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Full text of "The fungi"

THE FUNGI 




•»■ * f* _ - 






^ 









The father of American mycology. 






THE FUNGI 



IN TWO VOLUMES 
Volume II 



By Frederick A. Wolf and Frederick T. Wolf 

DEPARTMENT OF BOTANY DEPARTMENT OF BIOLOGY 

DUKE UNIVERSITY VANDERBILT UNIVERSITY 



New York: JOHN WILEY & SONS, Inc. 

Chapman &- Hall, Limited 
London 



Copyright, 1947 

BY 

John Wiley & Sons, Inc. 



All Rights Reserved 

This book, or any part thereof, must not 
be reproduced in any form without 
the written permission of the publishers. 



PRINTED IN THE UNITED STATES OF AMERICA 




PREFACE 

This treatise on fungi is intended as a reference and textbook. 
Its content falls naturally into two portions. The first portion, 
included in Volume I, is a consideration of the developmental 
morphology and taxonomy of fungi and is basic to any compre- 
hensive study of the fungi. The second portion, included in 
Volume II, deals more specifically with the activities of fungi. 
It must be borne in mind, however, that we have attempted 
throughout the treatise to stress the need for more emphasis on 
problems relating to fungus activities. 

The content of Volume II is concerned with metabolic and 
reproductive activities, the modification of these activities by 
environment, and the relationship of fungi to the welfare of 
man. Consideration is also given to certain fungi for which 
habitat is largely the basis of grouping. This volume may be 
spoken of as physiological and ecological in its emphasis. It does 
not purport, however, to constitute a well-rounded "physiology 
and ecology of fungi" for the reason that experimental data are 
still too meager to permit the preparation of such a textbook. 
This explanation is made at the outset to guard the reader against 
eventual disappointment. The need for a volume on the physi- 
ology of fungi is keenly felt by all who seek such information in 
textbooks on plant physiology, only to find that such books are 
limited to consideration of the physiology of seed plants. 

Teachers may at first regard our departure from the tradi- 
tional emphasis on taxonomy and classification as too radical to 
put into practice. It should be remembered, however, that last- 
ing impressions come from contact with living, functioning or- 
ganisms. From experience we know that we remember with 
facility where and under what circumstances we first encoun- 
tered many different fungi in their natural habitats, and we re- 
call how intent we became as we watched their development 
and the changes which they induced. If, on the other hand, we 
had been presented with an herbarium specimen, as is common 

v 



vi PREFACE 

laboratory practice, and had been asked to perform an "autopsy" 
in order to arrive at an understanding of the structure and pos- 
sible functioning of the victim, long dead and mummified, little 
stimulation of thought or interest would have resulted. 

The further objection may be raised that knowledge of fungus 
activities is still too meager to be presented in an organized man- 
ner to students. All that need be said in rejoinder is that the 
seven-league boots of physics, physical chemistry, biochemistry, 
physiological chemistry, and colloid chemistry will enable the 
teacher and the student alike to wade far out into the depths of 
the vast mvcologic unknown. Beginnings must be made. 

Opinions have a limited value in the field of science. At times, 
ours are expressed. .Mycologists may not find themselves in 
accord with some of these opinions. Be that as it may, data will 
some day exist upon which ultimate truth will become securely 
established, and then, of course, opinions now expressed will lose 
all value. 

Our efforts have been concentrated on helping the student to 
understand fundamentals. We have chosen % to include data and 
conclusions from certain reports of researches and have omitted, 
without apparent reason for so doing, to mention others that are 
equally good and pertinent. No intentional discredit or lack of 
merit is implied in these omissions. In a volume of this scope it 
is simply impossible to consider each subject monographically. 
The relative importance of subjects is not reflected by the amount 
of space devoted to them. References to reports are given at the 
end of each chapter; these papers contain additional pertinent 
references to other researches, so that interested persons can gain 
a more comprehensive grasp of a particular subject. 

Most of the illustrations are adapted from those of others. If 
in any instance the author of the original drawing or graph has 
not been mentioned, the omission is unintentional. We herewith 
acknowledge again, with gratitude, the kindness of those who 
supplied us with certain illustrative materials, and of Mary H. 
Wolf for her assistance in the preparation of illustrations. 

Since the legends are intended to explain the illustrations ade- 
quately, mention of illustrations is omitted from the text. 

We are indebted also to Dr. L. E. Wehmeyer, who carefully 
read the entire manuscript, for his criticisms and suggestions and 
to Mrs. Fred T. Wolf for her help in reading proof and in the 



PREFACE vii 

preparation of the indices. Also we are keenly appreciative of 
the gift of a wood-cut picture of Louis David de Schweinitz, to 
whom these volumes are dedicated, from his great-granddaughter, 
Dr. Adelaide L. Fries. 

F. A. Wolf 
F. T. Wolf 
March, 1947 



CONTENTS 

1. NUTRITION OF FUNGI 1 

Mineral nutrition of fungi, 2. Organic nutrients of fungi, 
16. Growth factors, 24. Influence of osmotic pressure, 
29. Implications, 29. 

2. ENZYMES AND ENZYM1C ACTIVITIES OF FUNGI . . 37 

3. RESPIRATION 53 

Historical material, 53. Types of respiration, 56. The 
respiratory ratio, 62. Respiratory systems, 63. Respiro- 
metry, 64. Inhibition of respiration, 65. Stimulation of 
respiration, 66. Implications, 66. 

4. BIOCHEMISTRY OF FUNGI 69 

Organic acids and other products having six or fewer 
carbon atoms, 70. Polysaccharides, 79. Fats, 79. Sterols 
and vitamins, 81. Amino acids, 83. Pigments of fungi, 

83. Other metabolic products, 86. Implications, 88. 

5. EFFECTS OF TEMPERATURE ON FUNGI 96 

Cardinal temperatures, 97. Resistance to low temperatures 
and high temperatures, 103. Influence of temperature on 
infection, 109. Temperature and reproduction, 111. 

Temperature and zonation, 114. Temperature coeffi- 
cients, 114. Temperature and oxygen tension, 117. Im- 
plications, 119. 

6. EFFECTS OF RADIATION ON FUNGI 123 

Morphogenic reactions, 125. Phototropism, 129. Lumines- 
cence, 137. Inhibitory effects, 138. Stimulatory effects, 
143. Effect on sporulation, 144. Effect of X-rays, 145. 
Induction of saltations, 145. Mode of action of short 
radiations, 147. Implications, 147. 

7. EFFECTS OF REACTION OF SUBSTRATE ON FUNGI . 151 



x CONTENTS 

8. SPORE DISSEMINATION 166 

Distribution of spores, 167. Structural adaptations for 
expulsion of spores, 179. Hygroscopic mechanism in 
Myxomycetes, 179. Spore expulsion among Phycomv- 
cetes, 180. Spore discharge among Ascomycetes, 186. 
Spore discharge among Basidiomvcetes, 194. Implica- 
tions, 205. 

9. GERMINATION OF SPORES 210 

Germination types, 210. Methods of testing spore ger- 
mination, 212. Hereditary factors and germination, 214. 
Water relations affecting germination, 218. Effects of 
temperature on germination, 221. Influence of reaction 
on (termination, 229. Influence of oxygen on germina- 
tion, 230. Influence of carbon dioxide on germination, 
231. Influence of light on germination, 231. Influence 
of nutrition on germination, 232. Resume, 232. 

10. HOST PENETRATION 236 

Direct penetration, 237. Stomatal penetration, 248. Pene- 
tration through wounds, 251. Haustoria and their sig- 
nificance, 252. Penetration by ectoparasites, 253. Im- 
plications, 254. 

11. PHYSIOLOGIC SPECIALIZATION AND VARIATION 

AMONG FUNGI 257 

12. ASSOCIATIVE EFFECTS AMONG FUNGI 279 

Antagonism, 280. Stimulation by associative interaction, 
287. General considerations, 292. 

13. MYCORRHIZAE AND MYCOTROPHY 297 

14. GENETICS OF FUNGI 317 

Sexual and asexual stages of fungi, 317. Homothallism 
and heterothallism, 319. Dominance and lethal factors, 
335. Resume, 336. 

15. POISONOUS AND EDIBLE FUNGI 339 

Poisonous fleshy fungi, 339. Food value of fleshy fungi, 
351. Ergot and ergotism, 354. Toxicity of Gibber ella 



CONTENTS xi 

sanbinettii (G. zeae) and Fusarium spp., 359. Implica- 
tions, 361. 

16. MEDICAL MYCOLOGY 364 

Historical material, 366. Coccidioides hmnitis, 367. 
Cryptococcus histolyticns, 368. Histoplasma capsulatum, 
369. Fhialophora verrucosa, 370. Malassezia ovalis, 372. 
Actinomyces bovis, 373. Sporotrichwn schenckii, 375. 
Monilia (Candida) spp., 377. Aspergillus fumigatus, 379. 
The Trichophytoneae or ringworm fungi, 379. Implica- 
tions, 390. 

17. GEOGRAPHICAL DISTRIBUTION OF FUNGI .... 395 

Distribution of Myxomycetes, 397. Distribution of Phy- 
comycetes, 399. Distribution of Ascomycetes, 402. Dis- 
tribution of Basidiomycetes, 405. Distribution of Deu- 
teromycetes, 410. Implications, 412. 

18. MYCOLOGY IN RELATION TO PLANT PATHOLOGY . 416 

Early concepts of disease in plants, 417. Contributory 
advances in bacteriology, 418. Signposts along the phyto- 
pathological path, 419. Developments in terminology, 
421. Fungi as antigens and plant pathology, 423. Pres- 
ent trends in mycologic and phytopathologic work, 424. 
Implications, 427. 

19. SOIL FUNGI 429 

Taxonomic studies, 429. Biochemical activities of soil 
fungi, 434. Soil-borne pathogens, 437. Implications, 437. 

20. FUNGUS-INSECT INTERRELATIONSHIPS .... 442 

Insects as vectors of plant-pathogenic fungi, 442. Fungi 
occurring on or within insects, 444. Biological control 
of insects, 448. Insects in relation to reproduction of 
fungi, 450. Fungi cultivated by insects, 451. Implica- 
tions, 455. 

21. MARINE FUNGI 458 

Historical background, 459. Marine Phycomycetes, 460. 
Marine Ascomycetes, 466. Marine Fungi Imperfecti, 468. 
Marine slime molds, 470. Implications, 470. 



xii CONTENTS 

22. FOSSIL FUNGI 474 

Geological time, 474. Age of fossil fungi, 477. The 
nature of fossilized fungi, 478. Preparation of fossils for 
study, 479. Classification of fossil fungi, 479. Fossil 
mycorrhizae, 487. Implications, 488. 

AUTHOR INDEX 491 

SUBJECT INDEX 502 




Chapter 1 
NUTRITION OF FUNGI 

Fungi are commonly regarded as unable to synthesize their own 
food; that is to sav, they are not autotrophic. It is customary 
therefore to consider them parasites and saprophytes; these group- 
ings are based upon whether they secure their food from living 
organisms or from dead and decaying plant or animal tissues. 
Parasitic fungi may better be spoken of as paratrophic, and 
saprophytic fungi as saprotrophic. A moment's contemplation 
will reveal, however, that these terms too are quite arbitrary and 
inadequate. For instance, experience has shown that certain fungi, 
such as the Peronosporaceae, Erysiphaceae, and Uredinales, are 
strictly paratrophic. Others— for instance, the Lycoperdales and 
Phallales— are strictly saprotrophic, and between these extremes 
all degrees of intergradation exist. In fact, such terminology be- 
comes confusing, because many plant pathogens are paratrophic 
during part of their annual cycle and saprotrophic during the 
remainder. 

Concepts regarding the nutritional relationships of fungi that 
underlie such terminology emphasize the fact that fungi lack 
chlorophyll, and thereby the impression is inferentially fostered 
that neither parasites nor saprophytes perform syntheses. As a 
consequence the metabolic changes they induce are not properly 
appreciated, and too little consideration is given to the determi- 
nation of how both parasites and saprophytes effect not only 
analyses (katabolism) but also syntheses (anabolism). 

In this discussion the term food is used herein in the broadest 
sense. Any substance is regarded as food which serves as a 
source of energy or is used for growth and repair or for the 
various metabolic processes of the fungus. This usage implies 
that both inorganic and organic materials play a role in the nutri- 
tional requirements of fungi and in this sense constitute food. The 
inherent implications in this usage of the term food permit dis- 

1 



2 NUTRITION OF FUNGI 

cussion of this subject under two headings: (a) inorganic or 
mineral nutrition of fungi, and (b) organic nutrition of fungi. 

MINERAL NUTRITION OF FUNGI 

Problems relating to the mineral nutrition of fungi appear at 
first to have been approached wholly by empirical methods. The 
experiences and techniques of bacteriologists constituted the 
foundation for these early studies. The investigators seem to 
have employed such chemical compounds and in such proportions 
as had been found to promote the growth of bacteria. The proper 
kind and amount of mineral elements were not sought by ex- 
tended series of experiments in which ash analyses were correlated 
with rate of growth or with amount of mycelial mat. 

Not only were these procedures employed with liquid media 
but also mycologists followed the bacteriologist in quite the same 
way in the use of the numerous kinds of semisolid media. It is 
not unusual to find now that a particular medium, compounded 
according to a certain formula, is a favorite with a given mycolo- 
gist and that he attempts to cultivate all species in which he may 
be interested on this particular medium. It will become apparent 
in the discussion which follows that this procedure may lead to 
erroneous conclusions regarding the nutrition of the fungi in- 
volved. 

Investigations of the mineral nutrition of fungi may be said to 
have beeun with the classical researches of Raulin (1869), a 
pupil of Pasteur. He employed Aspergillus niger as a test organ- 
ism and secured optimum growth in a medium, now known as 
Raulin's solution, having the following composition: 

Ammonium nitrate 4 grams Iron sulphate 0.07 gram 

Ammonium phosphate 0.6 gram Potassium silicate 0.07 gram 

Magnesium carbonate 0.4 gram Sucrose 70 grams 

Potassium carbonate 0.6 gram Tartaric acid 4 grams 

Ammonium sulphate 0.25 gram Water 1500 ml 

Zinc sulphate 0.07 gram 

He concluded that none of the minerals contained in this medium 
could be omitted if optimum growth was to be secured. 

Raulin's studies stimulated a series of investigations, the out- 
standing of which were those of von Naegeli (1880), Benecke 



MINERAL NUTRITION OF FUNGI 3 

(1895), Molisch (1892, 1894), and Wehmer (1895). Von 
Naegeli believed that sulphur and phosphorus are indispensable 
for all molds and that potassium and calcium are replaceable, 
potassium by rubidium or caesium, and calcium by magnesium, 
barium, or strontium. The experiments of Benecke and Molisch 
led them to conclude that magnesium is not replaceable by any 
other mineral element. Benecke secured luxuriant growth of 
many species of Aspergillus and Penicillium on a synthetic agar 
medium, the inorganic salts of which were ammonium phosphate, 
potassium chloride, and magnesium sulphate. Wehmer consid- 
ered especially the essentiality of iron and zinc, each of which 
was regarded as indispensable by Raulin (1869). 

Under the stimulus of studies on the mineral nutrition of green 
plants several other mineral nutrient solutions were compounded 
and employed not only with green plants but also with fungi. 
These included the following: 





Pfejfers Solution 




Ammonium nitrate 


10.0 grams 


Cane sugar 


50.0 grams 


Potassium phosphate 




Ferrous sulphate 


Trace 


(monobasic) 


3.0 grams 


Water 


1000 ml 


Magnesium sulphate 


2.5 grams 








Reaction: pH 


= 4.3 


- 




Richards' Soli 


■it ion 




Potassium nitrate 


1 gram 


Ferric chloride 


Trace 


Potassium acid mono- 




Saccharose 


3.43 grams 


phosphate 


0.5 gram 


Water 


100 ml 


Magnesium sulphate 


0.25 gram 








Reaction: pH 


= 4.2 






Uschinsky's Solution 




Ammonium lactate 


6.1 grams 


Sodium chloride 


5-7 grams 


Sodium asparaginate 


3-4 grams 


Calcium chloride 


0. 1 gram 


Potassium acid phosphate 


2-2 . 5 grams 


Glycerin 


30-40 grams 


Magnesium sulphate 


0.3-0.4 gram 


Water 


1000 ml 




Czapek's Solution 




Magnesium sulphate 


0.5 gram 


Sodium nitrate 


2.0 grams 


Potassium acid phosphate 


1 .0 gram 


Saccharose 


3-4 grams 


Potassium chloride 


0.5 gram 


Water 


1000 ml 




Reaction: pH 


= 6.8 





4 NUTRITION OF FUNGI 

Sulphur requirements. The results obtained with these nu- 
trient solutions make a voluminous literature. There appears 
little reason to doubt that these experiments prove the essentiality 
for all fungi of appreciable amounts of potassium, phosphorus, 
magnesium, and sulphur. Inorganic phosphates constitute en- 
tirely satisfactory sources of potassium and phosphorus. Mag- 
nesium sulphate serves well as the source of magnesium, but not 
all fungi are able to use sulphates as a source of sulphur. Arm- 
strong (1921) observed that persulphate, sulphite, and sulph- 
hydryl can be substituted for sulphates in the nutrition of Asper- 
gillus ?iiger, Penicillium glaucum, and Botrytis cinerea. Volkon- 
sky (1933, 1934) made similar observations with certain water 
molds, such as Achlya, Aphanomyces, Dictyuchus, Isoachlya, and 
Leptolegnia, and pointed out that each of these forms grows 
better on organic than on inorganic sulphur compounds. The 
results for inorganic sulphur were substantiated by Leonian and 
Lilly (1938), whose experiments show that the amino acid /-cys- 
tine is necessary for the growth of Saprolegnia mixta, Achlya 
conspicua, Aphanomyces camptostylus, and Isoachlya ?nonilifera. 
Schade (1940), on the other hand, found that Leptomitus lac- 
teas and Apodachlya brachynema fill their sulphur requirements 
by reducing sulphates. Steinberg's (1941) experiments show that 
Aspergillus niger utilizes both organic and inorganic sulphur. Of 
the organic sulphur compounds, alkyl sulphonates and alkyl sul- 
phinates are readily assimilated, but the alkyl mercaptans, sul- 
phides, and disulphides are not utilized. In the case of inorganic 
sulphur compounds, the sulphur is first reduced to sulphoxalate 
and then converted to organic sulphur. 

Calcium requirements. Whether calcium is essential for all 
fungi is still a controversial question that should be studied, the 
best techniques known for such tests being utilized. Molisch 
(1894) came to the conclusion that fungi do not require calcium. 
Mosher et al. (1936) have presented evidence to show that Tri- 
chophyton inter digitale requires calcium. Young and Bennett 
(1922) concluded that calcium is generally beneficial in the 
growth of most fungi and is certainly required by Fiisariinn 
oxysporum, Rhizopus nigricans, and Aspergillus niger. They 
also grew species of Ascochyta, Botrytis, Cercospora, Colleto- 
trichum, Dothiorella, Alacrosporium, Phoma, Rhizoctonia, Sclero- 



MINERAL NUTRITION OF FUNGI 5 

tinia, Sphaeropsis, and Vermicularia in Richards' solution with 
Ca(N0 3 ) 2 substituted for KNO3 and maintained, although their 
evidence is not conclusive, that best growth occurred in the solu- 
tions containing calcium. They attribute this phenomenon to 
the neutralization by calcium of the acids formed from sucrose. 
In support of this theory they demonstrated that growth, when 
inhibited by acids, can be renewed after neutralization of the 
acids. They also grew F. oxysporum, A. niger, and R. nigricans 
on similar solutions with the results shown in Table 1 . These data 
show, for each fungus, greatest growth in the presence of calcium. 

TABLE 1 

Comparative Growth of Fungi in KNO3 and Ca(NOs)2 

Weight of Mycelial Mat {grams) 



Richards' solution Richards' solution 

Organism with KNO3 with Ca(NOs)2 

Fusarium oxysporum 0.2094 0.2428 

Aspergillus niger 0.5450 0.8270 

Rhizopus nigricans . 201 5 . 2787 

Concentration and proportion of minerals. Manifestly 
chemical constitution is a factor of primary importance in the 
preparation of suitable mineral substrata for the growth of fungi, 
but account must also be taken of the proper balance of elements 
and of their concentration. Several important papers have ap- 
peared dealing with these factors. With Aspergillus niger 
Haenseler (1921) used the same three-salt mineral nutrient and 
quite the same procedure as has been utilized in physiological 
studies with green plants. The salts consisted of Ca(NO :i )2 or 
NaN0 3 , A4gS0 4 , and KH 2 P0 4 . Different concentrations of each 
salt, making total concentrations of 0.5, 2.1, and 4.2 atm, were 
employed, and to each culture flask were added equal amounts of 
sugar and other nutrients. The dry weight of the mycelial mat 
after 7 days served as Haenseler's basis for evaluating salt concen- 
tration and balance. The data in Table 2 show the plan employed 
by Haenseler in this type of study. 

Haenseler concluded that at concentrations equivalent to 4.2 
atm. growth is best, so that total concentration must be regarded 
as very important. Wide variation in the concentration of MgS0 4 
and KH0PO4 did not greatly modify growth. Better growth was 



NUTRITION OF FUNGI 



TABLE 2 

Growth of Aspergillus niger in a Three-Salt Nutrient Solution, Showing 
Plan of Varying Concentration (Molarity) of Each Salt and Yield 

of Mycelial Mat 

(M) 



Culture 
Number 




ft t / It k t v 1 * VI KJ t» *■* J 




Yield 


KH2PO4 


Ca(N0 3 )2 


MgS0 4 


{grams) 


Rl CI 


0.00888 


0.00625 


0.08648 


0.347 


Rl C2 


0.00888 


0.01250 


0.07567 


0.624 


Rl C3 


0.00888 


0.01875 


0.06486 


0.874 


Rl C4 


0.00888 


0.02500 


0.05405 


0.956 


Rl C5 


0.00888 


0.03125 


0.04234 


0.949 


Rl C6 


0.00888 


0.03749 


0.03243 


0.983 


Rl C7 


0.00888 


0.04374 


0.02162 


0.985 


Rl C8 


0.01776 


0.04999 


0.01081 


0.977 


R2C1 


0.01776 


0.00625 


0.07567 


0.351 


R2C2 


0.01776 


0.01250 


0.06486 


0.632 


R2C3 


0.01776 


0.01875 


0.05405 


0.865 


R2C4 


0.01776 


0.02500 


0.04324 


0.947 


R2C5 


0.01776 


0.03125 


0.03243 


0.969 


R2C6 


0.01776 


0.03749 


0.02162 


0.984 


R2C7 


0.01776 


0.04374 


0.01081 


0.991 


R3C1 


0.02664 


0.00625 


0.06486 


0.355 


R3C2 


0.02664 


0.01250 


0.05405 


0.610 


R3C3 


0.02664 


0.01875 


0.04324 


0.875 


R3C4 


0.02664 


0.02500 


0.03243 


0.957 


R3C5 


0.02664 


0.03125 


0.02162 


0.957 


R3C6 


0.02664 


0.04374 


0.01081 


0.976 


R4C1 


0.03552 


0.00625 


0.05405 


0.341 


R4C2 


0.03552 


0.01250 


0.04324 


0.603 


R4C3 


0.03552 


0.01875 


0.03243 


0.874 


R4C4 


0.03552 


0.02500 


0.02162 


0.960 


R4C5 


0.03552 


0.03125 , 


0.01081 


0.966 


R5C1 


0.04440 


0.00625 


0.04324 


0.354 


R5C2 


0.04440 


0.01250 


0.03243 


0.634 


R5C3 


0.04440 


0.01875 


0.02162 


0.867 


R5C4 


0.04440 


0.02500 


0.01081 


0.958 


R6C1 


0.05328 


0.00625 


0.03243 


0.364 


R6C2 


0.05328 


0.01250 


0.02162 


0.636 


R6C3 


0.05328 


0.01875 


0.01081 


0.886 


R7C1 


0.06216 


0.00625 


0.02162 


0.352 


R7C2 


0.06216 


0.01250 


0.01081 


0.625 


R8C1 


0.07104 


0.00625 


0.01081 


0.324 



MINERAL NUTRITION OF FUNGI 7 

secured with Ca(N0 3 ) 2 than with NaN0 3 , the differences being 
more pronounced at the higher concentrations. 

