken in the late fall. In the first week of the
following March, material was collected from the same tree, and all
the primordia now appeared in cross-section like figure 22. This is
the beginning of a new stage of development. The seat of activity
seems to have been removed from the ascogonium to the enveloping
hyphae. From this time on, the ascogonium degenerates. The dense
protoplasmic content gradually disappears, and now the contents are
represented either by ragged bridles across the lumen and irregular
masses around the walls, as shown in figure 22, or else the entire
contents draws up into a misshapen mass which stains very deeply
with safranin.

The behavior of the enveloping cells is quite the contrary. Their
contents now becomes more dense and retains the protoplasmic stains
more deeply than the ascogonial cells. Their nuclei also become more
prominent and apparently more numerous. Up to this time the in-
dividual hyphae can be traced, and there are open spaces between
them; but now they have increased both in size and in number, and
filled up the intervening spaces. They appear as a pseudoparenchy-

matous tissue instead of a coil of hyphae. The increased growth |

presses in the sides of the ascogonial cells which now have nothing
within to keep up their turgor.

The most important question at this time is in regard to the
branching of the ascogonium. Reasoning from analogy with many
other Ascomycetes, we would expect the ascognia to give rise to
ascogenous hyphae before their degeneration. Many hours were

29

spent searching for these hyphae. Only in a few cases was a con-
dition found which would lead one to believe that there were such
branches. Three of these cases are shown in figures 24, 25 and 27.
All of these, however, occurred when the ascogonium was about ready
to break down. <A distinct opening between the ascogonia and these
cells could be made out. The cells of these “apparent branches” differ
little from the surrounding cells except that the first cell is usually
almost devoid of contents, like the ascogonium. Since there is no
way of distinguishing them from the surrounding cells, their identity
cannot be determined in subsequent stages. In the vast majority of
cases, no such branches were found, but this may have been due to
a lack of sufficient material in the right stage for observation of this
point.

Beginning of the differentiation. The primordium now increases
very rapidly in size. The cells at the center grow more rapidly than
those at the periphery and at the same time the contents become more
vacuolar. The reciprocal pressure gives them more and more the
appearance of a pseudoparenchymatous tissue. The peripheral cells
on the other hand become elongated and flattened by the pressure
from the center, and at the same time are less vacuolar than the
central cells. This stage is shown in figure 23. As yet there is no
sharp differentiation of the wail cells. The crushed remains of the
ascogonium are occasionally seen at this stage but have not been
found later.

This period also marks the beginning of the neck, which is in-
itiated by a vigorous outgrowth of small cells at a point of the
periphery toward the exposed surface of the stroma, forming a blunt
cone (Fig. 23). The cells are very compact and have a dense pro-
‘toplasmic content with several small nuclei in each cell. It is not
possible at this time to trace individual hyphae in the young neck.
No canal is evident.

The next step marks a complete differentiation of the core cells
and the cells which are to form the wall of the perithecium. The
cells at the center become larger and still more vacuolated. The
membranes remain very thin. They form a perfectly spherical core
and are set off by an even line from the wall cells which have now be- -
come more distinctiy elongated and flattened. The membranes of
the latter cells become thicker and the contents still remain dense
so that it is now easy in stained sections to tell the exact dividing
line between wall and core. The distinctness of this line gives the
impression of two different tissues. A camera lucida drawing of
a few cells on either side of this line is given in figure 28. It will
be noticed here that one of the cells seems to be differentiating into
a core cell at one end and a wall cell at the other. Such a condition
indicates that these two tissues are not of different origin. The

30

core now measures about 135 microns in diameter and the wall is
composed of eight to twelve layers of cells and is about 35 microns
in thickness.

Pathological conditions. Peculiar pathological conditions of the
young perithecium are numerous at this as well as previous stages.
The delicate-walled core cells break down very easily and primordia
containing a central cavity, even before the beginning of the neck,
are common and misleading to any one searching for the normal be-
ginning of the cavity. Frequently very fine hyphae are found enter-
ing between the corecells and apparently living parasitically upon
them, causing them to break down and thus furnish a rich pabulum
for the invading hyphae. Soon a dense, deeply stained tangle of these
hyphae fills the lower part of the cavity. These are not the asco-
genous hyphae, as the writer suspected when he first saw them, and
such perithecia develop no further but may often be found crushed
out of shape between the naturally maturing perithecia.

