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Frank Lincoln Stevens.

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GIFT OF
Professor W*ASetchell




BIOLOGY LIBRARY



W1LUAM A. SETCHELL



STATE OF ILLINOIS

DEPARTMENT OF REGISTRATION AND EDUCATION
DIVISION OF THE

NATURAL HISTORY SURVEY

STEPHEN A. FORBES, Chief

Vol. XIV. BULLETIN Article V.

The Helminthosporium Foot-rot of Wheat, with

Observations on the Morphology of

Helminthosporium and on the

Occurrence of Saltation

in the Genus

BY
F. L. STEVENS




PRINTED BY AUTHORITY OF THE STATE OF ILLINOIS



URBANA, ILLINOIS
June, 1922




Original isolations of the foot-rot Helminthosporium made

in May, 1919, from bits of tissue from wheat

grown in Madison county, Illinois.



STATE OF ILLINOIS
DEPARTMENT OF REGISTRATION AND EDUCATION

DIVISION OF

NATURAL HISTORY SURVEY

STEPHEN A. FORBES, Cheif

Vol. XIV. BULLETIN Article V,

The Helminthosporium Foot-rot of Wheat, with

Observations on the Morphology of

Helminthosporium and on the

Occurrence of Saltation

in the Genus

BY
F. L. STEVENS



PRINTED BY AUTHORITY OF THE STATE OF ILLINOIS



URBANA, ILLINOIS
June, 1922



LIBRA*!

a



GIFT OF





BIOLuuY



STATE OF ILLINOIS

DEPARTMENT OF REGISTRATION AND EDUCATION
W. H. H. MILLER, Director



BOARD OF

NATURAL RESOURCES AND CONSERVATION
W. H. H. MILLER, Chairman



WILLIAM TRELEASE, Biology
JOHN M. COULTER, Forestry
ROLLIN D. SALISBURY, Geology
WILLIAM A. NOYES, Chemistry



JOHN W. ALVORD, Engineering

KENDRIC C. BABCOCK, Representing the
President of the University of Illi-
nois



THE NATURAL HISTORY SURVEY DIVISION
STEPHEN A. FORBES, Chief



(52929-1200-7-21)



CONTENTS

PAGE

Introductory 77

I. A foot-rot of wheat and its causal fungus:

Symptoms 78

Fungi present 78

Growth of the causal fungus on various media 79

Various agars as media 80

Summary concerning growth on agars 85

Rice and similar substances as media, with special note of color phenomena 86

Summary concerning growth on rice and similar substances 88

Miscellaneous vegetable media 88

Summary concerning the foregoing vegetable media 90

Cereal shoots grown from aseptic seeds as media 90

Environmental factors which induce variation 91

Quantity of nutriment 91

Inhibitory influences . . 93

Humidity of media 93

Humidity of air > 94

Temperature relations 98

Light 99

Carbohydrates 100

Nutrients as affecting conidial length, septation, and shape 101

Summary concerning environmental factors which induce variation . . . 102

Morphology of the foot-rot fungus 103

Mycelium 105

Senescence phenomena of aerial mycelium 107

Conidiophores 109

Conidia 110

Etiology of foot-rot:

Evidences of etiological relation of H. No. 1 124

Constant presence of the pathogene 124

Absence of other constant parasites 124

Identity of pathogene proved by culture 124

Evidence of infectiousness 124

Conidia produced in moist-chamber culture 125

Evidence from inoculation 125

Recovery of organism 128

Infection phenomena on wheat 128

Susceptibility of various hosts to infection 137

Summary concerning etiology of foot-rot 139



IV

II. Evidence and discussion of the occurrence of saltation within the genus Helmin-

thosporium : PAGE

Introductory 139

Characters of saltants as shown in transfers 141

Tendencies in saltation 145

Stability of the saltants 145

Stability of the saltants through the conidia 146

Apparent reversions 147

Supposititious causes of the variant sectors 147

Saltations from single conidia 149

Frequency of saltation 150

Saltations occurred on various media 152

Saltations and modifications occurring in test-tube cultures . . . . . . 152

Saltations in nature . 154

Notes concerning selected individual saltants 155

General discussion of saltation 157

Taxonomy 164

Conclusion ^ 168

Summary 168

Literature cited 171

Appendix:

Methods 179

List of Helminthosporiums used for purposes of comparison 181

Discussion of foregoing list with several brief descriptions 184

Graphs: Figures A Y.

Plates: VII XXXIV.



ARTICLE V. The Helminthosporium Foot-rot of Wheat, with Observations
on the Morphology of Helminthosporium and on the Occurrence of Saltation in
the Genus. By F. L. STEVENS.