Young and Bennett (1922) also employed the triangle system in 
determining the optimum concentration of Ca(N0 3 ) 2 , KH 2 P0 4 , 
and MgS0 4 in solutions of these three salts. The solutions were 
made up by molarity so that their osmotic concentrations were 
equal. Sufficient carbon to make 3.43% was supplied from su- 
crose. Fiisarhim oxysporum, Macrosporhim sarcinaeforme, and 
Phoma apiicola were the test organisms. After 15 days' growth 
on the solutions the mycelial mats were removed, carefully dried, 
and weighed. The results indicate that a proper balance of inor- 
ganic constituents is essential but that each organism appears to 
require a different medium that can be determined only bv trial 
and by techniques of the kind which Young and Bennett used. In 
addition, calcium and zinc should be incorporated in synthetic 
solutions, and it may be necessary to test several sugars before 
the most desirable one can be known. 

Mann (1932) also employed the triangle method, with Pfeffer's 
solution, to determine the influence of varying concentrations of 
the three salts, ammonium nitrate, monopotassium phosphate, and 
magnesium sulphate. She concluded that magnesium is absolutely 
essential, although good growth of Aspergillus niger and Penicil- 
lium sp. was secured at all concentrations greater than 0.0001 gram 
molecule per liter of culture solution. Spectroscopic analysis 
showed that calcium was present as a contaminant in proportions 
less than 1 part per 25 million. Calcium chloride added to the 
three salt solution caused no pronounced increase in the growth 
of A. niger. 

More recently Talley and Blank (1941) performed a carefully 
planned series of factorial experiments on the response of Phyma- 
totrichum ormiivorum to the three salts K 2 HP0 4 , MgS0 4 , and 
KC1 and on the effects of changes in the concentration of each 
salt on responses to the others. Certain of their data which demon- 
strate these interactions are assembled in Table 3. 

Optimum growth of P. omnivorum was secured when glucose 
and nitrogen were not limiting. Solutions containing 0.008 M 
dibasic potassium phosphate, 0.003 M magnesium sulphate, and 
0.002 M potassium chloride were not improved, as indicated by 
the growth of P. omnivorum, by increasing or by decreasing the 
concentration of any one of the salts or of their ions. 



8 



NUTRITION OF FUNGI 



TABLE 3 
Growth Responses of Phymatotrichum omnivorum on Combinations of K2HPO4, 

MgSC>4, AND KC1 AFTER INCUBATING FOR 21 DAYS AT 28° C 

(All solutions had 4% glucose, 0.0125 M NH4NO3, and 2 ppm of Fe, of Mn, and 

ofZn.) 



Solution 


Molar Concentration of Salts 


/.H 1 


Mean 
Weight 


Number 


K2HPO4 


MgS0 4 


KC1 


Original 


Final 


of Mat 
{milligrams) 


1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 


0.004 
0.004 
0.004 
0.004 
0.004 
0.004 
0.008 
0.008 
0.008 
0.008 
0.008 
0.008 
0.012 
0.012 
0.012 
0.012 
0.012 
0.012 


0.0015 
0.0015 
0.0030 
0.0030 
0.0060 
0.0060 
0.0015 
0.0015 
0.0030 
0.0030 
0.0060 
0.0060 
0.0015 
0.0015 
0.0030 
0.0030 
0.0060 
0.0060 


0.000 
0.002 
0.000 
0.002 
0.000 
0.002 
0.000 
0.002 
0.000 
0.002 
0.000 
0.002 
0.000 
0.002 
0.000 
0.002 
0.000 
0.002 


6.74 
6.78 
6.81 
6.84 
6.77 
6.78 
6.92 
6.93 
6.83 
6.82 
6.72 
6.72 
6.93 
6.92 
6.88 
6.88 
6.85 
6.85 


4.81 
4.55 
4.30 
4.16 
4.30 
4.01 
4.87 
5.56 
4.82 
4.68 
4.84 
5.14 
5.58 
5.33 
5.19 
5.35 
5.27 
5.24 


4.53 
4.90 

4.67 
5.90 

5.82 
5.58 
4.24 
4.63 
5.77 
5.59 
6.12 
5.74 
3.60 
3.49 
4.88 
4.77 
5.87 
6.45 



1 These figures represent averages of the number for pH of each of the replications 
and are not, of course, the average pH. 

These observations furthermore indicate that a proper balance 
between K 2 HP0 4 and AIgS0 4 in the growth of P. omnivorum is 
equally as important as the direct effect of either salt. This proper 
balance is maintained without significant change in the amount 
of mycelial growth even though the concentration of K 2 HP0 4 
and A'1jtS0 4 is decreased one-half or increased fourfold. If the 
concentration of either salt is increased, however, the concentra- 
tion of the other must be increased accordingly to maintain this 
proper balance. The potassium ion is of more imoortance in main- 
taining balance than is the phosphate radical. 



MINERAL NUTRITION OF FUNGI 



The support and maintenance of optimum mycelial growth do 
not constitute the only requirement of fungi for a proper balance 
of salts. Pratt ( 1945) demonstrated that the synthesis of penicillin 
by Fenicillium notatum is also conditioned by salt balance. By use 





100 



70 



60 50 40 

NaN0 3 ( percentage ) 



30 



20 



10 







Fig. 1. Graph of triangle system, showing percentage composition of differ- 
ent solutions to be tested. The apex of each triangle represents the constant 
molarity. Each number within a triangle corresponds to a culture or a 
series of identical cultures. (After Pratt.) 

of the triangle system he made up a series of 65 different nutrient 
solutions containing KH 2 P0 4 , MgS0 4 , and NaN0 3 in different 
proportions and in such way as to have a total molar concentration 
of 0.04. Each solution contained in addition lactose, corn-steep 
liquor, zinc sulphate, and phenylacetic acid, and the pH was ad- 
justed with NaOH to be the same as that of every other solution. 
As a result Pratt found that the best yields of penicillin were 
secured in solutions containing not less than 8 millimoles of 
KH 2 P0 4 per liter and not more than 20 millimoles of NaN0 3 per 
liter. The absolute concentrations of the three salts in the series 






W NUTRITION OF FUNGI 

of solutions giving the best yields were as follows: KH 2 P0 4 , 
0.019 M; MgS0 4 -7H 2 0, 0.002 M; and NaNO s , 0.019 M. 

Difficulties encountered in studies of mineral nutrition. 
Lest it be thought that the elements potassium, phosphorus, mag- 
nesium, sulphur, and calcium comprise all the minerals requisite 
for the proper nutrition of fungi, it is pointed out at this juncture 
that evidence has been gradually accumulating that iron, zinc, 
manganese, and copper are among other elements now known to 
be absolutely essential for the normal physiological processes of 
fungi. The experiments from which these conclusions are drawn 
will be considered subsequently. It should first be pointed out 
that the amounts of these elements, as Raulin (1869) first showed 
for iron and zinc, are so minute that the term trace elements has 
come to be applied to them. This terminology does not serve any 
useful purpose, since the amount of a particular element is no 
index or measure in determining essentiality. 

The conclusions from studies of the nutritional use of these ele- 
ments—for example, iron, calcium, zinc, manganese, and boron- 
are, as mi^ht be expected, contradictory. This situation has come 
about because it is now known that certain of the elements occur 
in distilled water or come into solution from test tubes, Petri 
dishes, and culture flasks in amounts sufficiently large to meet the 
metabolic needs of the organism. Water redistilled from Pyrex- 
glass stills should be utilized. Pyrex glass may be quite satisfac- 
tory, particularly if it has previously been used in such a way as 
to leach out the zinc. In addition, the sugars and C.P. nutrient 
salts contain various elements as impurities. By spectroscopic 
methods Steinberg (1937) identified in C.P. reagents commonly 
used in nutritional experiments with fungi the elements listed in 
Table 4. 

The presence of appreciable quantities of various elements in 
C.P. chemicals constitutes an important difficulty, especially in 
studies that involve elements utilized by fungi in small amounts, 
as are iron, zinc, boron, copper, and manganese. Repeated re- 
crvstallization of the nutrient salts has proved to be wholly un- 
satisfactory [Roberg ( 1928) ] in purifying them. Steinberg (1919, 
1935) has devised methods to effect practically complete removal 
of such elements. He heats the nutrient solution in the presence 
of excess CaCO l3 for 15 minutes at 15-lb pressure. The increased 
alkalinity in the presence of heat causes the undesirable heavy 



MINERAL NUTRITION OF FUNG! 11 

TABLE 4 

Elements Shown to Be Present by the Use o? Spectroscopic Methods 

of Analysis 

Chemical Reagents Contaminants 

NH4NO3 Ca, K, Mg, Na 

K2HPO4 Al, Ag, Cu, Mg, Na, Pb 

MgS0 4 -7H 2 Cu, Na 

ZnS0 4 -7H 2 As, B(?), Cu, Fe, Mg, Mn, Si, Sn(?) 

CuS0 4 -5H 2 Cu, Fe, Mg, Mn, Pb, Si 

MnS0 4 -2H 2 Al, Ca, Cu, Cr, Fe, Mg, Na, Si, V 

Na 2 Mo0 4 Al, Ca, Cu, Fe, K, Li, Mg, Mn, Na, Ni, Si, V(?) 

Dextrose Al, Ag, B, Ca, Cu, Fe, K, Li, Mg, Mn, Na, Ni, 

Rb, Rh, Si, Sn, Sr 

metals to be precipitated as phosphates, hydroxides, or carbonates. 
In some cases Mo;C03 may be substituted for heating;. 

Bortels (1927) and Roberg (1928) used activated charcoal as 
an adsorbing agent after adding (NH 4 ) 2 S as a precipitant. Some 
heavy-metal contaminants may be removed bv electrolysis. Wolff 
and Emmerie (1930) removed copper from their nutrient salts by 
electrolytic methods. 

The spores of Aspergillus oryzae were found by Aso (1900) 
to have iron among their ash constituents. Copper and doubtless 
other metals as well occur in the spores and mycelium of other 
fungi. These observations show that in studies involving the min- 
eral nutrition of fungi an appreciable metal contamination may be 
attributed to the inoculum. 

Steinberg (1935) determined that the quantity of the elements 
essential for optimum growth of Aspergillus niger is 0.20 mg of 
iron, 0.14 mg of zinc, 0.06 mg of copper, and 0.03 mg of manga- 
nese per liter of solution. The amount of growth was approxi- 
mately doubled with as little as 0.001 mg of zinc per liter. 

The weight of evidence indicates that these metal contaminants, 
especially zinc, copper, iron, and manganese, serve in the nutrition 
of fungi, not as structural materials, but as substances that modify 
physiological activities. For this reason they have come to be re- 
garded as biocatalysts. This conception is fostered by the numer- 
ous publications of Bertrand and of Javillier, which are reviewed 
by Foster (1939), and by the papers of Bortels (1927), Steinberg 
(1934), and Stern (1938). The heavy metals become organic 
components of respiratory enzymes. Stern (1938) has stressed 



12 



NUTRITION OF FUNGI 



this fact in connection with iron in Warburg's cytochrome, in 
other heme-respiratorv pigments, in catalase, and in peroxidase. 
Copper appears to act similarly in the oxidase molecule. Manga- 
nese is well known to act powerfully as a coenzyme. 

Differences exist in methods of measurements of fungal growth 
on semisolid substrata. Some workers measure dailv radial incre- 



R8 A 0.324 



Rl 




CI 



C2 



C3 



C4 C5 

KH 2 P0 4 



C6 



C7 



Fig. 2. Diagram illustrating triangle system of varying the concentrations 
of three-salt nutrient solutions used in nutritional studies and showing the 

relative vields. (After Haenseler.) 

ments; others measure ring area. In a recent report Worley 
(1939) indicates that several criteria should be considered in 
growth measurements, namely: (1) growth in a radial direction, 
(2) growth in a tangential direction, (3) number of units con- 
tributing to the subsequent growth, (4) the relationship of new 
increments of growth to the substrata being compared, and (5) 
the relative importance of radial and tangential growth quantities. 
The radial method of measurement ignores the effect of criteria 
2, 4, and 5. The ring-area method omits criteria 3 and 4 and mag- 



MINERAL NUTRITION OF FUNGI 13 

nifies or minifies criterion 5. Worley proposes therefore a sector- 
area method to use in comparing the effect of substrata on growth 
for any given time interval. 

In making measurements of mycelial growth on semisolid media 
it must be borne in mind that growth is three-dimensional. On 
one medium the growth by a given species may be appressed, on 
another profuse and cottony. Measurements of growth on differ- 
ent media cannot with fairness be compared, regardless of whether 
radial measurements, ring increments, or sector areas are used. 

Iron as nutrient. Raulin (1869) w 7 as perhaps the first to main- 
tain that iron is indispensable for fungi. This hypothesis was con- 
firmed by Molisch (1892), Benecke (1895), and many others, and 
as a result it is now firmly established that this element is an 
integral part of fungus protoplasm. Using Aspergillus niger, 
Steinberg (1919) secured a scant mycelial mat in solutions lacking 
iron; he obtained 43.7 times as much in the presence of iron. In 
similar studies with this fungus Roberg (1928) secured 75 times 
more mycelium in cultures containing iron. Benecke (1895) 
found that iron is essential both for growth and for sporulation 
among species of Aspergillus, Penicillium, and Mucor, an obser- 
vation which Bortels (1927), using refined techniques, was able 
to verify. 

Little is known about the functions of iron in fungi. Richards 
(1899) reported an increased efficiency in the use of sugar by 
Aspergillus niger and Yemcillium glaiicnm in the presence of 0.1% 
FeCl 3 . Wehmer (1891) found that in darkness the presence of 
iron in sugar solutions being fermented by Aspergillus results in 
decreased production of oxalic acid. Chrzaszcz and Peyros 
(1935), on the other hand, reported markedly increased produc- 
tion of citric acid by Aspergillus and Penicillium when the sugar 
solutions being fermented contain a small quantity of FeCl 3 . Simi- 
larly, others have recorded contradictory results, and this lack of 
agreement can be expected to prevail until the proximate function 
of iron is better understood. 

Copper as nutrient. When the growth of fungi in copper- 
containing nutrient solutions and in solutions lacking copper is 
compared, as Waterman [Foster (1939)], Bortels (1927), and 
McHargue and Calfee (1931) have done, all investigators are in 
accord in ascribing to copper the role of an essential element. The 
striking feature of studies of this nature is that minute amounts 



14 NUTRITION OF FUNGI 

produce such profound growth responses. The best evidence of 
the quantitative essentiality of copper is offered by Wolff and 
Emmerie (1930), who electrolytically purified the culture media 
in which thev attempted to grow Aspergillus niger. They secured 
no growth in the complete absence of copper. When they added 
0.2 y of copper per 250 ml of medium, growth occurred; conidia 
were produced only if a minimum of 0.3 y per 250 ml was added. 
In a copper-free medium, as produced by Metz (1930), A. niger 
was able to produce conidia, but Metz determined them to be 
twice as numerous in copper-containing media as in copper-free 
media. 

Wolff and Emmerie (1930) also showed that there is no pig- 
mentation of conidia of A. niger if the amount of copper provided 
is the minimum for conidial production, as is maintained also by 
Steinberg (1934). In 1938 Mulder [Foster (1939)] showed that 
maximum pigmentation in this fungus requires a minimum of 
6.25 y of copper per 100 ml of nutrient solution. Mulder also 
ascribed to copper an influence in the formation of acids during 
fermentation. 

Manganese as nutrient. The status of knowledge regarding 
manganese as a nutritive element for fungi has been reviewed by 
Foster (1939). A series of studies by Bertrand and Javillier 
[Foster (1939)] and by Steinberg (1936) shows that much less 
manganese is required than either iron or zinc. In fact, manga- 
nese in a concentration of 1 part in 10 billion is definitely stimu- 
latory to Aspergillus niger. Bertrand believed his evidence to 
show that iron, zinc, and manganese must have a certain balance 
and that thev function together synergeticallv. With a certain 
proportionality of manganese, iron, and zinc, he secured a sparse 
production of conidia by A. niger; with a larger amount of man- 
ganese, however, the conidia were developed in abundance. Stein- 
berg (1935) was able to confirm these observations to an extent 
by showing that a lack of manganese in the nutrient either sharply 
reduces or entirely inhibits conidial production. 

Stimulation of growth, as indicated by increased dry weight, 
followed the addition of manganese to cultures of A. flaws and 
Rhizopus nigricans by McHargue and Calfee (1931). 

Zinc as nutrient. As has been indicated by Foster (1939), zinc 
is the element of first choice in studies dealing with the mineral 
nutrition of fungi. Numerous experiments involving representa- 



MINERAL NUTRITION OF FUNGI IS 

tives of each class of fungi show that the presence of zinc in nu- 
trient media stimulates growth [Metz (1930)]. Alosher et al. 
( 1936) regard zinc as essential for the dermatophyte Trichophyton 
inter digit ale. In comparing the growth of Aspergillus niger in 
media lacking zinc with that in media containing zinc salts, Porges 
(1932) noted that a scanty, thin, smooth pellicle develops when 
zinc is lacking, whereas in its presence a heavy, wrinkled mat is 
produced. Steinberg (1919) secured an increase of mycelial mat 
of A. niger amounting to as much as 230,900%, the increases being 
correlated with the quantity of zinc present as an impurity in the 
salts; as he indicates (1934), increases of this magnitude can hardly 
be interpreted as "stimulatory" effects. 

Zinc does not uniformly influence conidial production in the 
same manner in all fungi. Roberg (1928) and Porges (1932) 
found that zinc inhibits sporulation of A. niger. Zinc represses 
sporulation of Trichoderma koningii but stimulates conidial pro- 
duction by Fiisariwn oxyspornm [Niethammer (1938)]. 

Pigmentation in fungi, as modified by the presence of zinc, has 
been considered by Bortels (1927), Roberg (1928), and Metz 
(1930). Metz's experiments involved species of Aspergillus, 
Penicillium, Fusarium, Macrosporium, and Botrytis. He found 
that the growth in zinc-deficient cultures is abnormal in color. 
The problem was further complicated, however, because, al- 
though mycelial growth is dependent primarily on zinc and to a 
lesser degree on iron and copper, it is essential that each of these 
heavy metals be present to produce normal colors in a particular 
fungus. 

The profound effect which zinc exercises on the growth and 
sporulation of fungi is an index of the influence which this ele- 
ment exerts on digestive and respiratory activities. That this 
fact has long been appreciated is evident from the work of 
Richards (1899) and Watterson (1904). 

The formation of organic acids as waste products in fungus 
metabolism is briefly considered in Chapter 4, but emphasis is not 
placed upon zinc as a modifying factor. Zinc has been shown to 
prevent the accumulation of different acids in cultures. This ef- 
fect has been demonstrated by Bortels (1927) and Wassiljew 
(1935) with oxalic acid production by A. niger, by Bernhauer 
(1928) and Chrzaszcz and Peyros (1935) with citric acid produc- 
tion by the same fungus, by Lockwood, Ward, and May (1936) 



16 NUTRITION OF FUNGI 

and Waksman and Foster (1939) with gluconic acid production 
by Rhizopus. 

Other elements as nutrients. A lame number of other ele- 
ments have been tested to determine whether they are essential 
for the metabolic activities of fungi. The role of boron for 
Penicillhnn glaucum and Aspergillus niger was investigated by 
Boeseken and Watermann (1912). Molybdenum and gallium 
have been found to be essential for A. niger by Steinberg (1936, 
1937, 1938), and the same investigator (1920) determined that 
uranium and cobalt can partly replace iron and zinc for this spe- 
cies. Lockwood et al. (1934) found that columbium, chromium, 
molybdenum, and tungsten are favorable for the production of 
fats by Penicillium javanicum. Steinberg (1938) tested 76 chemi- 
cal elements with the result that iron, zinc, copper, manganese, 
gallium, and molybdenum comprise all that may be regarded as 
essential for A. niger. Other extended systematic studies, espe- 
cially those of Pirschle (1934, 1935), involve the effects upon 
growth of many elements. Javillier (1913) concluded that co- 
lumbium and beryllium cannot replace zinc, which, when present 
in concentrations of 1 to 2 ppm, increased the amount of growth 
of A. niger 58 times. 

ORGANIC NUTRIENTS OF FUNGI 

Those investigators who laid the foundations for an understand- 
ing of the mineral requirements of fungi also contributed to the 
establishment of bases for comprehending the organic compounds 
utilized in the growth of these organisms. They noted that fungi 
vary in response to the addition of different carbon compounds 
employed to fortify synthetic media. They arrived at this con- 
elusion by what is commonly designated the "trial and error 
method." Apparently Pfeffer (1895) was the first to study this 
problem with the planned purpose of determining the quantitative 
acceptability to a particular fungus of various organic substances. 
In nearly all subsequent studies either of two purposes has been 
maintained: (1) to find whether the given fungus would grow 
upon the proffered carbon compound, in order to determine its 
enzyme-producing ability, or (2) to measure the comparative rate 
of growth of the fungus on different substrata, using the weight 
of the mycelial mat or the increase in diameter of colonies as a 



ORGANIC NUTRIENTS OF FUNGI 11 

criterion. These studies, as might be expected, have determined 
that cosmopolitan species of Aspergillus and Penicillium thrive 
on a wide variety of substrata. Specialized pathogens, on the 
other hand, either grow poorly in artificial culture on organic sub- 
strata or may even fail to grow at all. A survey of this situation 
clearly indicates that the underlying reasons for these differences 
in organic food requirements of fungi should be sought by inten- 
sive studies. 

Carbon requirements. Those carbon compounds that can be 
oxidized with the least expenditure of the energy stored in the 
compound or can be assimilated most readily appear to constitute 
the food of first choice for fungi. Evidence indicates that, in 
general, fungi, like bacteria, prefer carbohydrates as food sources, 
with proteins as second choice, and that few species thrive well 
on fats. 

Fungi grow more rapidly in proportion to their body weight 
than do green plants, and consequently expend relatively more 
energy in converting their food into an assimilable form. With 
molds an increase in body weight amounting to a thousandfold 
within a 10-day period, such as occurs in Phy corny ces nitens and 
Aspergillus niger, is not uncommon. 

The method that has been generally employed to determine the 
food value of carbon compounds is to grow the fungus in a basal 
mineral-nutrient solution and to vary the carbon or the nitrogen 
added. By preliminary trials the time required to attain maximum 
growth can be determined. The mycelial mat, if removed at the 
end of this period, can be desiccated and weighed. In comparison, 
another figure, which is the result of an analysis to determine the 
amount of compound that has been used by the mold, can be con- 
sidered. Unfortunately, as investigators have indicated, inaccu- 
racies appear as a consequence of the formation and accumulation 
of by-products, such as acids, alkalis, staling products, and toxins, 
and of the autolysis and utilization of dying and dead parts of the 
mycelium. 

Another method that has been employed to only a limited extent 
makes use of microrespirometers. This method, considered in 
Chapter 3, is adapted for use in determining whether the given 
carbon compound is acceptable and also the rate at which it is 
consumed. 



18 NUTRITION OF FUNGI 

Diversity in ability to use carbohydrates is indicated by numer- 
ous reports, but in general the literature shows that glucose is the 
favorite. It may be removed first from a solution containing a 
mixture of sugars, or it may even be the only one removed. Su- 
crose, if present, may be first inverted into dextrose and levulose. 
If Aspergillus niger is the test organism, Alolliard (1918) found 
that, when all the dextrose has been utilized, five-sixths of the 
levulose still remains. 

Attempts to employ the amount of carbon dioxide evolved as 
the sole measure of utilization of the carbon source may lead to 
erroneous interpretations, for the reason that some of the products 
metabolized may be stored within the body of the mold, where 
they may be oxidized subsequently. Most species give better 
yields on hexoses than on pentoses, although Hawkins (1915) 
found that Glomerella cingulata utilizes the two pentoses, arabi- 
nose and xylose. Weimer and Harter (1921) tested the responses 
to glucose of Botrytis cinerea, Diplodia tubericola, Fusarium 
acuminatum, Mucor racemosus, Rhizopus tritici, Sclerotium bata- 
ticola, and Sphaeronema fimbriatnm, finding that each organism 
differs in the amount of this sugar required to produce a unit of 
dry weight. Brannon (1923) found that glucose and fructose 
are equally acceptable to Aspergillus niger and Fenicilliwn camevi- 
berti. Fusarium lini } however, is said to be unable to utilize glu- 
cose [Tochinai (1926)]. 

In a series of studies that may well serve as a model, Raistrick 
et al. (1931) found that glucose, fructose, and sucrose are excel- 
lent sources of food for many molds. They evaluated these 
sugars by keeping "balance sheets" on the amount of sugar 
utilized and the amount of certain metabolic products formed 
or of mycelium produced. By means of such techniques differ- 
ences in the nutritive values of various carbon sources can be 
determined. Horr's (1936) observations show that both galactose 
and mannose constitute poor sources of carbon for Aspergillus 
niger and Fenicilliuvi glaucum. This observation regarding galac- 
tose is confirmed by Steinberg (1939), who added that lactose 
supports practically no growth of A. niger, that glycerol results 
in poor yields, and that dextrose, fructose, sucrose, and /-sorbose 
are equally effective. 