The cavity and paraphyses. The normal formation of the cavity
appears about the time the length of the neck equals the diameter
of the perithecium. A portion of the cells in the lower part of the
core—not on the periphery of the core but inward by about two to
four layers of cells—begins to break down, and in this cavity are
now found only scattered, irregular masses of protoplasm, degener-
ated nuclei and occasionally a part of a wall. Sometimes an entire
cell may remain intact even after all the cells about it have broken
down. But there is never a large cavity at any one time. As soon
as a few cells are broken down, the cells which border on the cavity
below begin a new period of activity. Even at this time they can be
distinguished by more prominent and numerous nuclei; the walls
are more distinct and the contents increases slightly in density.
These are the initial cells of the paraphyses which are now pushed
out into the cavity and follow its receding upper limit. Their origin
is shown in figure 29. They very soon become septate and at subse-
quent stages their origin would be hard to determine. They are
composed of short, plump cells, very rich in protoplasm, staining
very deeply, and containing several nuclei. The paraphyses branch
frequently and are very crooked, and, hence difficult to trace indi-
vidually in thin sections. Not only do they extend upward into the
cavity, but some of them run around the periphery and send out
frequent vertical branches into the cavity. They line only the bottom
and never come from the roof, at which place the core-cells remain
intact for a long time. A perithecium in a rather young paraphyses
stage is shown in figure 30. It is now about 200 microns in diameter.
There are no ascogenous hyphae or young asci at this time. The outer
wall has become more pronounced and is distinctly divided from
the bases of the paraphyses by several layers of large, clear core cells.

31

As the paraphyses become older, their component cells become more
elongated and slender. When the young asci appear they begin to
lose their dense contents and are soon not easy to distinguish.
But even after the first asci are mature, they may be seen as slender
filaments devoid of contents except for the nuclei, which persist for
a long time. Their function is probably to nourish the growing asci.
The asci. The writer was unable to determine the origin of the
ascogenous hyphae. The young asci arise as branches of a system
of hyphae which appear among the bases of the paraphyses, but
which cannot be distinguished from the paraphysogenous hyphae
by staining reactions or otherwise. They are undoubtedly a different
system and in no case has an ascus and a paraphysis been seen
coming from the same hypha. At the time the asci first appear the
perithecium is about 250 microns in diameter, and the neck is near-
ing the surface of the stroma but has not yet begun to turn black. So
far as could be determined from the material examined, the asci arise
as ordinary lateral or terminal branches. The young ascus is broadly
clavate. In the uninucleate stage, the protoplasm is gathered -about
the large nucleus, which is usually at the center, the ends being less
dense and therefore taking less stain. By three successive divisions,
eight nuclei are produced and the protoplasm about them becomes
clear and is soon closed off from the epiplasm by a membrane. But,
at the same time, the nucleus is dividing again and by the time the
wall can be distinguished, there is also a distinct septum in the
spore. This condition, in which there is a single nucleus in each
end of the spores, does not persist very long but soon there is another
division, making two nuclei in each end and frequently, by successive
divisions, the mature spore has three or four nuclei in each end, as
previously stated. The details of the nuclear divisions and the
cutting out of the spores in the ascus, being purely cytological and
outside the scope of this work, were not followed more closely.
Mature asci with the spores in place are shown in figures 34, 35
and 36. The arrangement of the spores in the ascus is irregularly
uniseriate or subbiseriate. There is, however, no uniformity in their
arrangement and two asci can hardly be found in which the spores
are placed alike. The epiplasm is still very distinct, especially where
it tapers to a point at the top of the ascus. There is a thickened
ring—reminding one of a doughnut—about the upper extremity of
the lumen of the ascus which is very prominent and shows peculiar
Staining reactions. It has been suggested that it is at this point
that the top of the ascus breaks off to free the spores. This explanation
is at least, plausible, but the writer has never been able to find the
asci in the process of liberating the spores, and is therefore, unable
to confirm the theory. When the ascus is lying flat on the side—as
is practically always the case in water mounts, the ring appears in

32

cross section as two highly refractive disks such as is shown in
figures 35 and 36. As figure 34 shows, the spore-bearing part of the
ascus is only about three-fourths of its total length. But in dried
specimens the point draws down until the ring is very close to the
spores as shown in figure 36. The natural shape is not recovered at
once on placing the ascus in water. This fact should be taken into
account in making measurements. It is best to use only fresh speci-
mens. Murrill (4), gives the dimensions of the ascus as 45—50x9
microns. The average of one hundred and fifty measurements made
by the writer was 51.2 x 8.9 microns.