INTRODUCTCXRIT / , -,^

The present study of wheat disease is^baseti upon Vfo6t-fot/6r rot of
the basal portion of the stems, of wheat plants, as it occurred in Madison
county, Illinois, in 1919 and subsequently. This disease was first reported
in United States Government publications as " take-all" ( Ophiobolus gram-
inis] ; later, merely as "take-all," no cause being assigned; and for some time
past, in Government publications it has usually been designated as "so-
called take-all." An annotated bibliography of nearly one hundred
titles concerning foot-rot disease of wheat, prepared by the writer, was
presented before the Cereal Pathologists of America at St. Louis in June,
1919, and this, expanded to one hundred and eighty-eight titles, was
published in October, 1919 (116). As early as May, 1919, cultural studies
quite clearly pointed to Helminthosporium as the true cause of the disease,
and at the December meeting of the American Phytopathological Society
I announced this fungus as the probable cause. In May, 1920, in a note in
Science (117), I published the statement that it had been conclusively
established that this foot-rot of wheat is caused by Helminthosporium.
One purpose of the present paper is to present the evidence on which
the foregoing conclusion is based and certain facts concerning the mor-
phology and parasitism of the fungus; but far transcending in interest the
disease itself which now appears to be one of much less alarming nature
than was at first feared is the fact that very striking phenomena of
variability are found in this and related fungi. In the following pages,
therefore, appear (I) an account of the Illinois foot-rot of wheat and its
causal fungus; and (II) evidence and discussion of the occurrence*of salta-
tion within the genus Helminthosporium.

ACKNOWLEDGMENTS

In this study I have been assisted financially by grants from the
Illinois Natural History Survey and from the University of Illinois. I
am indebted for specimens to persons mentioned in the list of species
used for comparison (pages 181-184), and to W. P. Snyder for compu-
tation of data embodied in several of the graphs. I wish also to express
my thanks to Prof. J. A. Detlefsen, who kindly read the manuscript and
offered valuable suggestions regarding genetic questions.



78
I. A Foot-rot of Wheat and its Causal Fungus

SYMPTOMS

As the name implies, the most obvious symptom is a rotting of the
basal portion of the stem of the wheat plant, that is, the lowesr portion
of the lowest internode. In earlier stages than that of actual rotting,
minute yellow or brown lesions occur on the stem (PI. VII), while
the roots, if diseased, are slightly yellowed and largely or quite devoid
of functional root-hairs. No weft of superficial mycelium or black incrus-
tation, such as is so frequently described in articles concerning take-
all, was seen. The diseased tissues, however, were invariably ramified
by an internal mycelium. Certain cases of diseased wheat came under
observation in which the plants had attained a nearly normal growth
and were eighteen inches high, when they suddenly died throughout.
In such cases there was a slight darkening of the lower node and a mycelial
invasion at this point. The opinion of those who observed this wheat
in the field was that the death was due to frost injury. It is probable
that the actual cause of death was foot-rot following the frost injury.

FUNGI PRESENT

Direct microscopic observation of the diseased tissues, in all cases
of foot-rot examined, revealed the presence of an internal hyaline or
faintly tinted mycelium in great abundance permeating the diseased
tissues. Mycelium of different character was also occasionally found,
but so inconstantly as apparently to have no actual relation to the disease.
Isolations of the fungi present in the diseased tissues were made by two
methods:

1. Direct planting of bits of diseased tissue on poured agar (corn-
meal agar or wheat-straw agar). The diseased tissue was secured in as
clean condition as possible by stripping back the enclosing sheaths, ex-
cising the diseased part with sterile tools, and tearing it apart in a sterile
Petri-dish.

2. Direct planting of similar bits of diseased tissue after surface-
sterilization in mercuric chloride (1-1000, 10 min.).

Dilution plating was unsatisfactory owing to the paucity of conidia
and the presence of numerous soil bacteria, particularly "spreaders."

As might be expected, the methods employed gave rise to colonies
of many genera and species, including Phyllosticta, Septoria, Fusarium,
Epicoccum, Alternaria, and Helminthosporium. A striking fact, however,



79

was that with the exception of the Helminthosporium, these fungi were
very rarely present, and then only a single colony or part of a mixed colony
on occasional plates. Alternaria occurred with remarkable rarity; only
two or three colonies among several thousand. Fusarium was found in
only a few colonies and so mixed that it was isolated with difficulty. Ep-
icoccum occurred in two colonies; Phyllosticta also in two colonies (two
species) .