Variation among species in ability to utilize sources of carbon 
is further shown by the inability of Achlya prolifera, A. racemosa, 



ORGANIC NUTRIENTS OF FUNGI 19 

Saprolegnia jerax, and S. monoica to utilize sucrose [Pieters 
(1915) ] and by the utilization of galactose by Trichophyton inter- 
digitale [Mosher et al. (1936)] and Aspergillus fischeri [Wenck 
etal (1935)]. 

Schade's (1940) observations show that Apodachlya brachy- 
nema grows well on dextrose, levulose, and sucrose but is unable 
to utilize maltose and galactose, whereas Leptomitus lacteus uses 
none of these sugars. 

By respirometric methods Wolf and Shoup (1943) noted that 
Allomyces arlniscula, A. javanicus, A. moniliformis, and A. cysto- 
genns are able to use dextrin. Allomyces arbuscula uses maltose 
and sucrose; none utilizes d-arabinose, /-arabinose, cellobiose, glu- 
cose, galactose, lactose, levulose, mannitol, or starch. 

Certain pathogens possess wide capabilities for utilizing carbo- 
hydrates, whether mono-, di-, or polysaccharides, as is illustrated 
by Moore's studies (1937) of Thymatotrichum omnivornm. She 
determined that this organism uses dextrose, levulose, galactose, 
maltose, sucrose, lactose, mannite, xylose, inulin, dextrin, starch, 
glycerin, and cellulose and introduced another factor into the 
problem by varying the oxygen tension. Decrease in oxygen 
tension was accomplished by the removal of oxygen with pyro- 
gallic acid; increase, by the introduction of oxygen from a storage 
cylinder. Oxygen at normal atmospheric concentration was 
found optimum for growth. 

Foster et al. (1941) studied the direct utilization of C0 2 by 
Aspergillus niger and certain other molds. By employing radio- 
active carbon (C n ), they were able to show that carbon is me- 
tabolized into cellular material and organic acids. It may achieve 
a role in respiratory changes connected with the formation of 
oxaloacetate from pyruvate and C0 2 . The oxaloacetate thus 
formed may in turn give rise to fumaric acid or to succinic and 
citric acids. Earlier workers had suggested that C0 2 enters into 
the metabolism of fungi, but proof was not forthcoming until 
Foster and his associates made use of labelled carbon. 

Careful consideration, beginning perhaps with von Naegeli's 
(1880) investigations in 1880, has been given to certain organic 
acids as sources of carbon. A4uch remains, however, to be accom- 
plished. The work of Camp (1923) with citric acid serves to il- 
lustrate the ability of fungi to utilize organic acids. He compared 
the growth of certain fungi on media containing citrates as a 



20 NUTRITION OF FUNGI 

sole source of carbon with their growth on media containing 
both citrates and dextrose. The organisms tested included those 
commonly associated with the decay of citrus, namely Penicillium 
digitatum, P. stoloniferuvi, Diplodia natalensis, Phomopsis citri, 
Alternaria citri, Oospora citri-aurantii, and Sclerotinia libertiana. 
All of them were able to grow in orange juice (pH 3.8), but only 
P. stoloniferum, O. citri-aurantii, and 5. libertiana achieved a fair 
amount of growth on lemon juice (pH 2.5). None of these fungi 
grew luxuriantly when citrate was the sole source of carbon. 
After P. stoloniferum and 5. libertiana had been started in dextrose, 
they could, if transferred to media containing citric acid, achieve 
fairly good growth. In general, these organisms attained better 
growth in the solutions containing citrate plus dextrose than in 
dextrose alone, only Penicillium digitatum and Phomopsis citri 
being unable to utilize citrate as a supplement. 

Leptomitns lacteus and Apodachlya brachyneina utilized all 
the straight carbon-chain fatty acids up to and including capric 
acid, with the exception of formic acid and propionic acid 
[Schade (1940)]. 

A very different approach to the problem of utilization of or- 
ganic compounds was made by Tamiya (1932), who attempted 
to determine the relationship of chemical structure to assimilabil- 
ity. For this purpose he employed 123 organic compounds with 
Aspergillus oryzae as the test fungus and noted whether the com- 
pounds were favorable for spore germination, were suitable for 
subsequent growth, and were utilized in respiration. Of those he 
studied, only 51 were found suitable to promote mycelial growth 
and were respired by A. oryzae; 8 others were used in respiration 
although they did not support growth. Tamiya concluded that 
the carbohydrates and polyatomic alcohols constitute the best 
sources of carbon. 

The aromatic series of alcohols and the monoatomic alcohols 
of the aliphatic scries, with the exception of ethyl alcohol, were 
not utilized. Aldehydes, ketones, and esters were unsuitable. 
Citric, lactic, malic, pyruvic, and succinic acids were among those 
utilized. Tamiya concluded that only those compounds are as- 
similated which possess certain characteristic atomic groups that 
he called "chief radicals." These chief radicals must be joined 
either in a ring or straight chain to "residual radicals"; and the 



ORGANIC NUTRIENTS OF FUNGI 21 

may, for example, CH 3 CHOH-, =CHCOH=, CH 3 CO-, 
CHoOH-CHo — , be split off in degradation. 

The relationship of molecular configuration of sugars to utili- 
zation in amino acid formation by Aspergillus niger has been 
elucidated by Steinberg (1942). This fungus was found to use 
all pentoses and hexoses having an /- 3 -carbon atom and a rf-4-car- 
bon atom except the epimers of d- xylose. 

Attention has been called by Steinberg (1939a, 1939b) to an- 
other factor that must be considered in tests to determine the as- 
similability of a given carbon compound. When A. niger was 
grown in the presence of lactose, galactose, glycerol, or mannitol 
alone, the yields were 75, 28, 350, or 34 mg, respectively. When 
the carbon source consisted of a mixture of mannitol and lactose, 
the yield was 234 mg; of mannitol and galactose, 393 mg; of gly- 
cerol and lactose, 458 mg; of glycerol and galactose, 155 mg; of 
mannitol and glycerol, 545 mg; and of lactose and galactose, 17 mg. 
Steinberg interpreted these improved yields from mixtures to 
better proportion of molecular groups. 

An introduction to the information concerning the use of fats 
and oils as sources of carbon may be secured from reports of 
Tausson (1928) and Hopkins and Chibnall (1932). Tausson 
found that Aspergillus flaviis consumes olive oil, cocoa butter, 
beeswax, tripalmitin, and higher paraffins. His data show that 
591.4 mg of paraffin was utilized in 35 days and that 289.1 mg of 
mycelial mat was formed as a result. Hopkins and Chibnall found 
that the higher paraffins with chains not exceeding C 34 H T o were 
assimilable by A. versicolor. In the breakdown of these substances 
evidence indicates that ketones first arise and on further oxidation 
yield fatty acids. Among other vegetable oils that have been 
found to be consumed by molds arc linseed oil and walnut oil. 

Nitrogen requirements. The numerous studies that have 
dealt with the nitrogen requirements of fungi have been primarily 
directed toward finding;- the most suitable sources of nitrogen. 
The results of this work until 1930 were largely summarized by 
Czapek (1930). Later Robbins (1937) classified fungi into four 
groups on the basis of the form of nitrogen they are capable of 
assimilating. One group utilizes organic nitrogen alone; the sec- 
ond, both organic nitrogen and ammonia; the third, not only or- 
ganic nitrogen and ammonia but also nitrate nitrogen. The fourth 
"group is capable of fixing elemental nitrogen and can also utilize 



22 NUTRITION OF FUNGI 

any or all of the other forms. On this basis it appears that fungi 
must be regarded as differing among themselves fundamentally in 
metabolic potentialities as far as usage of nitrogen is concerned. 

It might be anticipated that amino acids would constitute the 
nitrogen form of first choice for the reason that they can be 
utilized in the synthesis of proteins with the least need for energy. 
Evidently, how ever, there are other factors involved in the choice 
of nitrogen. At anv rate, amino acids are well suited to a large 
number of fungi, and Bacto-peptone serves well as a source for 
many species. Boas (1919) interpreted his experiments to show 
that amino acids can be used only after they have been deaminized. 
In this process energy is required, and Boas regarded ammonium 
salts as the most suitable nitrogen source, they being much superior 
to peptones. 

In general there is a paucity of convincing experiments on the 
best source of nitrogen for specific fungi. In their trials with 20 
plant pathogens Young and Bennett (1922) found that each spe- 
cies could utilize nitrate nitrogen; thus they fall into the third 
of the groups proposed by Robbins. In 1910 Hagem [Steinberg 
(1939a)] noted that Mucor cbristianensis, M. griseocyamis, M. 
racemosas, M. sphaerospora, and M. spinosus utilize either NH 4 + 
or NOr with glycerol. Aspergillus fischeri, according to 
Wenck, Petersen, and Fred (1935), uses either NH 4 + or organic 
nitrogen, thus falling into the second of Robbins' groups. Ophio- 
bohis graminis requires organic nitrogen [Fellows (1936)], and 
Basidiobolus ranarum and Saproleguia parasitica need amino acids 
[Leonian and Lilly (1938)]. Leonian and Lilly also used 23 other 
organisms in tests in which various amino acids, singly or in com- 
binations, were substituted for ammonium nitrate, they did not 
obtain any evidence of ability to utilize these amino acids. If they 
added thiamin (vitamin Bi), however, proper amino acids induced 
growth in 14 of the 23 species. Observations by Klotz (1923) 
showed that Aspergillus viger, Diplodia uatalensis, and Spbae- 
ropsis vialorum can utilize amino nitrogen. Neither Apodachlya 
brachynema nor Leptomitus lacteus is able to utilize nitrate nitro- 
gen or ammonia, but both find rf, /-alanine and /-leucine suitable 
for growth and, if acetate is present, utilize also glycine and 
asparagine [Schade (1940)]. 

Alloviyces arbuscula makes use of peptone, alanine, aspartic 
acid, asparagine, arginine-HCl, cystine, glutamic acid, and leucine 



ORGANIC NUTRIENTS OF FUNGI 23 

[Wolf and Shoup (1943)]. Similarly, Wolf and Shoup found 
that A. jcruanicus employs peptone, aspartic acid, asparagine, cys- 
tine, and glutamic acid; whereas A. moniliformis and A. cysto- 
genus utilize only peptone, alanine, aspartic acid, and glutamic 
acid. None of these species was able to use the amino acids gly- 
cine and tyrosine. 

Nielsen and Hartelius (1938) grew yeast in beer wort, with 
added thiamin, as a basic medium and then added /2-alanine, aspara- 
gine, aspartic acid, lysine, and arginine singly and in combina- 
tions. They found that alone each was toxic but that growth was 
improved when all were added. 

Claims that certain fungi employ NH 4 + to the exclusion of all 
other nitrogen sources and therefore may be called "ammonia or- 
ganisms" are not fully supported. Among other factors account 
has not been taken generally in these studies of the influence of 
pH. This subject was given special consideration by Rippel 
(1931). Pirschle (1934) found that ammonia organisms will uti- 
lize NO3"" provided that the cultures are aerated. This fact is 
shown by certain of his data that contrast the weight of yeast 
in nonaerated and aerated cultures. With ammonium sulphate, 
the dry weights in nonaerated and aerated cultures were 2.568 
and 6.348 grams, respectively; with calcium nitrate 0.703 and 8.089 
grams, respectively; with potassium nitrate 0.443 and 5.296 grams, 
respectively. Smaller growth from the nitrates than from the 
ammonium nitrogen may be accounted for by HN0 2 formation, 
since the consensus of opinion is that nitrites are toxic. Removal 
by aeration of this toxic effect in Pirschle's cultures is evidence 
that this hypothesis is valid. 

Whether any fungi are entitled to be grouped among the nitro- 
gen fixers has been the subject of much controversy. In 1892 
Frank [Duggar and Davis (1916)] maintained that Hormoden- 
dron cladosporioides, grown on -nitrogen-free media, fixes nitro- 
gen. The following year Berthelot made a similar claim [Duggar 
and Davis (1916)] for Aspergillus niger and Alternaria tenuis. 
The same ability was attributed to fhoma betae, A. niger , and 
Mucor stolonijer by Saida in 1901 [Duggar and Davis (1916)]. 
Latham (1909), working with A. niger, also reported the fixation 
of appreciable quantities of nitrogen, but Pennington (1911) was 
unable to verify her observations. 



24 NUTRITION OF FUNGI 

The studies of Duggar and Davis (1916) on fixation of atmos- 
pheric nitrogen showed gains in cultures of Fhoma betae on 
manqel-wurzel decoction and on suqar-beet decoction of 3.022 
mg and 7.752 mg of nitrogen, respectively. Under the same 
conditions there were no gains in cultures of Aspergillus niger, 
Macrosporium commune, Penicillium digitatum, P. expansum, 
and Glomerella gossypii. 

Aspergillus niger was employed in experiments involving fixa- 
tion of atmospheric nitrogen by Schober (1930), but no evidence 
of any increase in nitrogen in the culture flasks was obtained. 
Roberg (1931) got negative results of fixation not only with 13 
strains of A. niger but also with 13 other species of Aspergillus. 
Further confirmation of the inability of A. niger to fix atmos- 
pheric nitroq-en was supplied by Allison, Hoover, and Morris 
(1934). 

Certain symbiotic fungi, however, are able to fix nitrogen, as is 
shown by the work of Ternetz (1904). She isolated varieties of 
mycorrhizal fungi belonging to fhoma radicis from ericaceous 
plants and compared their nitrogen-fixing capabilities with those 
of Azotobacter chroococcum and Clostridium pastorianum. The 
strains of P. radicis gave yields of 18 to 22 mg of nitrogen per 
gram of dextrose used. 

The nitrogen requirements of fungi appear worthy of further 
study. Techniques patterned after those employed by the bac- 
teriologist should prove most serviceable. Sources of error in 
the interpretation of results of such studies should include those 
which have been mentioned valid in the interpretation of data 
involving carbon sources. 



GROWTH FACTORS 

The discovery by Wildiers (1901) in 1901 of the need of a 
growth factor for the cultivation of yeast on synthetic media 
marked the beginning of studies on accessory growth substances 
for fungi. Wilders attempted without success to use a medium 
which Pasteur maintained was adequate. When he employed as 
inoculum a few yeast cells, no growth occurred with this medium; 
but when he added a sizable mass of inoculum, the yeast grew. 
He interpreted these results as showing the need for a sufficient 
amount of a substance that he called "bios." As a result of this 



GROWTH FACTORS 25 

discovery the interest of other workers was directed toward de- 
termining- whether Wildiers' results could be confirmed, toward 
learning whether other fungi have similar requirements, and 
toward making attempts to discover the nature and properties 
of bios. 

As the result of various researches it gradually became apparent 
that bios is a complex, containing several growth factors. These 
are now known to include biotin (vitamin H), thiamin (vitamin 
Bi), pyridoxine (vitamin B ), /-inositol, /^-alanine, pantothenic 
acid, and possibly sterol. 

The specific function of each of the bios components is not yet 
satisfactorily known, although the complex has been subjected to 
considerable study. From the attempts to learn their functions, 
however, it may be concluded that they regulate respiration, re- 
production, and rate of growth and that some act as coenzymes 
and are essential in chemical syntheses effected by a particular 
fungus. Not all species, however, seem to have identical require- 
ments for growth factors. This observation has been interpreted 
to indicate that the particular factor either is elaborated by the 
fungus or else is not required at all. 

Copping (1929) found that certain wild yeasts, in a vitamin- 
free synthetic medium, are able to elaborate their own growth 
factors, whereas "domesticated" or "tamed" yeasts require that 
the bios substances be supplied. 

Williams and Rohrman (1936) maintain that the minimum com- 
plement of growth accessory factors required by yeast includes 
aspartic acid, pantothenic acid, /-inositol, /3-alanine, and thiamin. 
In their studies favorable growth responses with Trichophyton 
inter digit ale occurred only upon the addition to the media of 
pantothenic acid, riboflavin (vitamin B 2 ), thiamin, and /-inositol 
[Mosher et al. (1936)]. 

Robbins and his associates (1942) found that Trichophyton 
discoides, pathogenic to calves, suffers from complete deficiences 
of thiamin, pyridoxine, and /-inositol. When from 1 to 10 m/x 
moles of thiamin and pyridoxine and 0.1 to 0.5 mg of inositol were 
supplied, maximum growth was secured. 

Hawker (1936) studied the influence of /-inositol isolated from 
stale cultures of Botrytis cinerea and Gloeosporhnn fructigenum. 
She also utilized baryta and an extractive of lentils to secure a pre- 
cipitate containing /-inositol and a filtrate free from this factor. 



26 NUTRITION OF FUNGI 

Both fractions were found essential for the growth of Nemato- 
spora gossypii, but /-inositol was not necessary for the growth of 
Melanospora destruens. Hawker further found that inositol pro- 
duced a sporulation response with Sordaria fmiicola, Rosellinia 
necatrix, and Zygorhynchns vwelleri. 

Schopfer (1936) demonstrated that Thy corny ces blakesleeamis 
will not erow in a nutrient solution containing mineral salts, as- 
paracrine, and dextrose unless thiamin is added. Kogl and Fries 
(1937) secured favorable growth responses from the addition 
of thiamin to cultures of P. blakesleeamis, Phytophthora cactoruvu 
Nectria coccinea, Sclerotinia cinerea, Poly poms adustus, P. abie- 
tinus, and Fovies pinicola, but no benefit to the growth of Lenzites 
saepiaria Mas apparent. Biotin and inositol were each beneficial 
to the growth of Neviatospora gossypii and Lophoderinium 

pinastri. 

Robbins and Kavanaugh (1938) observed that the following 
species show increased growth in the presence of thiamin: Phy- 
tophthora capsici, P. cinnamorm, P. cryptogea, P. drechsleri, P. 
palm'rcora, P. parasitica, P. boehmeriae, P. cactorwn, P. cambivora, 
Phycoviyces nitens, Pythium arrhenovianes, P. poly clad on, 
Sphaerulina trifolii, Schizophylhnn commune, Sclerothnn del- 
phinii, and 5. rolfsii. Schopfer (1938) showed the need for thia- 
min by several Mucorales, including Absidia ramosa, Chaetocla- 
* dhnn brefeldii, Choanephora cucurbit arum, Dicranophora fulva, 
Mucor ramannianus, Parasitella simplex, Phycoviyces blakesle- 
eamis, and Pilaira anovmla. Quantz (1943) found that 1 y of 
thiamin per 100 ml of solution was optimum for Allomyces 
kniepii and Blastocladiella variabilis. 

Thiamin increased the production of dry matter by Colly bia 
velutipes 400% [Marczvnski (1943)] and was definitely benefi- 
cial to Stereum frustulosmn [Noecker and Reed (1943)]. Both 
riboflavin and pvridoxine, however, were ineffective with these 
two wood-destroving species; when biotin in amounts of 5 y 
per 26 ml of medium was supplied, definitely increased growth 
was noted with S. frnstulosum. 

Attention has also been directed in the studies of growth factors 
to methods of assav of thiamin, biotin, pantothenic acid, inositol, 
and other substances. These methods depend upon the need 
for an external source of accessory substance by a particular 



GROWTH FACTORS 21 

fungus. Phycomyces was used by Bonner and Erickson (1938) 
to assay thiamin. Williams and his associates at the University 
of Texas, who have devised several ingenious methods, em- 
ployed yeast and various bacteria. The nutritional need for vita- 
mins by fungi has been employed in bio-assavs of the thiamin 
content of green plants [Burkholder and McVeigh (1940)1. 
When they grew Floy corny ces blakesleeanns in solutions contain- 
ing minerals, glycine, and glucose with additions of crystalline 
thiamin, they were able to substitute small quantities of plant 
tissues for thiamin. By comparison of the weights of the mycelial 
mats in the cultures supplied with crystalline thiamin with those 
of the mats in the cultures in which plant tissues were substi- 
tuted, they could calculate the thiamin content of the green- 

i & 

plant tissues. 

The work of Robbins and Kavanaugh (1938) shows that some 
organisms are able to synthesize thiamin, which is composed of 
pyrimidine and thiazole; others can carry out the synthesis if 
either or both constituents are furnished them; a third group must 
be supplied with the intact compound if they are to grow nor- 
mally. Robbins and Kavanaugh found that Floy corny ces nitens 
will grow in a nutrient solution containing dextrose, asparagine, 
and mineral salts if 30 units of pyrimidine and thiazole are added, 
but that neither of these intermediates alone is effective. Quite. a 
different reaction was noted with Floytoplotloora fagopyri, Fytlo'mm 
butleri, P. polycladon, Sclerotium delphinii, S. rolfsii, and Sploaeru- 
lina trifolii. Each of these species grows well in this nutrient solu- 
tion if 30 units of pyrimidine are added, but thiazole alone is in- 
effective. By Allomyces kniepii and Blastocladiella variabilis, too, 
thiazole alone is not utilizable [Quantz (1943)], but a mixture 
of thiazole and pyrimidine is equally as good as thiamin. 

Such synthesizing capabilities are further exemplified by the 
researches of Leonian and Lilly (1940). They report that 
Fusarium niveum and Rloizopus minus possess the ability to syn- 
thesize thiamin when grown on a substrate of inorganic salt, amino 
acids, and dextrose. On the same medium Floy corny ces blake- 
sleeanns can also form thiamin, but only when furnished with 
pyrimidine and thiazole. Fytloiomorploa gonapodioides can elab- 
orate its own thiazole and, if supplied with pyrimidine, will link 
the two substances together to form thiamin. Finally, Mucor 



28 NUTRITION OF FUNGI 

ramannianus is able to produce pyrimidine and, if given thiazole, 
can unite the two to form thiamin. On the nutrient medium de- 
scribed above, Fusarium niveum, Mucor nrmannictmis, Fythio- 
morpha gonapodioides, and Rhizopus minus can synthesize their 
own biotin. Robbins and Ma (1941) observed upon Fusarium 
avenaceum a beneficial effect of biotin, present in amounts up to 
1 jug per gram of the agar used. If they employed crystalline 
biotin (methyl ester, CiiH 18 N L >0 :5 S) stimulation occurred with 
the addition of as little as 0.001 /xg. 

Graphium ulmi responds in liquid cultures to the presence of 
pyridoxine (vitamin B, ; ) [Burkholder and McVeigh (1942)]. 
.Marked increases in dry weight of mycelial mat followed the 
addition of 50 y of this vitamin per liter of basal mineral solution 
plus asparaqine and dextrose. On the other hand, this vitamin 
was found to be unimportant in the growth of Saccharomyces 
cerevisiae in media supplied with inositol, biotin, and pantothenic 
acid [Williams, Eakin, and Snell (1940)]. The interaction of 
biotin, inositol, pyridoxine, pantothenic acid, and thiamin in the 
<rrowth of yeast has been surveyed in a report by Williams ( 1941 ) . 

Certain amino acids are considered as growth accessory factors 
in yeast and in various fungi by Nielsen and Sing-Fang (1937). 
The relationship of vitamin deficiencies to the growth of many 
specific fungi is treated in a report by Robbins and Kavanaugh 
(1942). Work of this kind, of course, is dependent largely on 
the availability of vitamins and the synthesis and commercial pro- 
duction of some of them. Recently biotin was found to be 
identical with coenzyme R, and it can now be synthesized [Harris 
et ah (1943)]. The excellent treatise by Schopfer (1943) sum- 
marizes the fund of knowledge that has been derived from the 
researches of vitamins as related to the nutrition of fungi and other 
plants. 

Studies should also be directed toward determining more about 
the proximate function of growth factors in the physiology of 
fungi. Host specificity may be found to be correlated with re- 
quirements for these factors. Growth factors, if speculation is 
guided by the developments in recent years regarding their in- 
fluence on the physiology of animals and of chlorophyll-bearing 
plants, may be thought to be morphogenic or to regulate repro- 
duction. 



IMPLICATIONS 29 

INFLUENCE OF OSMOTIC PRESSURE 

Special attention has been devoted to the concentration of salts 
and nutrients in culture media because spores can be germinated 
and mycelium can be grown in solutions having high osmotic 
equivalents. Ordinarily the media are prepared with their con- 
stituents in such proportions that the osmotic pressure ranges 
from 0.5 to less than 10 atm. 

Knowledge of osmotic pressure finds practical application in 
food preservation with salt or sugar. Increased percentage of salt 
in brines, for example, is correlated with increased capabilities for 
preservation. Molliard (1918) found that conidial formation by 
Sterigmatocy stis nigra is prevented in a nutrient solution contain- 
ing 1% NaCl; within the range of 2 to 5% mycelial growth is 
retarded, and with 12% there is complete inhibition. 