Development of the neck. Even before the complete differentiation
of the core- and wall-cells, it is noticeable that the cells on the upper
side are pushing outward in a sort of a knob, and by the time the core
has become distinct, this structure has become a definite cone as
represented in figure 23. At this time the cells are small and very
compact, and distinct hyphae cannot be made out. The cone is a
perfectly solid mass, that is, there is no indication of a canal in the
center. But as the hyphae elongate toward the surface of the
stroma, they become less entangled, running almost parallel, converg-
ing toward the apex of the advancing cone and leaving an open canal
through the center. This advancing apex is shown in figure 31.
The hyphae, are slender, very densely filled with protoplasm and,
therefore, stain quite deeply. The arrangement is loose and indi-
vidual hyphae can be traced for long distances. The septa are far
apart. The converging apices are usually somewhat swollen. As
the apex pushes toward the surface, the stromatic hyphae are not
destroyed but are merely wedged apart to make room for the neck.
At a distance of about 50-75 microns from the apex, it will be noticed
that the hyphae are increasing in diameter and new branches are
being inserted. This process continues until the wall of the neck is
composed of densely packed hyphae and is quite firm. The walls of
these cells also become thick, and about the time the apex has
reached the surface, they become black. The apices of the branches
which extend into the central canal, however, do not take on these
latter characters but remain thin-walled and loose. These are the
periphyses. They extend outward and upward and their apices
almost come into contact. They are shown in figure 32. They are
confined to the neck and never occur within the perithecium proper.
But as yet the canal in the upper part of the neck is separated from
the cavity of the perithecium by the upper wall of the latter and
the cells of the solid cone which formed the beginning of the neck.
About the time that the paraphyses are maturing in the cavity, the
cells in a direct line from the cavity to the upper canal begin to draw
apart and to react differently to stains. These cells have not become
thick-walled like the other cells of the perithecial wall. There is prob-

33

ably also a disintegration of some of the cells which formed the
perithecial wall, but not of the cells of the original cone. These
latter merely draw apart, and the cells left projecting into the canal
thus formed take on the character of periphyses. Also where the
canal breaks through the wall, some of the cells are left projecting
like periphyses. These periphyses in the lower part of the canal differ
from those in the upper part in their irregularity, and in not pro-
jecting upward at an acute angle. An early stage in the formation
of the lower canal is shown in figure 32.

It is impossible to tell whether the neck follows the course taken
by the trichogyne up through the stroma since the trichogyne has en-
tirely disappeared by this time. The stroma is usually much broader
at the bottom than at the place where it breaks through the cork
layer. For this reason the necks seem to converge at the top. The way
in which the necks bend to get through the cork layer is shown in
figure 53. Where a broad stroma has formed, however, and a large
area of the cork has broken away, the necks extend almost straight
upward. There is not naturally a distinct valsoid disk in which all
the necks converge. The arrangement is diatrypoid rather than val-
soid. This fact is of importance in placing the species in its proper
genus. The neck does not usually end flush with the stromatic surf-
ace, but extends beyond as a little papilla (Fig. 11). The distance
to which the papilla extends depends largely on the location of the
stroma and the conditons under which it grows. In a dry situation
with plenty of sunlieht, it may hardly project at all, while in
shaded places and esrecially where it is moist, it may project more
than a millimeter. Much longer ones may be produced by developing
them in moist chambers. These papillae are not composed entirely of
the hyphae which grow out from the wall of the perithecium but as
they push out beyond the surface, the rind tissue grows up about
them. A cross section of a papilla is shown in figure 33. If the ad-
vancing apex of the neck encounters a pycnidium in the stroma, it
grows directly through it or occasionally may curve slightly around
it

The mature perithecium. When mature, the perithecium measures
about 350-400 microns in diameter and is mostly spherical in shape
but the shape is often modified by pressure of other perithecia. As
seen under the hand lens, the wall is gray or lead colored but not jet
black and shining like the wall of the neck. In cross section, the
wall now appears thinner than when the perithecium was young,
and the cells are more flattened. The cell-walls are heavy. The struc-
ture of the perithecial wall is shown in figure 30. The layers of large
core cells which previously divided the contents of the cavity from
the wall, have now entirely collapsed and, as a result, the ascus
mass is only loosely attached to the wall, and usually pulls away in

3

a4

sectioning. The entire cavity is now tightly packed with asci. The
older ones, having been pushed up are at the center and in the upper
part, and the younger ones lining the walls. The writer has calculated
the number of asci in a full perithecium at 3600, or 28,800 spores.