A Helminthosporium, however, appeared in every plate and from
nearly every bit of tissue used, no matter how great the care in securing the
inoculum. On many plates this Helminthosporium (which throughout
this article I designate as H. No. 1) appeared in pure culture Thus it
may be said that the Helminthosporium was universally present in the
plates; that it was the only organism that was present with any constancy;
and that all other fungi were obviously strays.* Though conidia were
never found in great numbers on plants brought in direct from infested
fields, when the plants were placed in moist chamber for two or three
days conidia developed in abundance. This was also the case with portions
of wheat stems which had been placed in bichloride of mercury for ten
minutes and then placed in moist chamber for several days. In passing
it may be remarked that although great numbers of nematodes and amebae
appeared in the plates there is no reason to believe that they had any relation
to the disease under discussion or to any diseased condition.

GROWTH OF THE CAUSAL FUNGUS ON VARIOUS MEDIA

Since the characters exhibited by various Helminthosporiums when
growing in artificial culture have been considered as of importance as a
means of distinguishing one species, variety, race, or strain from another,
many media were employed in the present study. This was done in part for
the purpose of comparing the growth characters of the Helminthosporium
with characters reported by others in connection with other forms; in part
with the hope that some of the media tested might give emphasis to cer-
tain characters and thus serve to differentiate between species or strains
of the forms under observation.

The following notes are, in the main, statements of the characters
presented by the foot-rot Helminthosporium (H. No. 1), though for the
purpose of comparison notes are added regarding the growth of several



*A letter from Professor Hoffer written in May, 1919, tells me of a similar result from platings of wheat
foot-rot from Indiana, and similar reports reach me from several other sources.



80

species or strains of Helminthosporium. These are throughout referred to
by number rather than by name, partly for brevity and partly because
the species of many of the races had not been determined, while in some
cases the names were of more or less doubtful reliability. That the reader
may formulate his own judgment of these forms, introduced for comparison,
a complete list of them is given in the appendix (pages 181-184) together
with certain notes regarding them.

VARIOUS AGARS AS MEDIA

Corn-meal agar in Petri dishes. This medium, prepared after the di-
rections given by Shear and Stevens (104), was found to be admirably
suited to Helminthosporium and was the medium chiefly used.

The fungus grew rapidly, the colony being at first nearly hyaline
both in the submerged and aerial parts, but when a diameter of about 2-3
cm. was attained the whole colony became much darker. Profusion of
conidia was the chief factor in giving the dark hue to a colony, the slight
darkening of the mycelium having little to do with it. The aerial mycelium
varied largely with change of conditions, sometimes being very scant
and at other times 5-6 mm. high, with windrow effects corresponding
with the zones. After the colony was about 3 cm. in diameter zonation
became quite pronounced, the zones corresponding approximately with
the growth of each day. At room-temperature the colony attained a
diameter of about 4.5 cm. in six days. Conidia-production was quite
uniform over the surface of the colony unless checked by some growth-
inhibiting cause, as drying, cold, or the antagonism of another colony
near by, when it was much increased, as evidenced to the eye by black
bands in such regions. By transmitted light the mycelium, and to some
extent the conidia at certain ages, had a distinctly greenish tinge. H. No. 1
could be distinguished from H. Nos. 5-8, which were paler and produced
fewer conidia. H. No. 6 approached nearer to H. No. 1 in these regards
than did the others. H. Nos. 3, 4 (see PI. IX), 6, 15-17, and 18 typically
developed more aerial white mycelium than did H. Nos. 1 and 14. H. No.
36 was of very distinct character owing to large development of aerial
mycelium (see PI. X).

Corn-meal agar in Freudenreich flasks. The flasks, of about 100 c.c.
capacity, each received 50 c.c. of agar and were slanted. The large amount
of nutriment available and the sustained moisture gave noteworthy
characters. At 7 days, with H. No. 1, the surface of the slant was com-



81



pletely overgrown and of an even black color, largely curtained by an
abundant, even overgrowth of white aerial mycelium. At contact with
the glass a sharp, black line gave clear evidence of the black surface-coat.
No clumps or balls of mycelium were present. At 22 days a few clumps
developed, though not so many as on H. Nos. 9, 13-16.

Cultures of H. No. 1 on corn-meal agar in large flasks, as those of
Kolle or of Piorkowski (PL XI-XIII) gave colonies very different from those
on the ordinary Petri dish, due presumably to the larger quantity of
nutrient available and to different humidity relations. These flasks
gave increased density of colony and conidia-formation, more aerial
mycelium, and some clumping of the mycelium. Though colonies of H.
No. 3 and H. No. 1 differed in these characters in these flasks (PI. XII,
XIII), portions of the colonies of these strains were indistinguishable.