Fungi differ greatly in their tolerance to salts with high osmotic 
pressures. Raciborski (1905) grew Torula sp. in a saturated solu- 
tion of sodium chloride or of sodium nitrate. Hawkins (1916) 
grew certain plant pathogens, including Botrytis cinerea, Diplodia 
tubericola, Fusarium radicicola, F. oxysporum, Sclerotinia cmerea, 
and Sphaeropsis malorum, in potassium nitrate solutions with a 
calculated diffusion tension of 47 atm. Certain molds, such as 
Aspergillus niger and Femcillium glauciim, have been grown in 
solutions having an osmotic pressure equivalent of 157 atm. 

Similarly the preservation of jelly, jam, syrup, and such foods 
against molds is correlated with the osmotic concentration of the 
sugar used. Heald and Pool (1908) found that a mold which they 
named Torula saccharina achieved optimum growth in Pasteur's 
nutrient solution containing 45% sucrose. Slight growth occurred 
in 75 to 80% sucrose solutions. 

IMPLICATIONS 

From the foregoing account it is manifest that species of fungi 
differ from each other in nutritional requirements. Some grow 
well on almost any substrate that is employed and for this reason 
may be regarded as "domesticated" or "tamed" fungi. Others, 
on the other hand, may barely survive on these same media, may 
grow poorly on a limited number of substrates only, or may not 



30 NUTRITION OF FUNGI 

live on artificial media after the reserve within the spore has be- 
come exhausted. Such species, in contrast, cannot be domesti- 
cated. Strange diets, never encountered in their natural habitats, 
are forced upon them in captivity in the test tube. Perhaps the 
mycologist who attempts to study their physiology in artificial 
media may actually be studying their pathology. 

Many published accounts dealing with the growth of a given 
species on a wide variety of media are quite pointless and con- 
tribute nothing fundamental to an understanding of the nutritional 
requirements of the species. Similarly the compounding of nu- 
trient formulae may be a misguided procedure, and the use of such 
formulae may yield only sterile knowledge. The making and 
using of formulae can be condoned only if their purpose is to 
reveal the necessity of some factor that conditions a metabolic 
activity of the fungus. 

In future studies more attention should be given to the utiliza- 
tion of specific organic and inorganic materials in particular meta- 
bolic activities. This necessity is indicated by the fact that some 
substances do not support growth, although they are respired. It 
is further indicated by the fact that many fungi grow well in the 
presence of a given food but do not reproduce. Evidence shows 
that some specific element, vitamin, or other growth factor is 
essential for reproduction but may not necessarily limit any other 
metabolic activity of the given fungus. 

More should be known regarding the mineral requirements of 
fungi. Account should be taken in such studies of the ash content 
of the fungus at the conclusion of the growth period, in com- 
parison to the known ash content of the nutrient before the 
fungus was allowed to grow upon it. It is indicated, furthermore, 
that fungi may well be used in analytic procedures, especially in 
the determination of trace elements [Niklas and Toursel (1941)1 
or of vitamins. 

It appears that a more adequate understanding of the nutrition 
of fungi would result if the terms parasitic (paratrophic) and 
saprophytic (saprotrophic) largely disappeared from the teacher's 
vocabulary. More emphasis would then be placed upon the abil- 
ity of fungi to synthesize foods as well as a variety of other sub- 
stances. As a consequence, the fact that fungi do not possess 
chlorophyll would be of little concern to the teacher, and the 



IMPLICATIONS SI 

student might then come seriously to question whether fungi were 
derived from al^ae by degradation. 



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32 NUTRITION OF FUNGI 

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Lock wood, L. B, G. E. Ward, and O. E. May, "The physiology of Rhizopus 

oryzae" J. Agr. Research, 55:849-857, 1936. 
Mann, Mary L., "Calcium and magnesium requirements of Aspergillus 

niger? Bull. Torrey Botan. Club, S9: 443-490, 1932. 



LITERATURE CITED 33 

Marczynski, M., "Studies on the nutrition of Collybia velutipes (Curt.) 
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170, 1943. 

McHargue, J. S., and R. K. Calfee, "The effect of manganese, copper, and 
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Metz, O., "Uber Wachstum und Farbstoffbildung einiger Pilze unter dem 
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1930. 

AIolisch, H., Die Pflanze in ihrer Beziehung zu Eisen. Jena. 1892. 

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AIolliard, M., "Influence de certaines conditions, sur la consommation, com- 
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Moore, Elizabeth J., "Carbon and oxygen requirements of the cotton-root- 
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AIosher, W. A., D. H. Saunders, L. K. Kingery, and R. J. Williams, "Nu- 
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Naegeli, C. von, "Ernahrung der niederen Pilze durch Kohlenstoff- und 
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277, 1880. 

Nielsen, E., and F. Sing-Fang, "Vergleichende Untersuchungen liber Wuch- 
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Nielsen, E., and V. Hartelius, "Wuchstoffwirkung der Aminosauren. III. 
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Niklas, H., and D. Toursel, "The determination of trace elements by means 
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Pennington, L. H., "Upon assimilation of atmospheric nitrogen by fungi," 
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Pfeffer, W., "Uber Election organischer Nahrstoffe," Jahrb. wiss. Botan., 
28: 205-268, 1895. 

Pieters, A. J., "The relation between vegetative vigor and reproduction in 
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Pirschle, K., "Biologische Beobachtungen iiber Hefe-wachstums mit be- 
sonderes Berucksichten von Nitraten als Stickstoffquelle," Biochem. Z., 
27^:412-445, 1930. 



34 NUTRITION OF FUNGI 

Pirschle, K., "Yergleichende Untersuchungen iiber die physiologische 

Wirkung der Elemente nach Wachstumsversuchen mit Aspergillus 

niger (Stimulation und Toxizitat)," Planta, 25:177-224, 1934. 
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Porges, N., "Chemical composition of Aspergillus niger as modified by zinc 

sulphate," Botan. Gaz., 94: 197-205, 1932. 
Pratt, R., "Influence of the proportions of KH2PO4, MgS0 4 , and NaNOs 

in the nutrient solution on the production of penicillin in surface cul- 
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Robbins, W. J., "The assimilation by plants of various forms of nitrogen," 

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growth of certain fungi," Am. J. Botany, 25: 229-236, 1938. 
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1942. 
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Vitamin B a auf Phycomyces," Ber. deut. botan. Ges., 52: 308-312, 1936. 



LITERATURE CITED 35 

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1926. 



36 NUTRITION OF FUNGI 

Volkonsky, M., "Sur les conditions de culture et le pouvoir de svnthese de 

Saprolegnia sp. Etude qualitative de l'alimentation carbone, azotce, et 

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Chapter 2 
ENZYMES AND ENZYMIC ACTIVITIES OF FUNGI 

Enzymes and catalysts. Enzymes may be defined as organic 
catalyzers, with specific powers of reaction, that are formed by 
living cells but are capable of functioning independently of them. 
This definition conveys a clear concept only if the reader has a 
working understanding of catalyzers. It has long been known 
that relatively small amounts of certain substances modify the 
velocity of chemical reactions. Finely divided platinum, for in- 
stance, greatly speeds up the decomposition of hydrogen peroxide 
into water and molecular oxvffen. Alteration of rate of reaction 
may not always be in the direction of acceleration; it may be in 
the direction of retardation instead, so that the velocity of reaction 
may be either accelerated or retarded by catalysts. 

According to the old conception, catalysts are mysterious 
chemical substances which are unable to initiate a reaction but 
can change the rate of one already in progress; they do not appear 
in the end-products, nor are they used up during the reaction. 
From the newer viewpoint catalysts are to be regarded as sources 
of surface energy. They are capable of functioning provided 
that the spatial configuration of atoms in the surface of the 
catalyst is such as to cause certain oriented adsorption relation- 
ships, thus permitting the catalyst to contribute surface energy to 
the system. Presumably this surface energy induces electron dis- 
placements in the adsorbed and oriented molecules, which are, 
as a result, chemically active. If in catalysis surface-energy forces 
and oriented adsorption are the important features, it can be 
understood that diverse chemicals can be used to catalyze a 
given reaction. Such a concept, moreover, affords a logical ex- 
planation for believing that catalysts, by inducing electron shifts 
in the reacting molecules, may themselves initiate reactions. 

According to this newer conception, even though the catalyst 
may not actually appear in the end products of the reaction, the 

31 



38 ENZYMES AND ENZYMIC ACTIVITIES OF FUNGI 

catalytic surface may have entered into the reaction, or there 
may have been an oriented adsorption at the catalyst's surface 
with the result that the reacting molecules are brought within the 
sphere of chemical attraction and reactivity. If then the products 
of the reaction are attracted to the reacting materials more strongly 
than to the catalyst, there is a continuous migration from the 
catalyst to the reacting materials until the transformation has been 
completed. 

Within certain limits the degree of acceleration of a reaction is 
proportional to the concentration of catalyst present, although 
the final equilibrium is entirely independent. With a smaller 
amount of catalyst, however, a longer time may be required to 
produce a definite equilibrium. 

Water constitutes one of the most important catalysts known. 
Its effect is evident in the case of pure chlorine gas and hydrogen 
gas, which will not combine to form HC1 at a measurable rate 
except in the presence of water. 

Our knowledge of enzymes begins with the observations of 
Pay en and Persoz, who in 1833 made the observation that germi- 
nating seeds contain a substance which transforms starch into 
sugar. They called this substance diastase, although amylase is 
the preferred name. This discovery marks the beginning of 
studies of "organized ferments," which were thought to be the 
agents that carried on catalytic processes within living cells, as 
distinguished from "unorganized ferments," which did not re- 
quire the presence of living cells. Confusion in the use of the 
word ferment led Kuhne in 1867 to suggest the name enzyme for 
all organized ferments. The distinction between organized and 
unorganized ferments completely broke down, how ever, when 
Buchner in 1897 isolated diastase from yeast. He crushed yeast 
cells with sand, pressed out a straw-colored fluid, filtered it to 
free it from living cells or their fragments, and found that the 
clear fluid was capable of producing alcoholic fermentation. He 
also demonstrated that this alcohol-forming substance was pre- 
cipitable by alcohol and easily destroyed by heat. Since then an 
enormous literature on enzymes has come into existence, much 
too copious to be summarized in one chapter or even one volume. 
For brief presentations Waksman and Davison (1926) or Tauber 
(1937) is very useful, and for a more elaborate summary the set 
of eight volumes edited by Nord and Weidenhagen (1932-1939) 



CHEMICAL PROPERTIES OF ENZYMES 39 

and of five volumes edited by Nord and Werkman (1941-1945) 
is recommended. 

Classification of enzymes. Several plans have been proposed 
as bases for the classification of enzymes. The simplest of these 
is to group them into extracellular or digestive enzymes and intra- 
cellular or respiratory enzymes. The two classifications are also 
sometimes designated as exoenzymes and endoenzymes, respec- 
tively. Exoenzymes occur in secretions which pass to the ex- 
terior of the living cell through the protoplasmic membrane and 
cell wall. Ptyalin, the amylolytic enzyme in saliva, pepsin, the 
proteolytic enzyme in the gastric juice, and sucrase, the inverting 
enzyme of yeast, are extracellular, and much of our knowledge of 
enzymes has been gained by study of their activities. Little or no 
energy that is available to the cell is liberated by these enzymes. 
The endoenzymes act inside the living cell and are not excreted 
into its environment. Such enzymes are incapable of diffusing 
through the cell membrane. Some of them can react when re- 
moved from living cells, whereas others produce their character- 
istic reactions only in vivo. In contrast to exoenzymes, they 
liberate large quantities of energy to provide for the metabolic 
activities of the cell. In Myxomycetes and certain animals that 
ingest their food, digestion is intracellular instead of extracellular, 
as is generally the situation among fungi. 

Enzymes may also be classified according to the type of chemi- 
cal changes produced, that is, whether they are oxidative, hydro- 
lytic, reductive, or synthetic, or on the basis of the type of 
chemical decomposed, for example, whether it is carbohydrate, 
protein, fat, glucoside, or pigment. In general, the name of each 
specific enzyme is formed from the name of the substrate by 
substituting ase for the last syllable. The list on page 40 includes 
a few of the better-known enzymes occurring in fungi and the 
end-products of the enzymic reactions. 

Chemical properties of enzymes. Concerning the chemical 
nature of enzymes there are two schools of thought. One is 
typified by the researches of Willstatter, Oppenheimer, and Wald- 
schmidt-Leitz, and the other by those of Sherman, Northrop, 
Sumner, and others. 

The first of these theories is that enzymes contain a special 
reactive or prosthetic group which possesses a specific affinity 
or ability to combine with definite groupings in the substrate. 



40 



ENZYMES AND ENZYMIC ACTIVITIES OF FUNGI 



Name 




of Enzyn\e 


Substrate 


Esterase 


Esters 


Lipase 


Fats 


Lecithinase 


Lecithin 


Tannase 


Tannin 


Pectase 


Pectin 


Sucrase 


Sucrose 


Invertase 


Rarnnose 


Maltase 


Maltose 


Trehalase 


Trehalose 


Cellulase 


Cellulose 


Cytase 


Hemicellulose 


Diastase 


Starch and dextrins 


Inulase 


I nuli n 


Raffinase 


RarTinose 


Lactase 


Lactose 


Glucosides 


Glucosides 


Amygdalase 


Amygdalin 


Rennin 


Casein 


Emulsin 


/3-Glucosides 


Pepsin 


Proteins 


Trypsin 


Proteins, proteoses, pep- 




tones, peptids 


Erepsin 


Proteoses, peptones, peptids 


Urease 


Urea 


Phenolase 


Phenols 


Tyrosinase 


Tyrosine 


Peroxidase 


Peroxides 


Zymase 


Glucose, fructose, mannose, 




galactose 


Glycolase 


Sugars 


Fumarase 


Fumaric acid 


Catalase 


Hydrogen peroxide 


Luciferase 


Luciferin 



End-products 

Acids and alcohols 
Glycerol plus fatty acids 
Cholin and glycerophosphoric 

acid and fatty acids 
Glucose and tannic acid 
Pectic acid 
Fructose and glucose 
Fructose and melibiose 
Glucose 
Glucose 
Cellobiose 

Dextrins and monosaccharides 
Dextrins and maltose 
Fructose 

Fructose and melibiose 
Fructose and galactose 
Glucose and other products 
Gentiobiose and benzaldehyde 

plus hydrocyanic acid 
Paracasein 

Sugar plus nonsugar residues 
Proteoses and peptones 
Peptids and amino acids 

Amino acids 

Ammonium carbonate 

Quinones 

Melanins 

Active oxygen plus reduction 

products 
Alcohol and carbon dioxide 

Lactic acid 
Malic acid 

Water plus molecular oxygen 
Oxyluciferin and light by bio- 
luminescent species 

This reactive group is attached to a colloidal carrier, and specific 
action is determined in part by the colloidalitv of the aggregate 
and in part by the affinity of the reactive group for the substrate. 
The enzyme becomes inactivated, therefore, when the colloidal 
properties of the aggregate are destroyed. The second group of 
investigators believe that enzymes are specific, definite chemical 
compounds, probably proteins, and that enzyme specificity is de- 



COENZYMES 41 

termined by the arrangement of the groupings in the complex 
molecule. 

Evidence of the protein nature of enzymes rests upon such 
observations as the following: (1) Many enzymes may be di- 
gested by other enzymes, as occurs when pepsin in acid solution 
is brought into contact with trypsin. (2) It has been shown that 
certain amino acids, such as arginine, aspartic acid, cystine, glu- 
tamic acid, histidine, lysine, tryptophane, and tyrosine, compose 
pepsin [Calvery, Herriott, and Northrop (1936)]. 

The first enzyme to be obtained in purified crystalline form 
was urease, extracted by Sumner in 1926 from jack bean. Since 
then several others have been obtained in crystalline form, in- 
cluding pepsin, trypsin, chymotrypsin, papain, catalase, carboxy- 
polypeptidase, lipase, and the yellow respiratory enzyme of War- 
burg. Northrop (1935) regards all of these crystalline enzymes 
as specific proteins. In opposition to this theory it is maintained 
that these crystalline proteins are not the enzymes themselves 
but the adsorption compounds of the enzymatic component. In 
answer Northrop points out that no specific prosthetic group is 
known for pepsin, urease, trypsin, and carboxypolypeptidase. It 
seems well established, however, that certain catalytic enzymes 
are associated with a carrier and that the facts necessitate accept- 
ance of both viewpoints. The catalytic activity of hematin, for . 
example, is know T n to increase ten-millionfold when associated 
with the colloidal carrier, which leads to a question regarding the 
relative importance of the colloidal carrier and prosthetic group 
in this type of enzyme. 

Coenzymes. Some enzymes, as has been indicated, are "en- 
zyme systems," containing an assisting material that also has 
the power of catalyzing. These are termed coenzymes. Some 
coenzymes are organic, whereas others are inorganic. In 1905 
Harden demonstrated that the dialysate of expressed veast juice 
and the residue are separately inactive but, when combined, are 
again active. In this instance the dialyzable portion is the co- 
enzyme. Among coenzymes are cozymase (coenzyme I), co- 
enzyme II, cocarboxylase (vitamin Bi), riboflavin, nicotinic acid, 
and glutathione. Other vitamins and hormones have been postu- 
lated to function as coenzymes; this theory, if valid, may explain 
the essentiality of specific vitamins in the metabolism of certain 
fungi. Others are so loosely held as to be able to oscillate between 



42 ENZYMES AND ENZYM1C ACTIVITIES OF FUNGI 

different enzymes. Some behave catalytically in rendering oxygen 
active; others seem to function as carriers of oxvgen, of hydro- 
gen, or of phosphates. 

Specificity of enzymes. Much of the classification of enzymes 
is based upon the assumption that each enzyme can act upon a 
single definite chemical compound. In order clearly to compre- 
hend this interpretation, the lock and key analogy has been 
widely employed to illustrate enzyme specificity. The substrate 
is analogous to the lock, and the enzyme to the key. A certain key 
is required to turn each lock, and hence a certain enzyme to de- 
compose each substrate. This analogy is very serviceable but 
conveys the implication that certain enzymes may be master 
enzymes, since they act as master keys. Zymase, for example, 
can decompose the four stereoisomers J-glucose, J-mannose, 
J-levulose, and J-^alactose. Similarly maltase will hydrolyze the 
a-methvlo-lucosides, and emulsin, the #-methvlo;lucosides, but re- 
ciprocally these two enzymes are without hydrolytic ability. 

Attention may well be called to the fact that different enzymes 
may produce different end-products from the same substrate. If 
the trisaccharide raffinose, for example, is decomposed by inver- 
tase, melibiose and fructose are formed; if by emulsin, sucrose 
and galactose. The fact that emulsin may be a complex of sev- 
eral enzymes may account for this result. Similarly there is evi- 
dence that amylase, zymase, and tryptophanase are not single 
enzymes but enzyme complexes or systems. 

Influence of reaction, temperature, and time. In the light 
of our knowledge of the chemical nature of enzymes and of the 
modifying effects of pH, temperature, and time on chemical syn- 
thesis and analysis in general, it should be unnecessary to elaborate 
on this subject as applied to enzymic reactions. These three en- 
vironmental factors are minutely correlated, and none operates 
independently of the others. Each enzyme reacts best under a 
definite environmental set-up. With time and temperature con- 
stant pepsin shows its optimum activity 7 in a solution of approxi- 
mately pH 2.5, whereas trypsin manifests its greatest activity at 
approximately pH 8.0. With pH and time constant, amylase 
shows greatest activity not at body temperature but at 60° C. It 
would be anticipated that enzyme activity would double within 
a limited range for a 10° rise in temperature, as is postulated in 
van t Hoffs law. 



ENZYMES OF WOOD-DESTROYING FUNGI 43 

Production of enzymes by fungi. Scientific interest in the 
production of enzymes by fungi had its beginning in Pasteur's 
studies of the cause of fermentation. Of course, fermentation 
had been utilized by man for centuries before Pasteur's time, but 
no adequate explanation of the process had been offered. Pasteur 
contended that fermentation was a biological process, not a 
mechanical breakdown of the sugar molecule as Liebig believed, 
and that it required the presence of living yeasts. The first proof 
that enzymes produced by the yeasts induced alcoholic fermenta- 
tion was offered in 1897, when Buchner extracted a fluid from 
veast cells and caused sugars to be fermented w T ith this fluid. 
Since then many studies of the enzymic activities of fungi, deal- 
ing either with the enzyme-producing ability of certain species 
or with the utilization of this ability in the production of end- 
products of commercial importance, have been made. 

Methods for detection of enzymes. Two general methods 
have been employed to determine the production of enzymes: 
(a) the in vitro method, in which some portion of the fungus is 
extracted in water and the enzyme is precipitated, the precipitate 
then being dried to an "enzyme powder"; and (b) the in vivo 
method, in which the fungus is cultivated on some chosen sub- 
strate and in which utilization or nonutilization of the substrate can 
be determined. Each method possesses advantages and disadvan- 
tages over the other, and various modifications have been instituted 
to make each more suitable for the problem in hand. In general, 
the in vivo method, as described by Crabill and Reed (1915), is 
open to less valid criticisms than the in vitro method. Among the 
criticisms levelled against the in vitro method are: (a) extraction 
diminishes the activity of enzymes; (b) the proteases may decom- 
pose some of the other enzymes present in the extracted fraction; 
and (c) certain enzymes, especially intracellular ones, may not act 
outside the living cell; that is, the enzymic reactions characteristic 
of the living organism cannot be duplicated with enzyme extracts. 

Enzymes of wood-destroying fungi. A brief summary of 
essential knowledge regarding the enzymes produced by wood- 
destroying fungi has been prepared by Bose (1939), who indicates 
that Bourquelot and Herissey (1895) first directed attention to 
this problem in 1895 in connection with their studies of Poly poms 
sulphur eus. In 1899 Czapek (1899) discovered that Merulius 
lacrymans is able to digest lignin by virtue of an enzyme that he 



44 ENZYMES AND ENZYM1C ACTIVITIES OF FUNGI 

called hadromase. Since then a series of studies have appeared 
that form a basis for a better understanding of the metabolism 
of heartwood-rotting and sapwood-rotting fungi. 

Cellulose and lignin are the most important constituents of 
wood. Some species of fungi destroy the cellulose portions but 
are quite unable to utilize the lignin. They constitute a group 
called the u bro\vn-rot group," typified by Poly poms scbweinitzii. 
Other species, which attack lignin primarily but are also able to 
decompose cellulose, constitute the "white-rot group," typified 
by Tonnes pint and Stereum frustulosum. The enzymic activity 
of the brown-rot group is regarded as mainly hvdrolvtic; of the 
white-rot group, as both hydrolytic and oxidative. 

Although this grouping may be of value to the forest patholo- 
gist, it should be "interpreted to mean that a given species prefers 
either cellulose or lignin but yet may be able to use both com- 
ponents. Campbell "(1929, 1932) divided the white rots which 
he studied into three groups. Some attack lignin in early stages, 
and among them the incidence of attack on cellulose is delayed, 
as occurs with Polystictus versicolor. In another group, exempli- 
fied by Armillaria mellea, cellulose and associated pentosans are 
first attacked, and the utilization of lignin is delayed. In the last 
group both lignin and cellulose are utilized in varying proportions, 
as they are by Ganoderma applanatum, Poly poms adustus, P leu- 
rows ostreatus, and Polystictus abietimis. 

Bavendamm (1928, 1928a) devised a technique to determine the 
ability of a given fungus to utilize lignin. He prepared agar plates 
enriched with such substances as tannic acid, pyrogallol, hydro- 
quinone, resorcinol, guiacol, phloroglucinol, gallic acid, or tyro- 
sine in varying concentrations. On these media he planted 
Merulius lacrymans, Coniophora cerebella, Trametes radiciperda, 
and Stereiim purpureum. After 8 days' growth in the presence 
of most of these substances, red-brown to dark-brown zones of 
discoloration had developed in advance of the hyphal tips around 
colonies of T. radiciperda and S. purpureum. The production 
of these zones was ascribed to the secretion into the agar of 
catechol-oxidative enzymes similar to those that cause the cut 
surface of an apple or a potato to brown. These species use lignin 
primarily and are white rots. M. lacrymans and C. cerebella, on 
the other hand, did not develop brown pigment and hence utilize 
cellulose and are brown rots. 



ENZYMES OF WOOD -DESTROYING FUNGI 45 

Some appreciation of the scope of investigations regarding 
enzymes produced by wood-inhabiting fungi may be gained from 
Table 5, in which representative findings are assembled. The list 
is not inclusive; other enzymes have been demonstrated for some 
of the species listed, and most of the species have not been tested 
to determine whether they are capable of producing all the en- 
zymes mentioned. The most prominent feature shown by this 
compilation is that nearly all species are able to produce amylase, 
catalase, cellulase, emulsin, maltase, and sucrase. 