Ejection of the spores. Rankin (59) has discovered that the asco-
spores are forcibly ejected from the necks of the perithecia into the
air, and showed that this occurs only during periods of rain.
Heald and Gardner (76,93) demonstrated the effect of temperature,
showing that expulsion does not take place below 52° F., and that
after being subjected to lower temperature, it requires three or four
days of favorable weather to cause further ejection. The writer and
Babeock (95) studied the phenomena of ejection with especial refer-
ence to its bearing on dissemination. The most essential factor in
producing ejection was found to be an abundance of moisture. Under
the hand lens it will be noted that there is a film of water over the
tip of each active ostiole, and that at each discharge this film is
broken and usually eight spores are shot outward, that is, the con-
tents of one ascus. What causes these asci to leave the body of the
perithecium and come up to the mouth of the neck was not determined
at that time.

If a fresh stroma containing mature perithecia is cut across with
a razor, the cut surface will remain level except where the perithecia
were cut through. Here the viscous contents will bulge out in a
prominent bead, showing that there is a tension inside the peri-
thecium. This is the force which drives the asci up through the
canal. There are at least three factors which aid in producing this
pressure: (1) The asci do not all mature simultaneously. Young
ones are continually pushed up between the bases of the older ones.
As they become mature they are pushed up into the center and upper
part of the cavity which is soon densely packed, and new ones are
still pushing for space. The remaining layers of core cells are first
pressed out flat against the walls. (2) But when they would tend
to pass out the canal of the neck, the periphyses act as so many
little springs and press them back. (3) The most immediate cause
of the outward pressure, however, is the swelling of the asci them-
selves when they become moist. Figure 34 represents an ascus which
has been kept in water for several hours. When it is dry, the ascus
wall is drawn so tightly up around the spores that it can hardly
be distinguished at all except at the top. Figures 35 and 36 show
stages of this process. The entire structure occupies less than half
the space occupied by the distended ascus. Thus the sudden addition
of water, tending to double the volume of the perithecial contents,
would easily drive the asci up the neck to the surface. Prepared
sections of perithecia which were fixed during the process of ejection,
showed that up to the tip of the neck the spores are still in the

ascus. Since the asci are never ejected into the air, it follows that
they must burst and liberate the spores when they arrive at the
surface film at the tip of the neck.

SUMMARY OF RESULTS.

1. Hach pycnospore contains a single nucleus which divides several
times before germination, and a polar body at each end. The asco-
spore contains from one to four nuclei in each cell.

2. Ascospores germinate readily in water; pycnospores require a
nutrient medium. Pycnospores germinate on twigs of a large num-
ber of common forest trees. They also germinate in humus about the
base of the tree.

3. At summer temperatures, pycnospores germinate in 12-36 hours;
ascospores in 2-12 hours. Lower temperatures retard germination.

4. Both kinds of spores swell greatly before germination.

5. Pycnospores usually germinate by two tubes and ascospores by
four.

6. Ascospores in the perithecia and pyecnospores in the “horns”
retain their power to germinate at least a year. The longevity is
diminished when the spores are separated from each other and when
exposed to the air.

7. Winter weather conditions do not affect the vitality of either
kind of spores.

8. The cells of the mycelium are multinucleate under all condi-
tions. They are densely filled with protoplasm when young but be-
come vacuolated as they become older.

9. The mycelium and pycnospores are colored by a yellow pigment
belonging to the aurine group of compounds.

10. The mycelium does not invade the living tissue as individual
hyphae, but in flat fan-shaped mats.

11. The mycelium continues to grow in the bark even during the
winter months but much more rapidly in the summer. Its vitality
is not affected by winter temperatures.

12. The fungus may be carried over in the bark for a year or
more by the mycelium even when the bark is kept dry.

13. The pycnidium is produced symphiogenetically. In the simplest
type it is merely a loose tangle of hyphae, the central branches of
which become the sporophores. It has a indefinite ostiole.

14. The sporophores are branched and the pycnospores are produced
successively from their tips.

15. Pyenidia are not produced in the absence of light.

36

16. The pycnidium is started before the stroma is formed. It
occurs directly under the cork layer and bears no relation to the
lenticels. The stroma is formed about the pycnidium and typically
there is but a single pycnidium in each stroma.