Corn-meal agar made at various temperatures. Corn-meal agar was
made in the usual manner excepting that the temperature in three cases
was held at 43, 85, and 100 respectively, instead of at 60, before
filtering. Duplicate plates were made. The four resulting agars are
designated according to the temperatures held, and colony data for each
are presented in the following table.

CORN-MEAL AGAR MADE AT VARIOUS TEMPERATURES



Temperature


Growth in
8 days


Zonation


Density


Colors


43
43


7.5
7.2


distinct


thin


pale


60
60


5.5
6


sharp


thick


dark


85


6.5


In above characters, ranks between 43 and 60 agars


100
100


8
7.8


none


very thin


very pale



The 100 agar is most favorable to linear growth, 43 agar stands
next; 43 and 85 agars give growth of poorer color than 60 agar, but
100 agar ranks lowest in this regard. Color is directly due to quantity
of conidia, and it is uniform in the mycelium on the four agars. Nutrition
in 100 agar was very little better than in plain agar. In general, it



82

appears that their order of nutritive value for this fungus, from poorest
to best, is 100, 43, 85, 60. Evidently a temperature of 43 is in-
sufficient to extract the nutrient proteids sufficiently, while 100 pre-
cipitates too many of them. While leucosin, a prominent proteid of the
embryo, is largely precipitated at 52 and a second coaguIuriTgOes down
at 82, no more is precipitated even by boiling (Osborne, 89).

Graphs 1-4 (Fig. A), indicating conidial length on these four agars,
show that although the quantity of conidia produced varied materially, the
length and general variability are not greatly influenced by varying the
composition of the agar done in this case by change of temperature.
The conidial length of all these agars is, however, considerably less than that
on wheat shoots (cf. graphs in Fig. A and Fig. K). Graphs of conidial
breadth and septation on 43 and 60 agars given in Fig. B also show but
little influence of these agars on these two characters. A "Difco" corn-
meal agar, prepared according to my directions by the Digestive Ferments
Company, gave growth-characters almost identical with those of my
own 60 agar. On "Difco" beef-agar the conidia were short, and were
frequently deformed (M, 17.44.22, <r, 2.46.16, CV, 14.15.93).

Plain agar (shredded agar only, 12 g. per liter). The fungus grew
rapidly, and in 6 days the colony was 35-45 mm. in diameter, but was
thin and colorless, with but few scattered conidia, and only 1 to 3,
or at the most 7 to 10, conidiophores per low-power field, except at
growth-inhibition points, as at the edge of the dish or where two colonies
approached each other, where the number of conidiophores rose to about
12 per low-power field. The conidiophores bore only one or two conidia
each. Conidiophores, conidia, and mycelium as well, were very faintly
straw-colored, much paler than on more nutrient agar. No zonation
occurred. No difference in rate or character of growth was observable in
1.3% and 2.6% plain agar.

Growth on plain agar, on corn-meal agars, and on various combinations
of these nutrients. Corn-meal agar of various compositions was used,
12 c.c. to each Petri dish. On 25% corn-meal agar (made of 3 parts plain
agar plus 1 part ordinary corn-meal agar) the colony was much darker
and denser than on plain agar; was zoned more strongly; and conidia were
much more abundant, there being about 80 conidiophores per low-power
field, each with one to five conidia. The mycelium was much darker
than on plain agar. Colonies on 50% and 75% corn-meal agar showed
no essential difference from the colony on 25% agar. On full corn-meal



83

agar the colony was much darker and more dense and the mycelium was
darker. The relative rate of linear growth on these agars, as shown in
millimeters of colony diameter at the end of 9 days at room-temperature,
was as follows:



On plain agar 70 mm.

On 25% corn-meal agar. .68 mm.
On 50% corn-meal agar. .62 mm.
On 75% corn-meal agar. .57 mm.
On 100% corn-meal agar. .54 mm.



When the fungus was planted on plain agar, and pieces (1 cm. square)
of corn-meal agar of the above-mentioned compositions were laid on the
surface at the edge of a well-developed colony, both color and conidia-
production increased with the increase of nutrients. Variation in conidial
length on these agars is shown in Fig. C. On this series of agars conidial
length was least on plain agar and increased consistently with the strength
of the medium. It is to be noted that the coefficient of variability is
very high on the 75% corn-meal agar. In Graphs 9 and 10 of this Fig.
C, conidial length is seen to be appreciably lower than on full corn-meal
agar (Graphs 1-4, Fig. A), and markedly shorter than conidia grown under
standard conditions (see Graphs, Fig. K; also App., page 180). Again, corn-
meal agar was made in the usual way but the amount of agar was varied,
6, 12, and 25 grams per liter being used. In general, in Petri dishes, 12
grams per liter proved most suitable. Comparisons between H. No. 1
and H. No. 3 on these three media showed at 11 days each that H. No. 3
had grown more rapidly than H. No. 1, the ratio being 6.8: 8.5. There
was usually a marked difference between these two strains on 12-gram
corn-meal agar, more marked than on the others, H. No. 3 showing more
definite zonation and more aerial mycelium.