Whether fungi that are capable of producing many enzymes 
attack a wide variety of woods, whereas those with restricted 
enzyme-producing powers are limited to a single species of tree 
or to closely related species, is as yet unknown. Studies of this 
kind might be fruitful. Woods differ in nature, as is well known, 
in the amount and kind of nutrients present, aside from cellulose 
and lignin, and also in their content of toxic substances. The 
wood of angiosperms is notably higher in pentosan content than 
is coniferous wood. These nutritional factors may determine 
the specificity of fungi for woods. Evidence on this point has 
been submitted by LaFuze (1937). In cultures he found that 
Polystictus versicolor, a generalized species, was able to oxidize 
tannin, resorcinol, quinol, tyrosin, and guiacol, whereas Fomes 
pinicola, a specialized species, had very little oxidizing ability. 
Moreover, P. versicolor showed little selective ability for kinds of 
nutrients, but F. pinicola was sensitive to differences in carbohy- 
drates, growing poorly in the presence of pentoses, galactose, and 
sucrose. In regard to toxic substances in woods, he suggests that 
glucosides, alkaloids, resins, oils, terpenes, and phenolic groups 
may be inhibitive to growth. 

The complement of enzymes produced by the assimilatory por- 
tion of wood-attacking fungi may be different from that in the 
sporophores, as suggested by Nutman (1929). Evidence in sup- 
port of this contention is found in the fact that hyphal growth, 
so far as is known, is apical, and that many fungi are able to effect 
penetration of woody tissues not by way of the bordered pits but 
by making boreholes. Smith (1923) noted apical growth in 
Rhizopus nigricans, Phytophthora parasitica, Rhizoctonia solani, 
Botrytis cinerea, Pyronema confliiens, Aspergillus niger, and Peni- 
cillium expansum. Boreholes in wood were noted by Hartig 



46 



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48 ENZYMES AND ENZYM1C ACTIVITIES OF FUNGI 

and other early workers who studied the pathological effects of 
wood-inhabiting fungi. 

Enzymes of fungi associated with decay of fruits and vege- 
tables. The literature dealing with the decay of fruits and ve^e- 
tables abounds in studies on the enzymes produced by the causal 
fungi. An introduction to the problems involved may be obtained 
by consulting the publications of Reed (1912), Crabill and Reed 
(1915), Harter (1921), Harter and Weimer (1921, 1921a), Funke 
(1922), Muhleman (1925), Davison and Willaman (1927), and 
Menon (1934). In soft rots of fruits and vegetables, caused by 
species of Rhizopus, Sclerotinia, Botrytis, and Glomerella, the 
middle lamellae, or primary host-cell membranes, are attacked by 
pectinase, and the cells tend to separate intact. These organisms 
also possess carbohydrases, by means of which they are able to 
attack starches and sugars in the decay of root and stem crops 
used by man as food. 

Since soft-rot-producing fungi are of so much economic im- 
portance, considerable attention has been devoted to their enzymic 
activities. Studies by Davison and Willaman (1927) involved the 
pectic enzymes of Botrytis cinerea, Rhizopus tritici, Sclerotinia 
cinerea, Monilia fructigena, and M. oregonensis. They found, as 
had other workers, that pectic substances are complex carbohy- 
drate derivatives composed of three types of materials: ( 1 ) proto- 
pectin, the parent pectic material which is water-insoluble but 
which yields pectins on hydrolysis; (2) pectin, the water-soluble, 
methoxylated, hydrolytic product derived from protopectin; and 
(3) pectic acid, the water-insoluble, methoxy-free, hydrolytic 
product. In this hydrolysis three enzymes, protopectinase, pec- 
tase, and pectinase are involved. Protopectinase attacks the pectic 
constituents of the middle lamella, pectin is formed, and as a final 
result the plant tissues are macerated. Pectase is able then to hy- 
drolyze the pectin to pectic acid, methyl alcohol, and acetone, re- 
sults which show that pectin is an ester of pectic acid and that 
therefore pectase is an esterase. Some or all of the products of 
hydrolysis are utilized as food by the soft-rot-producing species. 

Reed (1912) studied the enzymes, other than pectic enzymes, 
produced by another soft-rot fungus of apple, Glomerella rufor- 
maculans and noted that it produced amidase, diastase, emulsin, 
ereptase, invertase, lipase, protease, and oxidase. 



OTHER ENZYMIC ACTIVITIES 49 

Other exzymic activities. The pathological effects induced 
by disease-producing fungi might be understood if, in such cases, 
the enzymic potentialities of the pathogen were known. The 
production of "shot hole" on Prunus by species of Coccomyces 
may be used to illustrate and clarify this point. Higgins (1914) 
found that amy^dalm, stored in the leaves of Prunus, may be 
utilized by species of Coccomyces which secrete an amvgdalin- 
cleaving enzyme. Glucose resulting from cleavage induces in- 
creases in osmotic pressure in invaded tissues. In consequence the 
cells become swollen, and an abscission layer is formed at the 
periphery of the invaded tissues. All tissues inside the abscission 
layer eventually collapse and fall away, and the leaves then have 
the appearance of having been perforated by a discharge of shot. 

Aspergillus oryzae, when cultured on moist sterilized bran for 
approximately 48 hours, produces sufficient growth so that the 
mycelium may be macerated and extracted in water, and the 
enzymes precipitated and concentrated. This extract, w T hich is 
strongly diastatic, has been used in a variety of ways. In the 
textile industry it is employed in the treatment of cotton fabrics 
before mercerization, bleaching, and preparation for printing. 

The material from apple pomace or from the peel of citrus 
fruit that is to become commercial pectin is turbid when ex- 
tracted and must be clarified by enzymic extract from A. oryzae. 
Furthermore, in clarifying syrups and fruit juices it is often 
necessary, in order to facilitate filtration, that the starch be re- 
moved by the addition of enzymes prepared from this same 
fungus. 

Diastase from A. oryzae and A. flavus is used in the preparation 
of soya sauce and in the fermentation of rice to make sake and is 
administered as a therapeutic agent to infants and invalids who ex- 
perience difficulty in digesting starchy foods. It is also used in 
analytical procedures to determine the amount of starch present in 
assays of organic materials. 

The desired flavors of certain cheeses, particularly Roquefort 
and Camembert, are due to the ability of certain molds to decom- 
pose constituents of the cheese. FemciUhim roquefortii, for ex- 
ample, hydrolyzes the butter fats, producing thereby such aro- 
matic fatty acids as butyric, acetic, capric, and caproic. 

Penicillium brevicaule is among the molds that have been used 
in the detection of suspected arsenical poisoning. When grown 



SO ENZYMES AND ENZYMIC ACTIVITIES OF FUNGI 

in a test tube containing a sample of the stomach contents, this 
fungus possesses the ability to transform metallic arsenic into tri- 
methylarsine, which can be detected by its pungent odor, remi- 
niscent of garlic. 

The enzymic activity of fungi is involved in the production of 
a variety of products, including alcohol, organic acids, pigments, 
fats, and carbohydrates, as is explained in Chapter 4. From the 
discussion in that chapter some appreciation can be gained of the 
influence of food supply, temperature, reaction, and O^ tension 
on the enzyme-producing abilities of fungi. 

Certain cosmopolitan molds, such as Aspergilhts niger, Penicil- 
Ynmi glaucinn, and Khizopns nigricans, are omnivorous by virtue 
of their ability to produce a large number of enzymes, represent- 
ing each of the groups: carbohydrases, proteases, lipases, oxidases, 
and reductases. 

Humus formation is associated with enzymic activities, as is evi- 
denced in striking manner by the decomposition of litter on the 
forest floor. Many species of soil-inhabiting molds are capable 
of transforming the cellulose and lignin portions that are rather 
resistant to decomposition. The activities of a few of them, not- 
ably species of Trichoderma, Chaetomium, and Aspergillus, have 
been studied in considerable detail. A comprehensive idea of 
these activities and of their importance in the economy of nature 
is summarized in The Microbiology of Cellulose, Heviicelluloses, 
Pectins, and Gums by Thaysen and Bunker (1927). 

General considerations. Although much has been learned 
about the ability of enzymes from fungi to effect analyses and 
svntheses, further knowledge of these matters should be sought. 
Problems of host-parasite relationship, of host specificity, of the 
synthesis of vitamins by fungi, and of antagonistic and synergetic 
relationships among species may all be elucidated when more is 
known about enzymes. The phenomenon of autodigestion among 
fungi invites further consideration. Pleomorphism, especially the 
tendency of species that are mycelioid in their natural habitat to 
become yeast-like on artificial media, or vice versa, may be cor- 
related with enzymic activity. Until techniques have been per- 
fected to the extent that it is possible to measure the activity of 
small groups of fungus cells or even of single cells and a body of 
pertinent data has been amassed, material progress with studies 
of this kind may be impossible. 



LITERATURE CITED 51 



LITERATURE CITED 

Bavexdamm, W., "Neue Untersuchungen iiber die Lebensbedingungen holz- 
zerstorender Pilze. Ein Beitrag zur Frage der Krankheitsempfanglichkeit 
unser Holzpflanzen. II. Alitteilung: Gerbstoffversuche," Zentr. Bakt. 
Parasitejik., 16: 172-227, 1928. 
"Uber das Vorkommen und den Nachweis von Oxydasen bei holzzer- 
storenden Pilzen," Z. Pflanzenk., 38: 257-276, 1928a. ' 

Bayliss, J. S., "The biology of Polystictus versicolor (Fr.)," /. Econ. Biol., 
3: 1-24, 1908. 

Bose, S. R., "Enzymes of wood-rotting fungi," Ergeb. Enzymforsch., 8: 267- 
276, 1939. 

Bose, S. R., axd S. N. Sarkar, "Enzymes of some wood-rotting polypores," 
Proc. Roy. Soc. London, B, 123: 193-213, 1937. 

Bourquelot, E., axd H. Herissey, "Les ferments solubles du Polyporus sul- 
fureus (Bull.)," Bull. soc. my col. France, 77; 235-239, 1895. 

Buller, A. H. R., "The enzymes of Polyporus squamosus Huds.," Ann. 
Botany, 20:49-59, 1906. 

Calvery, H. O., R. M. Herriott, axd J. L. Northrop, "The determination 
of some amino acids in crystalline pepsin," /. Biol. Chem., 273:11-14, 
1936. 

Campbell, W. G., "The chemistry of white rots of wood. I. The effect 
on wood substance of Polystictus versicolor (Linn.) Fr.," Biochem. /., 
24: 1235-1243, 1929. 
III. The effects on wood substances of Ganoderma applanation (Pers.) 
Pat., Tomes jomentarius (L.) Fr., Pleurotus ostreatus (Jacq.) Fr., 
Armillaria mellea (Vahl.) Fr., Trametes pini (Brot.) Fr., and Polyporus 
abietinus (Dicks.) Fr.," Biochem. J., 26: 1829-1838, 1932. 

Crabill, C. H., axd H. S. Reed, "Convenient methods for demonstrating the 
biochemical activity of microorganisms with special reference to the 
production and activity of enzymes," Biochem. Bull., 4: 30-44, 1915. 

Czapek, F., "tiber die sogenannten Ligninreactionen des Holzes," Hoppe- 
Seylefs Z. physiol. Chem., 21: 141-166, 1899. 

Davisox, F. R., axd J. J. Willamax, "Biochemistry of plant diseases. IX. 
Pectic enzymes," Botan. Gaz., 83: 329-361, 1927. 

Fuxke, G. L., "Researches on the formation of diastase by Aspergillus niger 
van Tieghem," Rec. trav. botan. neerland., 19:219-275, 1922. 

Garrex, K. H., "Studies on Polyporus abietinus. I. The enzyme-producing 
ability of the fungus," Phytopathology, 28: 839-845, 1938. 

Harter, L. L., "Amylase of Rhizopus tritici, with a consideration of its se- 
cretion and action," /. Agr. Research, 20: 761-786, 1921. 

Harter, L. L., axd J. L. Weimer, "Studies on the physiology of parasitism 
with special reference to the secretion of pectinase by Rhizopus tritici" 
J. Agr. Research, 27:609-624, 1921. 
"A comparison of the pectinase produced by different species of Rhi- 
zopus," /. Agr. Research, 22: 371-377, 1921a. 



52 ENZYMES AND ENZYM1C ACTIVITIES OF FUNGI 

Higgins, B. B." "Contribution to the life history and physiology of Cylin- 

drosporium on stone fruits," Am. J. Botany, 1: 145-173, 1914. 
LaFuze, H. H., "Nutritional characteristics of certain wood-destroying 

fungi, Polyporus betulinus Fr., Fomes pinicola (Fr.) Cooke, and Poly- 

stictus versicolor Fr.," Plant Physiol., 72:625-646, 1937. 
Lanphere, W. M., "Enzymes in the rhizomorphs of Annillaria viellea" 

Phytopathology, 24: 1244-1249, 1934. 
Mayo, J. K., "The enzymes of Stereum purpureum" New Phytol., 24: 162- 

171, 1925. 
McDonald, J. A., "A study of Polyporus betulinus (Bull.) Fr.," Ann. Ap- 
plied Biol, 2^:289-310, 1937. 
Menon, K. P. V., "Studies in the physiology of parasitism. XIV. Compari- 
son of enzvmic extracts obtained from various parasitic fungi," Ann. 

Botany, 48: 187-209, 1934. 
Montgomery, H. B. S., "A study of Fomes fraxineus and its effects on ash 

wood," Ann. Applied Biol, 23: 465-486, 1936. 
Muhle.man, G. W., "The pectinase of Sclerotinia cinerea" Botan. Gaz., 

£0:325-330, 1925. 
Xord, F. F., and R. Weidenhagen, Ergebnisse der Enzynrforschung, Bd. 

I-VIII. Leipzig, 1932-1939. 
Nord, F. F., and C. H. Werkman, Advances in enzymology and related 

subjects of biochemistry. Vols. I-V. Interscience Publishers, Inc., New 

York. 1941-1945. 
Northrop, J. H., "The chemistry of pepsin and trypsin," Biol Rev., 10: 263- 

282, 1935. 
Nutman, F. J., "Studies of wood-destroying fungi. I. Polyporus hispidus 

Fr.," Ann. Applied Biol, 75:40-64, 1929. 
Reed, H. S., "The enzyme activities involved in certain fruit diseases," Ann. 

Kept. Va. Agr. Expt. Sta., 1911-1912, 51-77, 1912. 
Schmitz, H., "Enzvme action in Echinodontium tinctorium E. and E.," /. 

Gen. Physiol, 2:613-616, 1920. 
"Enzyme action in Polyporus volvatus Pk. and Fomes igniarius (L.) Gill.," 

/. Gen. Physiol, 3:795-800, 1921. 
"Studies in wood decav. V. Physiological specialization in Fomes pinicola 

Fr.," Am. J. Botany', 12: 163-177, 1925. 
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Enzvme action in Annillaria mellea Vahl., Daedalea confragosa (Bolt.) 

Fr., and Polyporus lucidus (Leys) Fr.," Ann. Mo. Botan. Garden, 

6: 193-200, 1919. 
Smith, J. Henderson, "On the apical growth of fungal hyphae," Ann. 

Botany, 31: 341-343, 1923. 
Tauber, H., Enzyme chemistry. John Wiley and Sons. 1937. 
Thaysen, A. C, and H. J. Bunker, The microbiology of cellulose, hemicel- 
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Waksman, S. A.. \\i) W. C. Davison, Enzymes, xii + 364 pp. Williams 

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with special reference to enzyme activity," Ann. Mo. Botan. Garden, 
3:439-512, 1916. 



Chapter 3 

RESPIRATION 

Present-day concepts of the process of respiration in plants 
come largely from studies with green plants rather than chloro- 
phyll-less ones. It would appear that respiration among fungi 
is worthy of more extended study than it has been accorded in 
the past and that much of value should result from a better under- 
standing of this subject. Just as in green plants, respiration is 
manifested by the disappearance of food substances within the 
cells with resultant liberation of energy, by the absorption of 
oxygen, and by the excretion of carbon dioxide. Many other 
products besides carbon dioxide are excreted by funei. Quite a 
goodly number of these products are of economic importance, 
and in consequence fungi have been utilized industrially. This 
matter will be given special consideration in Chapter 4, which 
deals with the biochemistry of fungi. 

HISTORICAL MATERIAL 

Aluch information of value concerning the respiration of fungri 
has come from the gradual acquisition of knowledge regarding the 
respiratory process commonly known as fermentation. This phe- 
nomenon was undoubtedly known to the ancients lone before the 
days of written records. Although no attempt will be made to 
give an elaborate historical summary of the growth of information 
concerning fermentation, a few of the prominent landmarks 
should be indicated in order that the complex nature of this bio- 
logical process may be better understood. Among the early 
workers who contributed to scientific knowledge of fermentation 
was Fabbroni (1787), from studies of wine making. He believed 
that the sugar was decomposed by material of a glutenous vege- 
table-animal nature that was contained within the grapes. When 
the grapes were crushed, the glutenous material was free to in- 
duce fermentation. He showed that air was not essential to the 

53 



54 RESPIRATION 

process and regarded alcohol neither as a constituent of the 
grapes nor as a product of fermentation. Instead he considered 
it to arise by the reciprocal action of the sugar and the glutenous 
material. 

The chemical studies of Lavoisier (1789) on fermentation led 
him to conclude that sugar was merely separated into two constit- 
uents, carbon dioxide and alcohol, and that if the two were re- 
united, sugar would be reconstructed. He thought that one con- 
stituent was oxygenated at the expense of the other, that the oxy- 
genated portion became carbon dioxide, and that the deoxy- 
genated portion became alcohol. 

Thenard (1802-1803) made the interesting observation that a 
deposit resembling yeast occurred during fermentation of the 
juice of gooseberries, cherries, apples, or other fruits. When this 
deposit was mixed with fresh juice, fermentation was started. He 
was unable to determine whether this deposit came into existence 
from a soluble state or whether it Mas a product of fermentation. 

In 1838 the classical work of Cagniard-Latour (1838), in which 
he described his microscopic studies of yeast, appeared. He stated 
that the globules which he found in wine and beer constituted 
the yeast and belonged to the vegetable kingdom. He correctly 
described their propagation by budding, the buds at first being 
small and attached to the mother cell. 

While Cagniard-Latour was making his discoveries, Schwann 
(1837) examined the deposit in beer and in grape juice and came 
to the conclusion that this deposit was yeast and that yeast was a 
fungus. He clearly established the relationship of yeast to fer- 
mentation by the following phenomena: (a) the constancy of 
occurrence of yeasts during fermentation, and (b) the checking 
of fermentation by heat, chemicals, or other agencies that destroy 
living organisms. According to him, alcohol was a waste product 
left as the yeast drew its food from the sugary solution. Schwann 
must properly be credited with founding the germ theory or 
biological theory of fermentation. 

.Meanwhile the chemical theory of fermentation had its adher- 
ents in such capable chemists as Berzelius and Liebig. They held 
up to contemptuous ridicule the work of Cagniard-Latour, 
Schwann, and all others, notably Kiitzing, who believed that 
veasts produce fermentation. Liebig and his pupils [Bulloch 



HISTORICAL MATERIAL 55 

(1938)] in 1839 anonymously published a skit in which the yeast 
globules were caricatured as blind, toothless animalcules with 
bristly suctorial snouts and enormously developed genitalia. 
These animalcules devoured sugar, whereupon alcohol was voided 
from the anus and carbon dioxide bubbled from the genital or- 
gans. If certain alkaloids were present in the sugar solution, the 
animalcules were capable of emesis, and the vomitus contained 
fusel oil. Liebig (1839) also published a technical treatise in 
which he set forth his views on the whole matter of decomposi- 
tion. He vigorously maintained these views for thirty or more 
years. In his opinion all decompositions were brought about by 
chemical instability of a ferment. The ferment itself w r as not an 
actual chemical substance, but a nitrogen-containing carrier of 
activity or inciter of decompositions that could transmit its in- 
stability to other substances. In short, according to Liebig, yeasts 
were nitrogen-containing, but fermentation was not concerned 
with the life activities of the yeast itself. Instead the yeast was 
produced from the gluten. 

The name of Blondeau [Bulloch ( 1938) ] is also worthy of men- 
tion, since his contributions did much to lay secure foundations 
for Pasteur's researches on fermentation. Blondeau made a study 
in 1847 of lactic, butyric, and acetic fermentations and the decom- 
position of urea and concluded that the different types were in- 
cited by different fungi, notably Torula cerevisiae, Penicillium 
glaucum, P. globosum, and Mycoderma vim. 

Finally came Pasteur's epoch-making series of researches, in 
which he proved that the activity of living yeast is absolutely 
essential to fermentation, that alcohol and carbon dioxide are by- 
products of the respiration of yeast, and that sugar can be fer- 
mented in the entire absence of atmospheric oxygen. This last 
fact, it should be recalled, had been established by Fabbroni in 
1787. Pasteur studied not only alcoholic fermentation but also 
lactic, tartaric, and acetic fermentations. His zeal as a scientific 
crusader and his professional acumen and forcefulness are evident 
in certain memoires, notably those dealing with his findings on 
lactic and alcoholic fermentations [Pasteur (1857, 1858, I860)]. 
That the enzyme of yeast can act in the absence of living cells 
was first established by Buchner, in 1897, nearly two years after 
Pasteur's death. 



56 RESPIRATION 

TYPES OF RESPIRATION 

Ordinarily respiration is arbitrarily divided into two types, 
aerobic and anaerobic. Aerobic respiration occurs in the pres- 
ence of atmospheric oxygen. Anaerobic respiration, on the other 
hand, occurs in the absence of a supply of atmospheric oxygen 
and proceeds at the expense of the oxygen that is combined in the 
substance being respired. Presumably aerobic respiration is of 
most common occurrence among fungi, but many species possess 
the ability to respire either aerobically or anaerobically and are 
spoken of as facultative anaerobes. Few, if any, species are known 
to be strict anaerobes. The facultative anaerobes, because of the 
products of their respiration, for example, alcohol, acetic acid, and 
lactic acid, are of most interest and importance to man. 

Aerobic respiration. The most important reason that can be 
given for elaborating upon aerobic respiration in this volume is 
that such a discussion may help to clarify certain misunderstand- 
ings of this process that are all too commonly prevalent and that 
are sometimes transmitted from teacher to student. 

In the first place fungi, in common with all other living things, 
release energy for their own metabolic activities during the proc- 
ess of respiration. In the oxidation of glucose such aerobic release 
is conventionally expressed as follows: 

C 6 H 12 6 + 60 2 = 6C0 2 + 6H 2 + 673 Cal 

This equation, the precise reverse of the reaction for photosynthe- 
sis, is correct only in so far as it expresses the energy relations 
and the final products. It merely indicates that the complete oxi- 
dation of 1 molecule of glucose requires 6 molecules of oxygen 
and that, while 6 molecules of carbon dioxide and 6 molecules of 
water are formed, 673 calories of energy are released. Such an 
equation leaves the erroneous impression that at one instant glu- 
cose is present and at another, by some miracle, the sugar has be- 
come carbon dioxide, water, and liberated energy. As a matter 
of fact, the process is a complicated one, and intermediate prod- 
ucts are formed. For this reason it is indefensible to indicate aero- 
bic respiration as occurring in accordance with the foregoing 
equation. 

Anaerobic respiration. Consideration will be given subse- 
quently to some of the kinds of anaerobic respiration, products 



TYPES OF RESPIRATION 57 

formed being used as the basis of classification. Anaerobic respi- 
ration of glucose of the alcoholic type is conventionally expressed 
as follows: 

C 6 H 12 6 = 2C0 2 + 2C 2 H 5 OH + 25 Cal 

Alcohol 

Here again, end results alone are indicated, and no essential in- 
formation of the steps and mechanisms involved is conveyed. 
Moreover, this equation shows that only a portion of the potential 
energy of the glucose molecule has been released, yet in this re- 
spect it typifies the energy-release relationships of all other 
anaerobically respired compounds. 

Interrelations between aerobic and anaerobic respiration. 
It seems best at this juncture to indicate the existence of evidence 
to show that aerobic and anaerobic respiratory processes are inter- 
related and that both may be presumed to occur not only amono- 
fungi but also among green plants. Once this interrelationship is 
appreciated, it will be possible to return to the essential steps in the 
process. Kostytchew (1927) has schematically represented the 
relationship in the following manner: 

2C0 2 + 2C 2 H 5 OH + 25 Cal 

/ 
Zymase 
Intermediate prod- 7* T , 
C 6 Hi 2 6 + Zymase -» ucts of anaerobic X ] n absence of ° 2 

respiration \ In presence of ° 2 

+ 60 2 

Xj Oxidizing-reducing enzymes 

6C0 2 + 6H2O + 673 Cal 

In this scheme the zymase complex (long believed to be a single 
enzyme but now known to consist of glycolase, which converts 
hexoses into methylglyoxal, carboxylase, which splits out carbon 
dioxide from certain organic acids, and in addition certain co- 
enzymes) is supposed to convert the hexose into labile inter- 
mediate products as a first step in both aerobic and anaerobic 
respiration. This change is an anaerobic one in either case, as 
Kostytchew's scheme shows. Whether or not atmospheric oxy- 
gen is available determines the next step and also the course of 
the subsequent respiratory reactions. 