17. Stone cells, bast fibers and walls of the collenchyma cells are
contained in the basal parts of the stroma.

18. The perithecia are produced at the base of the stromata in
which the pycnidia are contained.

19. The beginning of the perithecium consists of a coil of large
cells—the ascogonium—surrounded by “enveloping hyphae.” The
ascogonium is continued up to the surface of the stroma in a promin-
ent trichogyne. :

20. The trichogyne is not functional as such.

21. The perithecium is differentiated from the “enveloping hyphae.”

22. The cavity is formed by the breaking down of the core cells.

23. Paraphyses grow out from the wall into the cavity and almost
fill it. They have almost disappeared when the asci are mature.

24. The asci arise as branches of hyphae among the bases of the
paraphyses.

25. The neck of the perithecium is produced by an outgrowth of the
hyphae on the periphery of the forming perithecium.

26. The spores, still in the asci, are forced out of the body of the
perithecium and up to the tip of the canal by (a) the continued
growth of young asci from the walls, (b) the swelling of the asci when
they become moist.

f.

10.

Lf:

12.

13.
14.

15.

16,

Lt.

37

BIBLIOGRAPHY OF THE CHESTNUT TREE BLIGHT.

06

06

06

06

708

09

Merkel, Herman W. A deadly fungus of the American
chestnut. Tenth Ann. Rep’t. of the N. Y. Zoological
Soc. for 1905; 96-103. Jan. 1906.

Murrill, W. A. A serious chestnut disease. Jour. N. Y.
Bot. Garden 7: 143-158. June 1906.

Murrill, W. A. Further remarks on a serious chestnut
disease. Jour. N. Y. Bot. Garden 7:203-211. Sept.
1906.

Murrill, W. A. A new chestnut disease. Torreya 6:186-189
Sept. 1906.

Taylor, W. A. Article.on chestnut. Cyc. of Am. Hort. by
L. H. Bailey and W. Miller 2:294-297. 1906.

Rehm, H. Ascomycetes exs. Fasc. 39, No. 1710. Ann.
Myce. 5:210. 1907.

Metcalf, Haven. The immunity of the Japanese chestnut
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20

33.

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09

09

10

“lh

lt

38

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ati

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71T

at

aint

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"12

AD,

"12

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40

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76.

81.

89.

90.

He

712

712

712

712

712

712

712

712

"12

as)

41

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"13

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18 Penna. Chestnut Tree Blight Commission; Report July 1

"13

"138

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NOTE,—Many titles were added to original proof sheets.

43

EXPLANATION OF PLATES*

12) bys\I RIB;
Figs. 1, 2. Initial stages in the development of the pycnidium,
x 230.
BLS 2733 Cross section of a pycnidium on agar before the begin-

ning of the cavity. x 400.

Fig. 4 Same as figure 3 but a little older. x 430.

Fig. 5 Beginning of the cavity in the pycnidium. x 430.

Fig. 6 Section of pycnidial wall showing conidiophores.
x 4380.

i Bi at Conidiophores. x 800.

Bre 22S; Mycelium from agar. x 800.

Biess.< 9: Section of a ray from the fans in the bark. x 480.

Fig. 10. Section of a stroma showing the rind layer. x 600.

Byres 11. Diagrammatic drawing of a stroma showing the rela-
tion to the cork layer and of the organs to each
other. “x 25.

Fig. 12. Superficial pycnidia. x 14.

Fig. 13. Section of a germinating pyenospore. x 700.

Fig. 14. The resting pycnospore. x 3500.

PLATE II.

Fig. 15, 16, 17, Initial stages of the carpogonium. x 650.
Fig. 18. to 21, Later stages of the ascogonium. x 650.

Fig. 22. Degeneration of the ascogonium and growth of the
enveloping hyphae. x 650.
Fig. 23. The young perithecium and the beginning of the stage

of differentiation. x 650.
Fig. 24, 25,27, Apparent branching of the ascogonium. x 650.
Fig. 26. Degeneration of the tricgogyne cells. x 650.

PARE Een:

Fig. 28. Wall and core cells. x 650.

rea: Beginning of the paraphyses. x 650.

Fig. 30. Perithecium in the young paraphyses stage. x 230.
Fig. 31. Advancing tip of the neck. x 500.

Fig. 32. Lower part of the canal in the neck. x 460

Fig. 33. Cross section of papilla showing periphyses in the

neck. x 260.
Fig. 34, 35, 36, Asci showing stages of drying up. x 650.

*All drawings made with the aid of camera lucida except 11 and 12.