In Freudenreich flasks, with the 6-gram agar, H. No. 3 made much
aerial mycelium on the watery surface; H. No. 1 made only a black pellicle
and no aerial mycelium. On 12-gram agar H. No. 1 made small growth
of aerial mycelium and the colony surface was black, while H. No. 3 had
much loose, woolly mycelium. At 11 days H. No. 3 on each agar had more
aerial mycelium and more clumps than did H. No. 1. The most conspicu-
ous difference was on 12-gram agar, while on 25-gram agar H. No. 1 had
no clumps and H. No. 3 a few.

There is a clear, definite tendency in H. No. 3 to make more aerial
mvcelium and more clumps than H. No. 1, but this is so dependent on con-



84

ditions of moisture, in air and medium, that it is far from being a reliable
character for separating the two.

Green-wheat agar. (Formula as for corn-meal agar, substituting for the
corn-meal live wheat leaves and stems which had been_ passed through
a meat-grinder.) H. No. 1 grew well, producing a dense colony, but with
weak zonation and with much woolly, white aerial mycelium, and but few
and scattered conidia. This medium, while apparently very nutritious,
favored abundant vegetation rather than sporulation, and was a poor
medium for the differentiation of races.

To determine the effect of reducing the nutrients in green-wheat
agar, this was combined, in varying proportions, with washed agar with
varying results. On washed agar the growth of H. No. 1 was scant, color-
less, and with no conidia, the colony diameter reaching only 5.5 cm. in
9 days. As the content of green wheat was increased, there was a gradual
increase in density of colony and of aerial mycelium. In 9 days the colony
diameters were as follows:



On 25% green- wheat agar. .9 cm.
On 50% green-wheat agar. .8 cm.
On 75% green-wheat agar. .7.5 cm.
On 100% green-wheat agar. .7.5 cm.



These colonies showed no dark color and only very weak zonation,
and in the two high concentrations the aerial mycelium developed into a
dense, closely felted mat. Conidia were produced scantily and varied
greatly from the shape found elsewhere, being less tapering, more nearly
cylindrical, and materially thicker (Graphs 13, 14, Fig. D). In some
instances septation differed markedly diminished (Graphs 15, 16, Fig. D).
Many large conidia had no septa at all, and others had irregular or incom-
plete septa. It is evident that this medium, even at 25% strength, in-
duces many abnormalities, and the very high coefficient of variability
is especially striking. The differences in septation here noted were not
constant on the same plate and were much more common at the drying
edge. On this agar conidial length was less than under standard conditions
(see appendix, p. 180).

Wheat-straw agar. (Fifty grams of old wheat-straw, boiled 20 minutes
and filtered.) Growth was poor.

"Difco" beef -agar. H. No. 1 grew slowly but was very dense; surface
even; little aerial mycelium.



85

Starch agar. This medium proved to be of but slight differential
value. The growth was of a dark color and of somewhat bluish tinge.

Bean agar. H. Nos. 1, 3, 5, 13, 20, grew well on bean agar, all de-
veloping a dense, woolly, gray aerial mycelium. Zonation was poor, or
obscured by the aerial mycelium. The five strains showed no differences
in growth on this medium, which was therefore poor for differential use.
In Freudenreich flasks there was a definite black surface-line and much
tawny aerial mycelium in clumps.

Brazil-nut agar. (Formula according to Spencer, 108.) H. No. 1 in
test-tubes grew very rapidly and luxuriantly with small development of
aerial mycelium and a distinct black basal line. The agar was rapidly
cleared of proteid precipitate by the development of a proteolytic enzyme.
In Petri dishes a thick, dense, woolly, snow-white aerial mycelium devel-
oped which entirely curtained the surface-blackening. The colony was
surrounded by a broad translucent zone due to proteolytic action. This
agar is valuable for the pure-white aerial mycelium that develops on it,
and to demonstrate readily the proteolytic action, though it did not,
even in these regards, prove to be differential, since all of fifteen strains
tested upon it gave nearly identical responses.

Oat agar. H. No. 1 at 10 days gave a distinct black surface-line
and very heavy aerial gray growth. H. Nos. 1, 4, and 14 were indistin-
guishable on it.

Apple-fruit agar. H. No. 1 gave a black basal line and abundant,


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