To support Kostytchew's theory of the course and sequence 
of events in respiration, the following facts have been marshalled: 



58 RESPIRATION 

1. Green plants, and unquestionably certain fungi also, if de- 
prived of oxygen, respire anaerobicallv. 

2. Glucose and the enzyme complex, zymase, are universally 
present in plant cells. 

3. Acetaldehyde as an intermediate product in the anaerobic 
respiration of glucose has been detected in plant tissues. 

4. Alcohol, an anaerobic respiratory product, has been found 
in higher plants and in certain fungi. Kostytchew (1908) found 
that Agaricus cavipestris formed alcohol if the mycelium was sub- 
merged, even in media lacking sugar. On the other hand, Asper- 
gillus niger, grown in similar media, failed to produce alcohol. 

.Mechanism of aerobic respiration. The mechanism of aero- 
bic respiration among fungi has been presumed to be like that 
among green plants, and in neither group of plants have the de- 
tails been fully substantiated. Palladin (1909) long ago postu- 
lated a theory whose general plan outlined the mechanism as 
follows: 

Intermediate 
CeH 12 06 + Zymase — > anaerobic + 6H 2 + 12A 

products • Hydrogen acceptor, i.e., 

respiratory pigments, 
cytochrome in fungi 

+ Dehydrogenase -* 6C0 2 + 12AH 2 

Reduced acceptor 

Then 

12AH 2 + 2 + Oxidase -► 12A + 12H 2 

This plan means, if elucidated, that after the intermediate an- 
aerobic products are formed, they are oxidized by the active oxv- 
gen that comes from the water molecules, and the freed hydrogen 
combines with the respiratory pigments. As a next step, the 
respiratory pigments in the presence of oxidase again acquire oxy- 
gen, but they take it from the atmospheric oxygen. In this proc- 
ess the sugar is completely oxidized, and the ratio of the volume 
of CO;, released to the volume of O l . utilized is unity. 

.Mechanism of an akrobic respiration (alcoholic fermenta- 
tion). Two theories have been propounded to explain the mech- 
anism of alcoholic fermentation. One of these, called the pyruvic 
acid theory, has been elaborated by Xeuber^ and his associates 
[Xeuberg (1922), Neuberg and Gottschalk (1924)]; and the 
other, commonly called the sugar-phosphate or the Harden theory 
[Harden (1932)], by .Meyerhof and Kiessling (1935). 



TYPES OF RESPIRATION 59 

According to the pyruvic acid theory, the following steps occur 
sequentially: 

a. The hexose molecules become "activated"; that is, highly 
reactive y-glucose or y-fructose comes into transitory existence. 
These sugars are not straight carbon-chain complexes, being best 
represented by a ring type of formula. 

b. The "activated" y-hexose is cleaved by glycolase into two 
molecules of methylglyoxal and two of water, formally expressed 
as: 

C 6 H 12 6 + Glycolase -* 2(CH 3 COCHO) + 2H 2 

Methylglyoxal 

c. As the next step, one molecule of methylglyoxal is reduced 
to glycerol, and the other is oxidized, by a Cannizzaro reaction, to 
pyruvic acid with the two molecules of water. A dehydrogenase 
may catalyze this reaction: 

CH 3 • CO • CHO CH 2 OH ■ CHOH • CH 2 OH 

+ H 2 + HoO Glycerol 

+ II ^ + 

o 
CH3COCHO • CH3COCOOH 

Pyruvic acid 

d. Immediately carboxylase splits the pyruvic acid into acetalde- 
hyde and carbon dioxide, as follows: 

CH3COCOOH + Carboxylase -> CH 3 CHO + C0 2 

Pyruvic acid Acetaldehyde 

The course of events is identical up to this point, as has been 
stated, whether the process is aerobic or anaerobic. 

e. If then anaerobic conditions prevail, the other molecule of 
methylglyoxal produced in step b reacts with the acetaldehyde 
molecule in step d, and by a Cannizzaro reaction a molecule of 
pyruvic acid and one of alcohol are formed in this manner: 

CH3COCHO "> CH3COCOOH 

Methylglyoxal O Pyruvic acid 

+ + I! - + 

H 2 
CH3CHO CH 3 CH 2 OH 

Acetaldehyde Ethyl alcohol 

It is of interest to note that no energy is released in the trans- 
formations that result in the formation of methylglyoxal, glycerol, 
and pyruvic acid. 



60 



RESPIRATION 



If the fermentation is produced bv Saccharomy ces cerevisiae 
and sodium sulphite is added to the culture solution, the acetalde- 
hvde is fixed, and its presence can be demonstrated. With the 
addition of a high percentage of sodium sulphite, glycerine is 
produced from the acetaldehyde, and the reactions will yield 
acetic acid and alcohol also. 

By the sulphite process as much as 37% of the sugar fermented 
by the yeast may be transformed into glycerol. This fact is of 
enormous interest and at the same time of great importance when 
it is recalled that in ordinary alcoholic fermentation the yield of 
glycerol is less than 3%. The sulphite modifies reduction of 
acetaldehyde by hydrogen, and hydrogen can thus act directly 
to reduce the intermediate compound, glyceric aldehyde, forming 
glycerol. 

These chemical changes may be shown briefly as follows: 



C 6 H 12 6 

Glucose 



CH 2 OH 

CHOH 

CHO 

Glyceric 
aldehyde 



CH 3 

i 

c=o 

CHO 

Methyl- 

glyoxal 



OH 



CH 3 


CH 3 


CH 3 


c=o - 


+ c=o - 


->CHO 

Acetal- 


C— H 


COOH 


dehyde 


/ \ 

OH 


Pyruvic 
acid 


+ 


Methyl- 
glyoxal 

hydrate 


+ 

H 2 


co 2 



If sulphite is added, it may unite with the acetaldehyde: 
CH 3 CH 3 

CHO + Na 2 S0 3 + H 2 + C0 2 -> C— H + NaHC0 3 

l\ 
HO SOoONa 



In this event the hydrogen is prevented from reducing the 
:etaldehyde to alcohol; instep 
hvde directly in this manner: 



acetaldehyde to alcohol; instead it can react with the glyceric alde- 



CH 2 OH CH 2 OH 

CHOH + H 2 -> CHOH 



CHO 

Glyceric 
aldehyde 



CH 2 OH 

Glycerol 



TYPES OF RESPIRATION 61 

The complexity of the respiratory reactions, as they have just 
been presented, indicates only a portion of the misconception that is 
conveyed by the formal expression for fermentation: C 6 Hi 2 Oe -» 
2C 2 H r ,OH + 2CO L ». This point is further emphasized by the 
following data of Rubner [Lutman (1929)1 on the products of 
fermentation by yeast of 100 grams of sucrose and the caloric 
value of the products formed: 

Kg-cal Value 

51.1 grams alcohol 358.36 
3.4 grams glycerin 14.38 
0.65 grams succinic acid 1.99 
1.3 grams miscellaneous products 5.15 

49.2 grams carbon dioxide 0.00 



Total Kg-cal value 379.88 

Kg-cal value, 100 grams sucrose 396.80 

Energy released, Kg-cal 16.92 

According to the sugar-phosphate theory, some phosphate, 
such as that of sodium or potassium, is necessary in alcoholic 
fermentation. The phosphate reacts with the hexose to give a 
diphosphoric acid ester. Apparently the phosphate is not a co- 
enzyme to make possible the working of zymase, but it acts as a 
catalytic agent. The formation of the diphosphoric acid ester is 
accompanied pari passu by a second reaction that again liberates 
the phosphate and the hexose. These reactions may be expressed 
as follows: 

a. 2C 6 H 12 6 + 2R 2 HP0 4 + Zymase -> 

Hexose Phosphate 

2C0 2 + 2C 2 H 5 OH + 2H 2 + C 6 H 10 O 4 (PO 4 R 2 ) 2 

Alcohol Glucose di- 

phosphate 

b. C 6 H 10 O 4 (PO 4 R 2 )2 + H 2 -> C 6 H 12 6 + 2R 2 HP0 4 

According to Meyerhof and Kiessling (1935), the hexose and 
phosphate react to form both glucose monophosphate and glu- 
cose diphosphate. Then by oxidation-reduction the monophos- 
phate becomes a molecule of glyceric aldehyde phosphoric ester 
and one of glyceric aldehyde, and the glucose becomes two mole- 
cules of glyceric aldehyde phosphoric ester. As a next step, the 
glyceric aldehyde phosphoric esters are hydrolyzed to glyceric 
aldehyde, and phosphate is again freed. The glyceric aldehyde 



62 RESPIRATION 

then may be oxidized to methylglyoxal, to be in turn transformed 
sequentially into pyruvic acid, acetaldehyde, and, as a final prod- 
uct, alcohol. The decarboxylation of pyruvic acid yields the 
carbon dioxide evolved in the process. 

THE RESPIRATORY RATIO 

As is well known and has been stated previously, the complete 
respiration of hexose yields a respiratory ratio of unity. Fungi, 
however, respire not only hexoses but also various fats and organic 
acids. When such substances are oxidized in the respiratory 
process, it may be anticipated that the ratio of 2 consumed to 
C0 2 released will differ from that shown by the respiration of 
hexoses. The anaerobic respiration of oxalic acid, for example, 
should yield a ratio of 4, as is indicated by the reaction 
2(COOH) + 2 -> 4CO, + 2H 2 + 60.2 Cal. Again, it should 
be anticipated that the ratio will be small if substances poor in 
oxygen are respired completely, as appears from the reaction 
involving the fat tripalmitin: 

C 5 iH 98 6 + 72.50 2 -> 51C0 2 + 49H 2 + 7590 Cal 

In this case the ratio is 5lC0 2 /72.50 2 , or 0.7. 

Richter's experiments with fermentation by yeast (1902) show 
that factors other than the character of the substrate enter into 
the problem of the respiratory ratio. He grew the organism 
in large, flat-bottomed, hermetically sealed flasks containing 50 ml 
of nutrient salt solution, consisting of K 2 HP0 4 , xMgS0 4 , and a 
trace of Fe. To this solution he added varying amounts of sucrose 
and peptone. In those to which he added 0.15 gram sucrose and 
0.25 gram peptone, the C0 2 /0 2 ratio after 24 hours was 4.26; 
after 48 hours, 2.25. In those to which he added 0.3 gram sucrose 
and 0.5 gram peptone, the C0 2 /0 2 ratio after 24 hours was 8.32; 
after 48 hours, 6.16. In those to which he added 0.75 gram sucrose 
and 1.25 grams peptone, the CCX, 2 ratio after 24 hours was 
11.16; after 48 hours, 27.46. From these experiments he concluded 
that yeast utilizes sugar in preference to peptone as a source of 
energy, but that the concentration of food in the substrate be- 
comes an important factor in modifying both the respiratory 
ratio and the rate of respiration. 



RESPIRATORY SYSTEMS 63 

Manifestly temperature is also a controlling factor in respira- 
tion, just as in almost all other biological reactions. The time 
factor, which is correlated with temperature, must also be meas- 
ured, as is indicated by Richter's experiments. The temperature 
effect, apart from time, is strikingly shown in the classical experi- 
ments of Muller-Thurgau [Lutman (1929)], involving Saccharo- 
myces cerevisiae, in which all other conditions were identical and 
fermentation was permitted to proceed until the maximum 
amount of alcohol had been Droduced. These experiments yielded 
the following results: 



Constant 


Maximum Alcohol 


emperature 


Content by Weight 


36° C 


3.8% 


27° C 


7.5% 


18°C 


8.8% 


9°C 


9-5% 



RESPIRATORY SYSTEMS 

The general subject of respiratory enzymes is summarized in 
an extensive compendium, Ergebnisse der Enzymforschimg by 
Nord and Weidenhagen (1932-1939). It need only be stated 
here that the main respiratory system in plant and animal cells 
is composed of the following: Dehydrogenase— Substrate— Cyto- 
chrome— Oxidase— Oxygen. Not only yeasts but also many, and 
presumably all, fungi that live aerobically contain cytochrome. 

There is evidence that some other system operates in certain 
anaerobic organisms. One of these systems is due to the presence 
of glutathione, which can function as an oxidation-reduction sys- 
tem. In the oxidized state it would appear thus: 

COOH COOH 

CHNH 2 ^ CHNH 2 

CH 2 CH 2 

CH 2 COOH COOH CH 2 

CO— NH— C— H H— C— NH— CO 



CH 2 — S — S — CH 2 

Glutathione (oxidized) 



64 RESPIRATION 

In the reduced form two molecules of glutathione give up the hy- 
drogen of the sulphhvdrvl groups thus: 

COOH COOH 



CHXH 2 CHXH 2 

CH 2 CH 2 

CH 2 COOH CH 2 

CO— XH— C— H + H— C— XH— CO 

CH 2 — S— |H HI— S— CH 2 



Glutathione (reduced) 



These observations indicate that the presence or absence of free 
oxygen conditions the respiratorv svstems in even the same species. 

RESPIROMETRY 

In recent vears use has been made of respirometers of a type 
called the Warburg apparatus and of its several modifications, 
which are especially adapted for use with germinating seeds, bits 
of tissue of special organs, blood cells, and bacteria. To date, 
however, all too little use of such apparatus has been made in the 
study of respiration in fungi. 

One such study, indicating the usefulness of this procedure, was 
made by Wolf and Shoup (1943). They employed a Fenn 
respirometer to test the ability of species of the water mold, Al- 
lomvces, to utilize certain carbohvdrates and organic nitrogen 
compounds. After a period of starvation to remove the reserve 
foods the several species were given various compounds singly 
with the following results: 













TABLE 


6 














Utilization 


of Organic 


Compounds bv 


Species of 


Allomyces 


















.45- 


As- 


Glu- 




Argi- 






Dex- 


Suc- 


Malt- 


Pep- 


Ala- 


parlic 


para- 


tamic 


Cys- 


ninc- 




Organism 


trin 


crose 


ose 


tone 


nine 


A cid 


gine 


A cid 


tine 


IJCl 


.1 


arbuscula 


+ 


+ 


+ 


+ 


+ 


+ 


+ 


+ 


+ 


+ 


,1 


j<i: aniens 


+ 


— 


— 


+ 


— 


+ 


— 


+ 


+ 


+ 


.1 


moniliformis 


+ 


— 


— 


+ 


+ 


+ 


— 


+ 


— 


— 


A 


cystogenus 


+ 


— 


— 


+ 


+ 


+ 


— 


+ 


— 


— 



INHIBITION OF RESPIRATION 65 

It may be indicated, in addition, that no species was able to use 
mannitol, if-arabinose, /-arabinose, glucose, levulose, galactose, lac- 
tose, soluble starch, cellobiose, glycine, or tyrosine. 




Fig. 3. A Fenn microrespirometer, having identical glass vessels of 15-ml 
capacity. A closed system is formed when the stopcocks are shut off. There 
are KOH wells for the absorption of C0 2 in each vessel. The fungus to be 
tested is placed in a buffered nutrient in one vessel and buffered solution 
alone in the other. The rate of movement of a. droplet of kerosene in 
capillary toward the vessel with the fungus indicates the rate of O2 

consumption. 

INHIBITION OF RESPIRATION 

Much information has been acquired concerning the influence 
of cyanides and carbon monoxide on the respiration of animal 
cells and bacteria. Little consideration, however, has been ac- 
corded the influence of these inhibitors on the respiration of fungi. 
Such studies, for some reason, seem to have been made quite in- 
cidentally. In a report by Tamiya (1942) the observation was 
made that the respiratory rate of Aspergillus oryzae is decreased 
26% in an atmosphere consisting of 95% CO and 5% 2 . He 
also noted that in liquid media submerged hyphae of this fungus 
are much more sensitive to cyanide than are aerial hyphae, as is 
shown in Table 7. 



66 RESPIRATION 

TABLE 7 
Inhibition of Hvphae of Aspergillus oryzae by Cyanide 

Concentration of Cyanide 



Hyphae 


0.001 M 


0.002 M 


0.01 M 


Aerial 


14 


18 


71 


Submerged 


78 


85 


• • 



STIMULATION OF RESPIRATION 

That it is possible to stimulate or increase the respiratory rate 
of fungi has been shown by a number of investigators. Pratt and 
Williams (1939) determined that thiamin and pantothenic acid 
increase the respiration of certain yeasts. Dammann et al. (1938) 
showed that Gibberella saitb'mettii, in the presence of thiamin, is 
able to ferment glucose at an increased rate and that this greater 
activity 7 is not correlated with increase in mycelial weight. 

Similarly Hawker (1944) demonstrated that thiamin (aneurin) 
in the amount of 10 y per 100 ml of medium increases the amount 
of glucose consumed per unit dry weight of mycelium by 
Melanospora destruens. 



IMPLICATIONS 

Problems related to dormancy of spores and to their germina- 
tion and early growth appear to be worthy of study by respirom- 
etry. The Warburg respirometer or some modification of it 
is also suitable for testing the ability of the selected fungus to 
utilize different nutrient complexes, for discovering its metabolic 
rate, and for determining the kind of enzymes that the organism 
is able to produce. It is possible that the modifying effect of such 
environmental factors as temperature, pH, and perhaps light might 
be better understood by respirometrv. In experiments of this 
sort caution must be exercised in interpreting the results, for the 
reason that several substrates may be oxidized simultaneously. If 
a number of oxidative changes are proceeding concurrently and 
at equal rates, the respiratory ratio cannot be known with any de- 
gree of accuracy. If the evidence indicates that one substrate is 
being oxidized to the extent that its respiration predominates, how- 
ever, the respiratory ratio becomes meaningful. Again, if the 



IMPLICATIONS 61 

total volume of C0 2 evolved is in excess of that anticipated by 
calculation of the quantity which should occur in the carbohy- 
drate being respired, the possibility of autodigestion of reserve 
glycogen, "mold starch," fats, or other reserves should be con- 
sidered, since many fungi are known to store foods and to utilize 
them during periods of stress. 

LITERATURE CITED 

Bulloch, William, The history of bacteriology. 422 pp. Oxford Univers- 
ity Press. 1938. 
Cagniard-Latour, Charles, "Memoire sur la fermentation vineuse," Ann. 

chim. phys., 68: 206-222, 1838. 
Dammann, E., O. T. Rotini, and F. F. Nord, "Mechanism of enzyme action. 

XVIII. Biochemistry of Fusaria. V. Enzvmic transformations by 

Fiisariimi graminacearum Schwabe (Gibberella saubinettii) . Mode of 

action of hydrocyanic acid and vitamin B^" Biochem. Z., 291: 184-202, 

1938. 
Fabbroni, Adamo, DeW arte di fare il vino. 264 pp. Firenze. 1787. 
Harden, A., Alcoholic fermentation, 4th ed. 194 pp. London. 1932. 
Hawker, Lilian E. "The effect of vitamin Bi on the utilization of glucose 

by Melanospora destruens Shear," Ann. Botany, 8: 79-90, 1944. 
Kostytchew, S., "Der Einfluss des Substrates auf die anaerobe Atmung der 

Schimmelpilze," Ber. dent. Botan. Ges., 20: 327-334, 1902. 
"Zweite Mitteilung iiber anaerobe Atmung ohne Alkoholbildung," Ber. 

deut. botan. Ges., 26: 167-177, 1908. 
Plant respiration, xi + 163 pp. P. Blakiston's Sons and Co., Philadelphia. 

(Translated and edited by J. C. Lyon.) 
Lavoisier, A. L., Traite elhnentaire de Chymie. Paris. 1789. 
Liebig, J. von, "liber die Erscheinungen der Garung, Faiilniss, und Ver- 

wesung und ihre Ursachen," Ann. Physik. Chemie, 2R, 18: 106-150, 1839. 
Lutman, B. F., Microbiology, x -f- 495 pp. McGraw-Hill Book Co., New 

York. 1929. 
Meyerhof, O., and W. Kiessling, "Die Umersterungsreaktion der Phos- 

phobrentztraubensaure bei der alkoholischen Zuckergarung," Biochem. 

Zeitschr., 281: 249-270, 1935. 
Neuberg, C, "Von der Chemie der Garungs-Erscheinungen," Ber. dent. 

chem. Ges., 55: 3624-3638, 1922. 
Neuberg, C, and A. Gottschalk, "Beobachtungen iiber den Verlauf der 

anaeroben Pflanzenatmung," Biochem. Z., 151: 167-168, 1924. 
Nord, F. F., and R. Weidenhagen, Ergebnisse der Enzymforschnng, I- VIII. 

1932-1939. 
Palladin, W., "tjber das Wesen der Pflanzenatmung," Bioche?n. Z., 18: 151— 

206, 1909. 
Pasteur, Louis, "Memoire sur la fermentation appelee lactique," Comp. 

rend., 45:913-916, 1857. 



68 RESPIRATION 

Pasteur, Louis, "Memoire sur la fermentation appelee lactique," Ann. chini. 

phys., 3 nit - ser., 52:404-418, 1858. 
''.Memoire sur la fermentation alcoholique," Ann. chim. phys., 3 me ser., 

58: 323-126, 1860. 
Pratt, E. F., and R. J. Williams, "The effect of pantothenic acid on respira- 
tion activity," /. Gen. Physiol., 22:611-6*1, 1939. 
Richter, Andreas, "Kritische Bemerkungen zur Theorie der Garung," 

Zentr. Bakt., Parasitenk., II Abt., 8: 787-796, 1902. 
Schwann, Theodor, "Vorlaufige Mitteilung betreffend Versuche iiber die 

Wcingahrung und Faulniss," Ann. Physik. Chenrie, 41: 184, 1837. 
Ta.miva, H., "Atmung, Garung, und die sich daran beteiligenden Enzvme 

von Aspergillus," Advances in Enzynwl., 2: 183-238, 1942. 
Thenard, Louis Jacques, "Sur la fermentation vineuse," Ann. chim., An. XI, 

46: 1802-1803. 
Wolf, Fred T., and C. S. Shoup, "The effects of certain sugars and amino 

acids upon the respiration of Allomyces," My col, 35: 192-200, 1943. 



Chapter 4 
BIOCHEMISTRY OF FUNGI 

Essentially all that is known regarding the biochemistry of fungi 
has come from investigations made since the turn of the present 
century, and the larger proportion of this knowledge has been 
acquired during the past few years. Interests in these matters have 
been divided, both the students with purely academic viewpoints 
and those concerned with industrial applications having been at- 
tracted. There has resulted from these studies of the biochemistry 
of fungi, including the yeasts and bacteria, a voluminous litera- 
ture. In one volume, much less in one chapter, it is impossible for 
one person to convey adequately the scope of these studies, to 
indicate the evidences in them of scientific acuity and perspicacity, 
or to venture prophecies on their implications and applications. 

Before the nineteenth century little about the biochemistry of 
fungi was common knowledge among mycologists, except per- 
haps that yeasts produce alcohol and carbon dioxide. Our pres- 
ent-day concepts of this subject admittedly had their beginning 
in Pasteur's epoch-making researches on fermentations as accom- 
plished through the agency of yeasts. To be sure, yeasts were 
used by man in the making of bread and the preparation of alco- 
holic drinks long before anything fundamental about them or 
about their biochemical activities was known. For the develop- 
ment of industrial uses of fungi, the initial impetus doubtless came 
from Hansen's classical work with yeasts and from the studies of 
Wehmer, performed about the same time, on the production of 
oxalic and citric acid by Penicillium. Such a mass of data on mold 
biochemistry is now available that only the intrepid would ap- 
praise it or venture to view it in perspective and to speculate on 
the many problems that have been brought into focus and that 
await solution. For an introduction to this subject the excellent 
summaries of Raistrick (1931), Raistrick et al. (1931), Raistrick 
(1938), Iwanoff (1932), IwanorT and ZwetkorT (1933, 1936), Bir- 

69 



10 BIOCHEMISTRY OF FUNGI 

kinshaw (1937), Lockwood and Aioyer (1938), and Tatum (1944) 
will be found very serviceable. 