44

Mature ascospores. x 900.

PGA Vi

Outline drawings of germinating pycnospores.

PLATES V AND VI.
Germination of pycnospores.
Germination of pycnospores.

PLATES VII AND VIII.
Germination of ascospores.
Germination of ascospores.

PLATE IX.
Canker showing atrophy.
Canker showing stromata.
PLATE X.
The blister stage.
Stromata showing papillae, indicating the perithecial
stage.
PLATE XI.
Spore horns on smooth bark.
Spore horns in crevices of rough bark.
PLATE XII.
Canker outlined with paint to indicate monthly
growth.
PLATH XIII.
Mycelial fans under the bark.

Jed byNOLID) DIE

Rings of pycnidia on chestnut agar cultures.

PLATE XV.
Photomicrograph of pycnospores.
Vertical section of a perithecium.
PLATE XVI.
Photomicrograph of pycnidium on wood.
Stroma containing labyrinthiform pyecnidium,
PLATE XVII.

Vertical section of stroma showing empty perithecia
and the black necks.

Vertical section of young pycnidium on agar showing
early stage in the formation of the cavity.

PLATE I.

Development of Pycnidium.

am hy:
te a
PAM ee

Taye!

PEAS y

le

PLATH II.

Development of Perithecium.

SN
SE SS wr

UAE =

oh Lf S
LOD ae =<
eh n0G RS
N Wig Z sonige TS
eA i Ry
RYE WA? sh
A elt 3

AS
Lx

=

TO

aor,

ar ace

.
ae
: oe
DUS
pe ee
ei

=
Ty
ae s

oy =
ObseaneaS Sars
Ly TS Poe

GAC ole

Development of Perithecium.

:
Ae

ne
roe

RESTING 2 AST.
SPORE

3°10 A.

ONE SPACE 18//UCRONS

PLATH V.

Germination of pycnospores.

:s
‘

CW Aad Mod 7

RESTING SPORE

6 44

S

XS
8 N
Eq = s
NY N

NS

:
&

&

g a NN

8 8 8 §
R s v S

N
8
ONC

ONE SPACE 285 MICRONS

Resting spore
19 PM on ao 2M LZ pM ae Ahm

6 PM 7PM
4PM 5PM

gq PM

1PM

to PM

lil =

1 space = 2 £54

PATE valle

Germination of ascospores.

Resting

Spore

ld fee)

124 5PM

6PM
3PM

unit}

1 space = 2.85

PLATE VIII.

Germination of ascospores.

IVILAMINIG, JON. PLATE IX.

Fig. 43.—Canker showing atrophy. Tig. 44.—Canker showing stromata.

PLATE X.
Fig. 45.—Blister stage of canker.

XG

PLATE

g parithecial stage.

in

at

indic

apillae,

§ Dp

in

a show

tromat

S

46 .—

Fig.

7* a an An v4) (Ol Rear py

re

ee ee ere

ie
ne

=
<i e

a - ee ne

oe
——-

_——

_

ey Soe,
“ ® Ray,

PLATE XI.

horns on smooth bark.

Fig. 47.—Spore

PLATE XI.
Fig. 48.—Sporehorns in crevices of rough bark.

a
oad WS

PLATE XII.
Fig. 49.—Outlined canker, indicating monthly growth.

Phu

- x} ais
ails |

PLATE XIII.
Fig. 50.—Mycelial fans under the chestnut bark.

‘7 teat 4 2 ae

Ce pos :
ak dee AY)

Crk ey Sar

hie

Two Week’ old culture
o¥ Endofhta pava sitica
on chestnut wqar

Showing ‘aap As Hidia tt
concernltrre « eC.

PEATE XLV.
Fig. 51.—Petri dish culture of pycnidia.

PLATE XV.
Vig. 52.—Photomicrograph of pycnospores.

PLATE XV.
Vig. 53.—Vertical section of a perithecium.

"a J © oe or

ae i.» : ee! \

“a cate BY -%s y Z

ry oh A 7 - .
a rs 7

Le ee Seis

rae haa :

PAG ACD eXeVele

Vig. 54.—Photomicrograph of pyenidium on wood.

PLATE XV?I.

Fig. 55.—Stroma containing labyrinthiform pycnidium.

PLATD XVII.
tig. 56.—Vertical section of stroma showing empty perithecia and black necks.

PLATH XVII.
Fig. 57.—Pyecnidium on agar showing early stage in the formation of the cavity.

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