In these biochemical researches it is of more than passing in- 
terest to note that members of the cosmopolitan genera Aspergillus 
and Penicillium have been very commonly employed. In fact, 
Aspergillus niger is the biological agent in so many tests that it is 
easy to understand why this species may appropriately be desig- 
nated the "fungus guinea pig." This epithet may be applied 
equally appropriately to Venicillium glaucum. The reason for the 
use of these species and of closely related ones lies in their ability 
to produce a wide variety of enzymes, making it possible for them 
to utilize many kinds of substrata as foods, as is indicated in 
Chapter 1. In the discussion that follows, emphasis will be placed 
on the metabolic products formed by fungi, only incidental at- 
tention being given to the influence of nutritional factors. (The 
nutrition of fungi is considered separately in Chapter 1.) Inade- 
quate emphasis must of necessity be placed upon the mechanisms 
by which these metabolic products come into being, mainly be- 
cause they are in many instances quite unknown or at least not yet 
fully understood. 

ORGANIC ACIDS AND OTHER PRODUCTS HAVING 
SIX OR FEWER CARBON ATOMS 

The foundations for our understanding of the genesis of organic 
acids by fungi were established between 1896 and 1897 by the 
classical studies of Wehmer. These studies involved the common 
carboxvlic acids, but later observers have devoted themselves to 
the production of acids belonging to other groups as well. Cer- 
tain essentials regarding the mechanisms in these fermentations 
have been elucidated by such workers as Bernhauer, Chrzaszcz, 
Butkewitsch, Neuberg, Cohen, Raistrick, and Birkinshaw and 
their associates and pupils. 

Oxalic acid. Wehmer (1891 ) observed that crystals of calcium 
oxalate are present in the mycelium of Aspergillus niger and in the 
culture medium in which this organism grows. He was first to 
recognize that this acid is a by-product in the fermentation of a 
variety of substrates and that with the addition of calcium carbo- 
nate to the substrate very large yields may be obtained. Subse- 
quently others have confirmed these findings with A. niger and 



ORGANIC ACIDS AND OTHER PRODUCTS 



11 



have shown that this acid is produced by fermentations induced by 
A. ochr ace ous and A. violaceiis-fuscus. Currie and Thorn (1915) 
described a species, which they named Penicillium oxalicum, that 
has the same ability. Indeed many mycologists have noted that a 
wide variety of fungi, grown in nutrient agars, induce the produc- 
tion of oxalic acid, evident as octahedral crystals of calcium 
oxalate. 

Butkewitsch and FedororT (1930) observed that Mucor stoloni- 
fer can convert acetates into oxalic acid, and they postulated that 
this conversion is possible by either of these two courses: 



a. CH 3 
COOH 

Acetic acid 

b. COOH 
CH 3 
CH 3 
COOH 

Acetic acid 



• 



o 



Q 

+ o CH 2 OH _ H2 I +0 

> - — > > 



-H 2 



COOH 

Glycolic acid 

COOH 



COOH 

Glyoxalic acid 

COOH 



CH 2 
CH 2 
COOH 

Succinic acid 



-H 2 



COOH 



CH 
CH 
COOH 

Fumaric acid 



COOH 



+H 2 
> 



COOH 
COOH 

Oxalic acid 

COOH 
CHOH 
CH 2 
COOH 

Malic acid 



-H 2 



C=0 +H2 COOH 

> 



CH, 



CH, 



COOH 

Keto- 
succinic acid 



COOH 

Oxal- 
acetic acid 



As another essential condition for oxalic acid production 
Chrzaszcz and Tiukow (1930, 1930a) found that the process varies 
with the amount and kinds of amino acids present. 

Citric acid. The production of citric acid from the fermenta- 
tion of hexose sugars was demonstrated by Wehmer in 1893. He 
identified the molds concerned as members of a new genus, Citro- 
myces, and named them C. glaber and C. pfefferiammi. He found, 
as with oxalic acid production, that improved yields can be ob- 
tained when calcium carbonate is present in the medium. Later 



12 BIOCHEMISTRY OF FUNGI 

he established that Penicillium luteum has the same fermentative 
ability. Subsequently P. expansum, P. divaricatiim, P. citrimmi, 
and P. spimilosum and several species of Aspergillus, including 
A. niger, A. clavatns, and A. parasiticus, were employed under 
similar conditions to produce citric acid. Wehmer separated 
Citromvces from Penicillium because of this ability to produce 
citric acid, but it soon became apparent that this physiological 
characteristic constituted an untenable generic basis. The species 
of Citromvces, totalling about twenty, have therefore come to be 
included in the Genus Penicillium. 

A number of studies have been concerned with the conditions 
required for the production of citric acid. Molliard (1922) re- 
ported that an insufficient quantity of nitrogenous material in the 
substratum supplied to Aspergillus niger was correlated with the 
accumulation of citric acid; this finding was not substantiated, 
however, in the experiments of Bernhauer (1926). 

Butkewitsch (1923) reported that both Penicillium glaucmn 
and A. niger must be grown in an acid medium to stimulate the 
formation of citric acid. If the medium was neutral, oxalic and 
nitric acid were produced; if it was alkaline, oxalic acid alone was 
formed. 

In his studies on citric acid production by A. niger Porges 
(1932) used an inorganic mineral nutrient to which sucrose was 
added as a source of carbon. He found that it was necessary first 
of all to secure a heavy mycelial mat over the surface of the 
nutrient solution. As a source of nitrogen NaN0 3 proved far 
superior to (NH 4 ) 2 S0 4 . Both Fe and Zn were essential. Sugar 
concentrations of 15 to 20% gave best yields. As a final condition, 
it was requisite that the mat be undisturbed in order to provide a 
partially anaerobic environment. 

In 1917 Currie (1917) observed that in sugar solutions fer- 
mented by the A. niger group the lag in acidity can be accounted 
for by citric acid, which made up the difference between total 
acidity and oxalic acid. 

Kostytchew and Tschesnokow (1927) noted, that so long as no 
nitrogen is being absorbed, citric acid is not accumulated. At the 
end of approximately 48 hours the mycelial mat of A. niger will 
have covered over the nutrient solution. This solution must then 
be replaced by a sugar solution that lacks mineral elements. After 
3 days' growth on such a medium A. niger will have produced a 



ORGANIC ACIDS AND OTHER PRODUCTS 



13 



maximum of citric acid and will have utilized 40 to 50% of the 
sugar present. This mineral-nutritional relationship is substanti- 
ated by Butkewitsch and Timofeeva's results (1935) with cultures 
deprived of phosphorus, sulphur, and nitrogen. 

Several mechanisms have been suggested to account for the 
formation of citric acid. Butkewitsch and Fedoroff (1929, 1930) 
and Chrzaszcz and Tiukow (1930, 1930a) maintain that it forms 
through acetic acid or from acetates of sodium or potassium. For 
the formation from acetic acid their scheme is: 



COOH 



COOH 



COOH 



COOH 



CH 
CH, 



3 _h 2 



CH 2 
CH, 



-H 2 



CH 
CH 



+H 2 



CH 2 
CHOH 



COOH 

Acetic acid 



COOH 

Succinic acid 

CH 3 
+ COOH —?* 

Acetic acid 



COOH 

Fumaric acid 

CH 2 • COOH 
COH • COOH 
CH 2 • COOH 

Citric acid 



COOH 

Malic acid 



Bernhauer and Siebenauger (1931) have shown that A. niger 
can convert ethyl alcohol into citric acid. Bernhauer and Bockl 
(1932) obtained yields of citric acid from alcohol up to 25% of 
the theoretical amount. They also showed another possible course 
of formation, in which aconitic acid appears: acetic acid — > suc- 
cinic acid — > fumaric acid — > aconitic acid -* citric acid. Their 
proof rests upon experiments in which they grew A. niger on 
2.4% potassium aconitate and obtained 23.2 to 25.8% citric acid. 

Even more convincing is Kinoshita's [IwanofT and ZwetkofT 
(1933)] evidence from the growth of A. itaconicns on a sugar 
solution containing calcium carbonate. Kinoshita got citric acid, 
which disappeared, and then itaconic acid thus: 

CH 2 (COOH)COH(COOH)CH 2 (COOH) ^> 

Citric acid 

CH 2 (COOH) • C(COOH) : CH(COOH) 

Aconitic acid 



-C0 2 



> CH 2 (COOH)C(:CH 2 )COOH 

Itaconic acid 



14 BIOCHEMISTRY OF FUNGI 

Raistrick and Clark (1919) maintain that the hexose first be- 
comes a-y-diketoadipic acid, that then acetic and oxalacetic acids 
arise bv hydrolysis, and that finally they combine to form citric 
acid. 

Optimum conditions for citric acid formation vary not only 
with the substrate but also with the mold concerned and with the 
pH. This variation is indicated by an optimum pH of 2.0 for 
A. niger and of 3.0 to 4.0 for Penicillium glaber. Of the sub- 
strates tested, the following carbohydrates have been found suit- 
able for citric acid fermentation: starch, sucrose, glucose, fructose, 
lactose, maltose, glycerol, and molasses. 

Whatever the mechanism, it has been found to be commercially 
practicable to produce citric acid by mold fermentation, several 
thousand tons being produced annually, in competition with citric 
acid extracted from natural sources. It requires an initial concen- 
tration of about 1 5 c o of sugar, a low concentration of ammonium 
nitrate, and a pH of 3.5. 

J-Gluconic acid. Gluconic acid is of value when used as a cal- 
cium salt in food and medicine. It was first isolated by Molliard 
in 1922 from among the by-products in fermentations induced 
by A. niger. Subsequent workers, notably Bernhauer (1924), 
Herrick and May (1928), May, Herrick, Thorn, and Church 
(1927), Mover, May, and Herrick (1936), and May, Herrick, 
Mover, and Hellbach (1929) have shown that a variety of other 
molds possess the ability to ferment this acid, among them being 
Aspergillus cirmamomeus, Penicillium glaucum, P. purpurogemim 
var. rubrisclerotvum, P. chrysogemim, and Finnago vagans. 

Gluconic acid arises from the fermentation of glucose as fol- 
lows: 

CH 2 OH CH 2 OH 

HCOH HCOH 

HCOH HCOH 

I ^ I 

OHCH OHCH 

HCOH HCOH 

CHO COOH 

(f-Glucose <f-Gluconic 

acid 



ORGANIC ACIDS AND OTHER PRODUCTS 



15 



Studies have also been directed toward finding optimum condi- 
tions for the formation of gluconic acid. Herrick and May (1928) 
secured good yields in 10-day-old cultures, incubated at 25° to 
30° C, in the following medium: 



Glucose 

Magnesium sulphate 
Disodium phosphate 



200.00 grams 
0.25 gram 
0. 10 gram 



Potassium chloride 
Sodium nitrate 
Water 



0.05 gram 
1 .00 gram 
1000.00 ml 



Kardo-Ssysojewa (1933) recorded increased yields from de- 
creasing total salts but increased yields if the nitrates were in- 
creased in acid media. He grew mats of A. niger in nutrient-sugar 
solution in the presence of calcium carbonate, and after pouring 
off this solution replaced it with one of 20% sugar to which he 
added calcium carbonate. After 4 days he secured 11.89 grams 
of gluconic acid from 11.36 grams of sugar, and after 5 days 12.19 
grams of gluconic acid from 11.76 grams of sugar. Traces of 
citric acid were present also, but no oxalic acid. 

Fumaric acid. In 1911 Ehrlich demonstrated that Rhizopus 
nigricans produces small amounts of fumaric acid from glucose 
and fructose. These observations have subsequently been con- 
firmed, and in 1918 Wehmer (1918) reported yields of 60 to 70% 
from a species of Aspergillus that he named A. fiimaricus. This 
organism gradually declined in fumaric acid-producing ability 
after repeated subculture and formed instead gluconic, malic, and 
citric acids. Galactose and arabinose have also been utilized in 
fumaric acid fermentation. Other fungi that have been found 
capable of producing this acid include Rhizopus oryzae, R. tritici, 
and Yenicillium griseo-jidvum [Raistrick and Simonart (1933)]. 

Gottschalk (1926) and Butkewitsch and FedorofT (1929) se- 
cured calculated yields of fumaric acid of approximately 50% 
from R. nigricans. They found that this acid may arise in an 
alcoholic fermentation as follows: 

CHoOH N 
2C0 2 + 2 



CrHioO 



+02 



12^6 



CH 3 

Alcohol 



/COOH 

21 



-H 2 



CH 3 

Acetic acid 



CH 2 • COOH 
CH 2 ■ COOH 

Succinic acid 



H 2 



CH • COOH 
CH • COOH 

Fumaric acid 



16 BIOCHEMISTRY OF FUNGI 

Malic acid. Although malic acid has for years been considered 
to be among the products formed by molds, proof was first pro- 
vided by Wehmer (1928) in 1928. He secured small yields from 
sucrose fermentation by Aspergillus fumaricus. Raistrick et al. 
(1931) have found that several other fungi, among them species 
of Aspergillus and Clasterosporium, can utilize glucose to form 
malic acid. 

Succinic acid. It has long been known that yeasts produce 
succinic acid during alcoholic fermentation. Moreover Fitz 
(1873) recorded its presence in solutions during alcoholic fermen- 
tation induced by Mu cor mucedo. Raistrick et al. (1931) showed 
that a species of Clasterosporium isolated from cotton pulp, as 
well as Fumago vagans, formed succinic acid from glucose. It 
has also been shown to be produced by Aspergillus t err em 
[Raistrick and Smith (1935)] and Penicillium aurantio-virens 
[Birkinshaw and Raistrick (1932)]. This ability is doubtless pos- 
sessed by a variety of fungi. 

Succinic acid, as has been shown, forms during fermentations 
that give rise to such other acids as citric, gluconic, and fumaric, 
with which it is chemically related; in consequence its origin may 
be accounted for by the oxidative breakdown of sugars. On the 
other hand, it may well arise from yeast proteins themselves or 
from mold proteins. Accord seems not to have been reached on 
the matter of the origin of succinic acid. 

Lactic acid. Until recently it was the general belief that only 
bacteria, especially such species as Streptococcus lactis, Lactoba- 
cillus acidophilus, and L. bulgaricus, are capable of causing lactic 
acid to be formed. More recently however, several workers have 
demonstrated this acid in sugar fermentations by species of Rhizo- 
pus and Alucor, including Rbizopus oryzae, R. chinensis, R. ele- 
gans, and R. tritici. A 40% yield from R. japonicus was reported 
when this species was grown on 10% sugar solution containing 
calcium carbonate. Waksman and Foster (1938) found a member 
of the R. arrhizus group to be a very efficient lactic acid former 
when grown in solutions containing glucose or starch. In the 
presence of calcium carbonate 70 to 75% of the carbohydrate 
was transformed into lactic acid. This yield is all the more re- 
markable in that the fungus is supposedly strictly aerobic. Waks- 
man and Foster, however, permitted it to form a film over the sur- 
face of the liquid substrate, and within such a film under reduced 



ORGANIC ACIDS AND OTHER PRODUCTS 77 

oxygen tension an intermediate substance is formed which be- 
comes converted into lactic acid, alcohol, and carbon dioxide, 
with little loss of potential energy. 

Ethyl alcohol. Sac char omyces cerevisiae has long been known 
for its ability to metabolize ethyl alcohol. As long ago as 1873, 
however, Fitz (1873) noted that Mucor racemosus is also capable 
of transforming sucrose into alcohol. Of the closely related genus 
Rhizopus, ethvl alcohol is known to be formed bv R. nigricans, 
R. tritici, R. arrhizus, and R. oryzae. 

Fusarium lini, the cause of flax wilt, gives yields of the same 
order of magnitude as those from cultivated yeasts [Letcher and 
Willaman (1926), White and Willaman (1928)]. It will ferment 
almost any hexose and in addition almost any pentose, the pentoses 
not being utilized in this manner bv baker's yeast. Many other 
species of Fusarium, moreover, are able to decompose glucose 
with the production of alcohol, each differing in relative yields; 
as might be anticipated, a variety of other products appear dur- 
ing the fermentation [Raistrick et al. (1931)]. 

Species of Aspergillus and Penicillium have been tested for their 
ability to produce alcohol. As a result it is known that 96 species 
or strains of Aspergillus and 75 of Penicillium possess this capa- 
bility. Among them are A. niger and several members of the 
A. glaiicus group and the A. flawis-oryzae-tamarii group. Yuill 
(1928) is among those who have studied alcoholic fermentation 
by A. flavits. Other notable alcohol-forming molds are Eidamia 
catemriata, E. viridescens, Trichoderma lignorum, and Helmintho- 
sporhnn gemadatiim. 

Ethyl acetate. This fruity ester was demonstrated by Rai- 
strick et al. (1931) to be formed by Fenicillium digitatum, a com- 
mon mold associated with the decay of citrus fruits, when grown 
on glucose solution. They state that it is not known to be formed 
by any other mold. Presumably ethyl acetate originates by a 
Cannizzaro reaction from acetaldehyde, an anaerobically formed 
respiratory product. 

Glycerol. Connstein and Liidecke (1919) considered the 
principles involved in the commercial production of glycerol by 
fermentation. It is a matter of common knowledge that this 
compound is formed during fermentation by yeasts, and as has 
been shown in Chapter 3, high yields can be secured by the addi- 



18 



BIOCHEMISTRY OF FUNGI 



tion of sodium sulphite to the nutrient solution. More recently 
the carbonate has been substituted for the sulphite with good 
results. Emmerling (1897) reported that Mucor vmcedo can 
metabolize glycerol from sucrose. Raistrick et al. (1931) showed 
that other molds, for example, Aspergillus wentii, a Clastero- 
sporium isolated from cotton pulp, and Helm'inthosporhim geni- 
culatwn, possess like capability. 

Kojic acid. This acid is a y-pyrone of the following constitu- 
tion: 

CO 

/ \ 
HOC , CH 



HC 



C-CHoOH 



O 

It is of special interest to the toxicologist because, when orally 
administered to dogs, it produces symptoms like those of epilepsy. 
Kojic acid was first isolated by Saito in 1907 from the mycelium 
of Aspergillus oryzae. This fungus was subsequently found cap- 
able of utilizing in the production of kojic acid not only sucrose 
but also maltose, dulcitol, succinic acid, and inulin. Raistrick et al. 
(1931) and Birkinshaw (1937) showed that this capability is 
possessed also by Aspergillus flaws, A. efjusus, A. parasiticus, 
A. tamarii, and Fenicilliwn daleae. The conditions for its produc- 
tion were studied by May, Moyer, Wells, and Herrick (1931), 
who secured yields of 45% of the glucose present. They varied 
the amount of nitrogen and sugar in the medium, getting best 
yields with approximately 20% sugar. The mode of its forma- 
tion is not established, but it may be as follows: 



HCOH- 
HCOH 



HOCH 
HCOH 

HC 

CH 9 0H 



O 



CHOH- 
HCOH 

c=o 

HCOH 



CH 



O 



HC 



CHoOH 



COH 

c=o 

CH 

C 

CH 2 OH 

Kojic acid 



o 



FATS 19 

Mannitol. Several workers have reported the occurrence of 
the hexahydric alcohol, mannitol, within the tissues of molds, and 
it has been regarded as a reserve product. Raistrick et al. ( 193 1 ), 
however, established that mannitol can be formed in Czapek-Dox 
glucose solution, where it appears as a product of fermentation. 
Aspergillus elegans, A. nidulans, A. wentii, Penicillium chrysoge- 
num, and Helminthosporium geniculatum were the organisms con- 
cerned in their experiments. Coyne and Raistrick (1931) found 
that Aspergillus can ferment glucose, mannose, and galactose with 
the production of mannitol, and that the pentoses, xylose and 
arabinose, can likewise be fermented in the same manner. For 
some reason not understood, Aspergillus did not form mannitol 
from fructose. 

POLYSACCHARIDES 

Many fungus structures have long been known to become blue 
when stained with iodine, a reaction used to establish the presence 
of "mold starch." Presumably the term mold starch applies to a 
group of closely related substances rather than to a single one. 
Boas (1917, 1922) found that Aspergillus niger can utilize various 
sugars, glycerol, mannitol, and several organic acids, such as citric, 
malic, oxalic, and tartaric, in producing mold starch, provided that 
high temperatures are maintained and free acids are present in 
the culture solution. 

Chrzaszcz and Tiukow (1929, 1929a) observed that many spe- 
cies of Penicillium produce mold starch. 

Dox and Neidig (1914) grew Penicillium expansion on Raulin's 
solution containing <i-glucose. From the mycelium they isolated 
a polysaccharide which they named mycodextran. From Asper- 
gillus niger grown on the same medium they isolated both myco- 
dextran and another polysaccharide that they called mycoga- 
lactan. 

Several other polysaccharides have been isolated [Raistrick 
(1938)], including luteic acid elaborated by Penicillium luteum, 
mannocarolose and galactocarolose by P. charlesii, and varianose 
by P. varians. 

FATS 

It is well known that many species of fungi store globules of 
fats within their spores and that fats may be present also within 



80 BIOCHEMISTRY OF FUNGI 

the mvcelia. According to Pearson and Raper (1927), the fresh 
mycelium of Aspergillus iiiger contains 2.4% fat and that of 
Rhizopus nigricans 5%. These fats in Penicillium aurantio-brun- 
neinn were studied by Strong and Peterson (1934) and were 
found to resemble butterfat. The analyses showed them to con- 
tain 40.2% oleic acid, 31.2% lineolic acid, 8.6% palmitic acid, and 
5.3% stearic acid; the remainder consisted of 9.1% glycerol, 1.9% 
ergosterol, and a non-fatty residue of 4.5%. Their analysis of 
the fats of Aspergillus sydoixii showed 8.8% palmitic acid, 11% 
stearic acid, 29.6% oleic acid, 16.3% lineolic acid, and a small per- 
centage of higher unsaturated acids. Analysis of the fats of 
Penicillium javanicum by Ward and Jamieson (1934) revealed 
them to consist of 69.5% palmitic acid, 28% stearic acid, and 2.5% 
7;-tetracosic acid. Nord and A lull (1945) indicated that Fusarium 
gramineum forms fats similar to those produced by yeasts. 

The factors that influence the amount of fat produced have 
been investigated by Lockwood et al. (1934) and Ward et al. 
(1934, 1935) in a goodly number of species of molds. They se- 
cured best production with Penicillium javanicum when it was 
grown on a medium containing 40° ' glucose. Sucrose, xylose, 
and glycerol also served well as carbon sources. These workers 
found that the mycelium contained up to 41.5% fat in old cul- 
tures. The studies of Prill, Wenck, and Peterson (1935), using 
Aspergillus fischeri, showed increased fat production with higher 
pH within the range 2.0 to 8.0, along with greater concentration 
of glucose. 

Even though the syntheses are not understood, the procedures 
are now so well known that they could be utilized industrially 
if a supply of animal fats could not be procured. In fact, one of 
the Endomycetales, Endomyces vernalis, has been used for some 
time in the commercial production of fats. 

Various hypotheses, briefly considered by Smedley-AIcLean 
(1936), have been proposed to explain the mechanisms involved 
in the transformation of a carbohydrate into a fatty acid. Con- 
densation of three hexose molecules to give the stem of stearic 
acid or of two pentose molecules and one hexose molecule to give 
palmitic acid has been suggested. In support of this hypothesis 
attention may be directed to the fact that in many naturally 
occurring fatty acids the number of carbon atoms is a multiple 
of six. 



STEROLS AND VITAMINS SI 

Another hypothesis is that acetaldehyde is produced from lactic 
acid as an intermediate substance and that by its repeated conden- 
sation the series of fatty acids from butyric upward is produced. 
The culturing of fungi on solutions of acetaldehydes has not 
strongly supported this hypothesis. 

Another hypothesis, which is looked upon with favor but has 
not been elucidated, is that the hexose is fermented to pyruvic 
acid, from which the fatty acid is formed. 

STEROLS AND VITAMINS 

Within the past few years an appreciation has been developing 
that sterols and vitamins occur in fungi. This recognition has 
come in part from the therapeutic use of yeast to correct dietary 
deficiencies. The dietetic value of yeast becomes all the more 
remarkable when it is remembered that the yeast plant, cultured 
on hexose solution containing ammonium chloride together with 
a few drops of wort in which yeasts have previously been grown, 
is able to synthesize not only a goodly complement of vitamins 
but also all the amino acids. All these syntheses by a simple 
plant! To date the function of sterols (ergosterol, CosH^O.oo, 
is the precursor of vitamin D) and vitamins in fungi remains 
largely unknown. The studies have centered largely on the oc- 
currence of these substances and on their employment in animal 
feeding. Evidently they are of wide occurrence among fungi. 
The factors which condition their formation were studied by 
Birkinshaw, Callow, and Fischmann (1931). In 1929 Heiduschka 
and Lindner [Birkinshaw (1937)] determined the ergosterol con- 
tent of Dematium pullirtans to be 0.3% of the dry weight; of 
Penicillium glaiicum, 0.75%; and of Aspergillus oryzae, 0.46%. 
Bernhauer and Potzelt (1935) found a variation in sterol content 
of 0.23 to 1.16% among 16 strains of A. niger. 

Preuss et al. (1931, 1932, 1932a) studied the sterol content of 30 
species of Aspergillus, 20 of Penicillium, and 15 of other species 
of fungi when grown on a synthetic medium containing 4° 7 glu- 
cose. The difference in sterol content among species is shown by 
the occurrence of 0.98% in Aspergillus oryzae, 0.4% in A. niger, 
0.35% in Penicillium expansum, and 0.16% in P. janthivellum. 
They found that different strains of the same species vary in 



82 BIOCHEMISTRY OF FUNGI 

ability to produce sterol since, other conditions being uniform, 
4 strains of A. oryzae yielded 0.54, 0.63, 0.76, and 0.98%. The 
duration of the period of cultivation was also found by these 
workers to influence the yield, since a given strain of A. oryzae 
after 10 days had produced 0.63%, and after about 50 days 1.07 
Reindel, Niederlander, and Pfundt (1937) found that best yields 
from Torula were produced in a molasses medium, increased 
yields being correlated with increased sugar concentration. Preuss 
et al. (1931) also administered in daily doses 10 mg of dried, finely 
ground fungus material to rachitic rats. They used A. niger, 
A. oryzae, Marasmius oreades, Hypholoma incertum, and Seco- 
tium acuminatum, with the result that each manifested antirachi- 
tic action. Among other of the higher fungi that have been found 

O CO 

to contain vitamin D are Psalliota campestris, Hehella esculenta, 
Boletus edidis, Cantharellus cibarius, C. clavatus, Hydnum ivibri- 
catum, and Ganoderma hicidimi [IwanofT and ZwetkorT (1936)]. 

Evidence has also been accumulated to show that other vita- 
mins are present in fungi and that some of them can be employed 
to enrich animal diets. Gorcica, Peterson, and Steenbock ( 1934) 
found vitamins Bi (thiamin), B 2 (riboflavin), and B 4 in Asper- 
gillus sydoivii. Scheunert and Reschke [IwanofT and ZwetkofF 
(1933)] found that Cantharellus cibarius is unusually rich in vita- 
min A (C20H30O). Lederer [IwanofT and ZwetkorT (1936)] 
studied the carotene (provitamin A) content of many yeasts and 
fungi. Its wide distribution among fungi is indicated by his 
finding it in the slime mold, Lycogala epidendrum, in the rust, 
Puccinia coronifera, in the jelly fungus, Tremella mesenterica, 
and in the near yeast, Torula rubra. 

Much interest also centers on the occurrence of growth sub- 
stances, notably heteroauxin, in yeasts and certain fungi. An in- 
troduction to this subject can be obtained from Kogl and Koster- 
mans' report (1934) of the existence of heteroauxin in Rhizopus 
nigricans and Aspergillus niger. It appears to be formed in the 
breakdown of tryptophane, since none is produced in mineral-nu- 
trient solutions. 

According to Xord and Mull (1945), a diet containing 10% 
Fusariuui lini as a source of vitamins, with crystalline vitamin Bi 
added, and 37% proteins from the same fungus serves excellently 
for growth, reproduction, and lactation by mice. 



PIGMENTS OF FUNGI 83 

AMINO ACIDS 

Apparently many fungi are able to synthesize their amino acids 
from inorganic nitrogen. Such synthesis may not be sufficiently 
rapid, however, for optimum growth, as is indicated bv the fact 
that more rapid growth occurs after amino acids are added to the 
substrate. 

Steinberg (1942) studied the utilization of amino acids as carbon 
and nitrogen sources for Aspergillus niger and interpreted his ex- 
periments as showing that amino acids may be formed from and 
reconverted to sugars. A mixture of proline, glutamic acid, and 
ornithine provided carbon and nitrogen almost as satisfactorily 
as did sucrose and ammonium salts. 

Biogenesis of specific amino acids, as of arginine and trytophane, 
especially by Neurospora crassa, has been given consideration 
[Tatum (1944)]. From such investigations the accumulated evi- 
dence indicates that the formation of primary amino acids in- 
volves oxidation of the a-hydroxy acid and amination of the keto 
acid. 

PIGMENTS OF FUNGI 

To almost any question regarding the pigments of fun^i the 
mycologist makes the embarrassed answer, "I don't know." iMany 
species are beautifully pigmented, and use is made of this fact 
in classification. Almost surely pigments serve some essential 
function in the metabolic activities of fungi, presumably in respi- 
ration, but this field of physiology remains quite wholly unex- 
plored. To date the studies on such pigments deal mainly with 
their chemical nature. 

Citromycetin and citrinin. These two pigments were iso- 
lated by Raistrick and his associates (1931), citromycetin being 
obtained from Pemcillium glabrum and citrinin from P. citrinum. 
The organisms were grown on modified Czapek-Dox medium 
(NaNO s , 2 grams; KH,P0 4 , 1 gram; KC1, 0.5 gram; MgSCV 
7H 2 0, 0.5 gram; FeS0 4 -7H 2 0, 0.02 gram; water, 1 liter; glucose, 
50 grams). From P. glabrum Hetherington and Raistrick [Rai- 
strick (1931)] extracted in 50% alcohol a substance that crystal- 
lized into lemon-yellow needle crystals, citromycetin, which is 
intensely olive green with ferric chloride. The reactions of this 



84 



BIOCHEMISTRY OF FUNGI 



dye indicate that it is related to the xanthone and flavone group. 
Its empirical formula is Ci-iHioOy^HjO, with the following 
structural constitution: 




C 4 H e O 



The other pigment, citrinin, also crystallizes as yellow crystals 
but is iodine brown in ferric chloride and discolors instantly in 
potassium permanganate. Its empirical formula is given as 
C13H14O5, with the following structural constitution: 

C 2 H 5 
0/S0H 



H 3 C 

H 3 C 

H 



COOH 
O 



Carotene. Certain Phy corny cetes, such as Phy corny ces blake- 
sleeamis, Mucor hievmlis, and Allomyces javanicus, contain /^-caro- 
tene, the precursor of vitamin A. The gametes of Allomyces 
macro gyna and A. moniliformis contain y-carotene [Emerson and 
Fox ( 1940) ] . Carotene occurs also in Pilobolus and is not uncom- 
mon among Ascomycetes and Basidiomycetes, especially rusts 
and jelly fungi. 

Other pigments. Clutterbuck et al. (1932) isolated from Penl- 
cillhim clnysogemim a yellow pigment whose empirical formula 
is CisH-.O,;. From Monascus purpureus two pigments, monasco- 
rubrin, C22H24O5, and monascoflavin, Ci 7 H_. L .0 4 , have been ob- 
tained. Monascorubrin is red and may be converted by hydro- 
gen peroxide into monascoflavin, which is yellow [Birkinshaw 
(1937)]. Two pigments, oosporin, CioH 14 0,;, and aurantin, 
C1r.H-.O3, have been obtained from Oospora aurantla. 

Gould and Raistrick (1934) isolated from members of the 
Aspergillus glaucus group three pigments; flavoglaucin, C19H28O3, 
auroulaucin, C^H^O.,, and rubro^laucin, Cir.Hi-O.-. 



PIGMENTS OF FUNGI 



85 



Kogl and Erxleben [IwanorT (1932)] have extracted pigments 
from a number of the higher fungi. From Amanita muse aria 
they extracted a red crystalline glucoside, muscarufin, C 2 5Hi 6 9 . 
From Hydmnn ferrugineum and species of Thelephora they got 
thelephoric acid, G>oHi 2 6 , whose crystals resemble in color po- 
tassium permanganate. 

A group of interesting pigmented compounds is produced by 
each of the more commonly known species of Helminthosporium 
pathogenic to grasses, some compounds being obtained from more 
than one species. Raistrick (1937) and his associates cultured 
these species of Helminthosporium on Czapek-Dox solutions. 
From such cultures of H. gramineum, H. cynodontis, H. catenar- 
ium, and H. tritici-vulgaris they isolated helminthosporin, Ci 5 - 
H10O5, consisting of very dark maroon crystals. From cultures 
of H. cynodontis, H. euchlaenae, and H. avenae, cynodontin, 
CisHioO,;, consisting of bronze leaf-like crystals, was obtained. 
From cultures of H. tritici-vulgaris, tritisporin, Ci 5 H 10 O 7 , consist- 
ing of reddish brown platelets, was obtained. Cultures of H. 
ravenelii, a fungus widely present in the southeastern United 
States on smut grass, Sporobolus sp., yielded ravenelin, Ci 4 Hi O 5 , 
an intensely yellow pigment. The following constitutions are 
assigned to these four pigments from Helminthosporium [Birkin- 
shaw (1937)]: 



OH CO 



OH CO OH 



— CH 



OH CO OH 

Helminthosporin 

OH CO OH 



HOCH 




OH 



OH 

Tritisporin 



-CH 3 



CO OH 

Cynodontin 

O OH 

V-CH 3 



CO OH 

Ravenelin 



Wood lying in moist situations may be discolored by Chloro- 
splemum aeruginosum. The pigment concerned is sylindein, and 
such wood, because of its beautiful verdigris-green stain, is util- 
ized in making ornaments and souvenirs. 



86 



BIOCHEMISTRY OF FUNGI 



Quite a goodlv number of other pigments have been isolated 
and studied, but the functions of most of them have not been given 
any consideration. Some of them catalyze oxidations, as does a 
red pigment, phoenicin, found in Penicillium phoenicttm and also 
in the bacterium, Pseudovwnas aeruginosa. This oxidative func- 
tion may be exercised by pigments that are associated with the 
discoloration of agarics and boletes that have been injured. Stro- 
bilomycol, a red pigment that turns black in the presence of the 
oxidizing enzyme laccase, has been isolated from Boletus (Stro- 
bilomyces) strobilaceus. From B. sat amis and B. luridus [Iwanoff 
and ZwetkorT (1930)1 crystals of boletol have been obtained. 
These crystals become blue on oxidation as they are transformed 



into isoboletol in the following manner: 



O 



O O 



HOOC 



OH 



HOOC 



OH 




OH 



O 

Boletol 



O O 

Isoboletol 



Evidence is being accumulated, furthermore, that many molds 
and yeasts contain glutathione, which can function in respiratory 
processes as an oxidation-reduction system, perhaps in conjunc- 
tion with pigments. Miller and Stone (1938) record the occur- 
rence of glutathione in Monilia sitophila and in species of Peni- 
cillium, Aspergillus, and Rhizopus. 



OTHER METABOLIC PRODUCTS 

Amonsr the products of outstanding interest produced by a 
species of Penicillium, presumably P. notatum, is a bactericidal 
substance. Attention was called by Fleming (1929) to this prop- 
erty of culture solutions in which an unnamed species of Peni- 
cillium had been grown. This solution inhibited the growth of 
various organisms taken from the throat and favored the growth 
and isolation of Hemophilus influenzae. Reid (1935) investigated 
the properties of this germicidal substance, now known as peni- 
cillin. Later Chain and his associates (1940) reported its thera- 



OTHER METABOLIC PRODUCTS 81 

peutic action against Streptococcus, Staphylococcus, and Clostri- 
dium septique in laboratory animals. From the same laboratory 
Abraham et al. (1941) purified penicillin and determined its action 
against body cells and against bacteria, indicating its therapeutic 
potentialities to replace sulfonamides. In fact, it was found to 
operate when sulfonamides are ineffective and to be without toxic 
effect against body tissues. It is bacteriostatic to Staphylococcus 
and Streptococcus in vitro in dilutions of one to a million. Its 
chemical formula, according to Meyer et al. (1942), is Ci 4 Hi 9 NO«. 

In a series of reports additional important findings by Raistrick 
and Smith (1941), Oxford, Raistrick, and Smith (1942), Oxford 
and Raistrick (1942), and Oxford (1942) were announced on the 
production by fungi of substances that inhibit the growth of 
pathogenic bacteria. From Penicillium citrinnm these workers ob- 
tained penicillin, and from P. cyclopium penicillic acid. Both 
substances are bacteriostatic to Staphylococcus aureus, and peni- 
cillic acid is inhibitory also to the typhoid and paratyphoid bac- 
teria. They isolated spinulosin from Penicillium spinulosum and 
fumigatin from Aspergillus fumigatiis. Fumigatin is especially 
potent against Bacillus anthracis, Staphylococcus aureus, and 
Vibrio cholerae. The same workers synthesized both spinulosin 
and fumigatin. 

Waksman and Schatz (1943) found that Aspergillus clavatus 
produces a potent bacteriostatic substance designated clavacin; 
differing amounts are produced by different strains. 

Kochalaty ( 1943) purified an antibacterial substance called pena- 
tin, produced by Penicillium notatum, to the extent that it in- 
hibited growth of 50 species of pathogenic and non-pathogenic 
bacteria in dilutions of one to ten millions or more. 

Waksman and Bugie (1943) concluded that the antibiotic activ- 
ity of Aspergillus flavus is due to two substances: (1) aspergillic 
acid, which is active against both Gram-positive and Gram-nega- 
tive bacteria, and (2) flavacin, which is active against Gram-nega- 
tive bacteria and may be identical with penicillin. In the produc- 
tion of these antibacterial substances three factors are involved: 
(1) differences in strains of A. flavus, (2) the composition of the 
substrate, and (3) the conditions of growth, especially aeration. 

Bergel et al. (1943) isolated clavatin (identical with clavacin) 
from solutions in which Aspergillus clavatus had been grown. 
Their analyses indicated for clavatin the empirical formula 



88 BIOCHEMISTRY OF FUNGI 

CtH ); 04. Furthermore they were able to show that this antibac- 
terial substance is probably identical with claviformin, isolated 
from Penicillium claviforme by Chain and his associates, and also 
with patulin, isolated from P. patulum by Raistrick and his asso- 
ciates. Trichoderma viride has been shown to produce a very 
potent pigment called viridin [Brian et al. (1946)]. Surveys re- 
veal that many fungi produce antibiotics fWilkins and Harris 
(1942, 1943, 1945)]. 

Growing Penicillium charlesii on Czapek-Dox nutrient with 
glucose added, Clutterbuck et al. (1934) isolated a group of re- 
lated substances. These included carolic acid, C9H10O4, carolinic 
acid, C9H10O7, carlic acid, Ci H U )Og, carlosic acid, CioH 12 (; , 
ramigenic acid, CioIT.oO,;, and verticillic acid, Clv.H^Oi-.. Peni- 
cillic acid, C^Hind, has been isolated from P. puberultmi and P. 
cyclophmi. Puberulic acid, C 8 H ( ,0,„ has been obtained from 
P. puberuluvi. Mvcophenolic acid is formed by both P. glaucuvi 
and P. stoloniferuvi. 

The ability of molds, especially P. brevicaule and Aspergillus 
sydowiij to react with arsenicals is of peculiar interest. These 
organisms liberate volatile arsenical products when growing on 
arsenic-containing wallpaper or when inoculated into the stomach 
contents of persons who have succumbed to arsenical poisoning. 
Challenger et al. (1933) found that trimethyarsine, which has a 
very pungent odor reminscent of garlic, is produced in this re- 
action. Among other fungi capable of producing a similar re- 
action are Aspergillus niger, A. virescens, Mucor mucedo and 
M. raceviosus. 

IMPLICATIONS 

Manifestly many problems in mycological chemistry await 
solution. In some instances, at least, it seems unfortunate that the 
details involved in the utilization of molds in industrial processes 
have remained trade secrets. In this period when vitamin defi- 
ciencies are so widely encountered, more should be known re- 
garding the possibilities of utilizing fungi as sources of vitamins. 
The extent to which vitamins are essential in the metabolism of 
funtn themselves is also deserving of further elucidation. 

The manufacture of citric acid and gluconic acid by mold 
fermentation has already been industrialized. Doubtless, when 
more is known regarding the fermentations which give rise to 
other organic acids, molds will come to be used in their commer- 



IMPLICATIONS 89 

cial production. Scarcely more than a beoinnino- seems to have 
been made in the study of fungus pigments and of their uses to 
man and to the mold itself. 

The study of toxin production by fungi is still in its infancy. 
Fungus toxins may eventually come to have an important place in 
therapy against pathogenic bacteria. The production of sera 
containing fungus antitoxins is deserving of more consideration. 

Lastly, the physiology of molds should be studied in quite the 
same manner as has been done with bacteria. A few studies of 
this kind, typified by that of Martin and Jones (1940), in which 
carbohydrate fermentations and colony characteristics were em- 
ployed to distinguish species of Candida, indicate the potentiali- 
ties of these procedures. By such studies and by the refinement of 
techniques a better understanding may be reached regarding the 
protein metabolism of fungi and the mechanism by which they 
are able to elaborate mannose, glycogen, toxins, and many other 
substances. 

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90 BIOCHEMISTRY OF FUNGI 

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LITERATURE CITED 91 

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1932. 
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Currie, J. N., "The citic acid fermentation of Aspergillus niger," J. Biol. 

Chem., 31: 15-37, 1917. 
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50:454-455, 1897. 
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mould tissue. V. Fractionation of the nitrogen in the mycelium of 

Aspergillus fischeri," Biochem. /., 2*; 504-511, 1934. 
Gottschalk, A., "Biochemische Synthese von Fumarsaure aus Brenztauben- 

saure," Hoppe-Seyler's Z. physiol. Chem., 152: 136-143, 1926. 
Gould, B. S., and H. Raistrick, "Studies in the biochemistry of micro- 
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glaucus series," Biochem. ]., 28: 1640-1656, 1934. 



92 BIOCHEMISTRY OF FUNGI 

Herrick, H. T., and O. E. May, "The production of gluconic acid bv the 
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Iwanoff, N. A I., 'The biochemistry of the fungi," Ann. Rev. Biochem., 
7:675-697, 1932. 

Iwanoff, N. M., and E. S. Zwetkoff, "The biochemistry of the fungi," 
Ann. Rev. Biochem., 2:521-540, 1933; 5:585-612, 1936. 

Kardo-Ssysojewa, E., "Uber die Bildung von Gluconsaure durch Asper- 
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Kochalaty, W., "Purification and properties of the second antibacterial 
substance produced bv PeniciUiuni notation" Science, 94: 186-187, 
1943. 

KouL, F., and D. G. Kostermans, "Hetero-auxin als Stoffwechselprodukt 
niederer pflanzlicher organismen. Isolierung aus Hefe," Hoppe-Seyler's 
Z. physiol. Chem., 228: 113-121, 1934. 

Kostytchew, S., "Der Einfluss des Substrates auf die anaerobe Atmung der 
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25:44-50, 1907; 25: 188-191, 1907a. 

Kostytchew, S., and W. Tschesnokow, "Bildung von Citronensaure und 
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Letcher, H., and J. J. Willaman, "Biochemistrv of plant diseases. VIII. 
Alcoholic fermentation of Fusarium //'///," Phytopathology, 16: 941-949, 
1926. 

Lockwood, L. B., and A. J. A [oyer, "The production of chemicals bv fila- 
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Lockwood, L. B., G. E. W\rd, O. E. May, H. T. Herrick, and H. T. 
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Zentr. Bakt. Parasitenk., II Abt., 411-425, 1934. 

Martin, D. S., and C. P. Jones, "Further studies on the practical classification 
of the Alonilias," /. Bact., 39: 609-630, 1940. 

May, O. E., H. T. Herrick, A. J. Mover, R. Hellbach, "Semi-plant-scale 
production of gluconic acid bv mold fermentation," Ind. Eng. Chem., 
21: 1198-1203, 1929. 

May, O. E., H. T. Herrick, C. Thom, and A I. B. Church, "The production 
of gluconic acid bv the PeniciUiuni luteum-purpurogenum group. I," 
/. Biol. Chew., 75:417-422, 1927. 

May, O. E., A. J. AIoyer, P. A. Wells, and H. T. Herrick, "The produc- 
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1931. 

Meyer, Karl, et al., "On Penicillin;' Science, 96:20-21, 1942. 

Miller, T. E., and R. W. Stone, "Occurrence of glutathione in micro-or- 
ganisms," /. Bact., 36:248-249, 1938. 

Molliard, M., "Sur une nouvclle fermentation acide produite par le Sterig- 
matocystis nigra" Comp. rend.. 774:881, 1922. 

Mover, A. J., O. E. May, and H. T. Herrick, "Production of gluconic acid 
by PeniciUiuni chrysogemtm" 'Zentr. Bakt. Parasitenk., II Abt., P5;311- 
324, 1936. 



LITERATURE CITED 93 

Neuberg, C, and Clara Cohen, "Uber die Bildung von Acetaldehvd und 
die Verwicklichung der zweiten Vergarungsform bei verschiedenen 
Pilzen," Biochem. Z., 722:204-224, 1921. 

Nord, F. F., and Robert P. Mull, "Recent progress in the biochemistry of 
Fusaria," Advances in EnzymoL, 5: 165-205, 1945. 

Oxford, A. E., "Antibacterial substances from moulds. Part III. Some 
observations on the bacteriostatic powers of the mould products citrinin 
and penicillic acid," Cheviistry & Industry, 61:48-51, 1942. 
"Part V. The bacteriostatic powers of the methvl ethers of fumigatin 
and spinulosin and other hydroxy-, methoxv-, and hvdroxymethoxy de- 
rivatives of toluquinone and benzoquinone," Cheviistry & Industry, 
61: 189-192, 1942. 

Oxford, A. E., H. Raistrick, and G. Smith, "Part II. Penicillic acid, a 
metabolic product of Penicillhtm puberulum Bainier and Penicillium 
cyclophnn Westling," Chemistry & Industry, 61: 22-24, 1942. 

Oxford, A. E., and H. Raistrick, "Part IV. Spinosin and fumigatin, meta- 
bolic products of Penicillium spinuloswn Thorn and Aspergillus fumi- 
gatus Fresenius," Chemistry & Industry, 61: 128-129, 1942. 

Pearson, L. K., and K. B. Raper, "The influence of temperature on the 
nature of the fat formed by living organisms," Biochem. J., 21: 875-879, 
1927. 

Porges, N., "Citric acid production by Aspergillus niger" Am. J. Botany, 
19: 559-567, 1932. 

Preuss, L. AL, H. J. Gorcica, H. C. Greene, and W. H. Peterson, "Wach- 
stum und Steringehalt gewisser Schimmelpilze," Biochem. Z., 246:401- 
413, 1932. 

Preuss, L. M., W. H. Peterson, and E. B. Fred, "Isolation and identifica- 
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5>7:483-489, 1932a. 

Preuss, L. M., W. H. Peterson, H. Steenbock, and E. B. Fred, "Sterol con- 
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90: 369-384, 1931. 

Prill, E. A., P. R. Wenck, and W. H. Peterson, "The chemistrv of mould 
tissue. VI. Factors influencing the amount and nature of the fat pro- 
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Raistrick, H., "Biochemistry of the lower fungi," Ergeb. Enzymforsch., 
1: 345-363, 1931. 
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Raistrick, H., et al., "Studies in the biochemistrv of micro-organisms," 
Trans. Roy. Soc. London, B, 220: 367 pp., 1931. 

Raistrick, H., and A. B. Clark, "On the mechanism of oxalic acid formation 
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Raistrick, H., and P. Simonart, "Studies in the biochemistrv of micro- 
organisms. XXIV. 2:5-Dihydroxybenzoic acid (gentisic acid), a new 
product of the metabolism of glucose bv Penicillium griseofulvum 
Dierckx," Biochem. J., 27:628-633, 1933. 



94 BIOCHEMISTRY OF FUNGI 

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"Antibacterial substances from moulds. Part I. Citrinin, a metabolic prod- 
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1941. 
Reid, R. D., "Some principles of a bacterial inhibitory substance produced 

by a mold," /. Bad., 29: 215-221, 1935. 
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Steinberg, R. A., "The process of amino acid formation from sugars in 

Aspergillus niger," J. Agr. Research, 6^:618-633, 1942. 
Strong, F. AL, and W. H. Peterson, "Chemistry of mould tissue. IV. 

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1944. 
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antibiotic substances. II. Aspergillus flavus-oryzae group," Proc. Nat. 

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Waksman, S. A., and J. W. Foster, "Respiration and lactic acid production 

by a fungus of the genus Rhizopus," /. Agr. Research, 51: 873-900, 1938. 
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56: 973-975", 1934. 
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von Beijma in large-scale laboratory apparatus," lnd. Eng. Chem., 

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1668, 1918. 
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Pilze," Biochem. Z., 191: 418^32, 1928. 
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LITERATURE CITED 95 

Wilkins, W. H., and G. C. M. Harris, "Investigation into the production 
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chem. J., 22: 1504-1507, 1928. 



Chapter 5 
EFFECTS OF TEMPERATURE ON FUNGI 

Temperature is one of the most important environmental factors 
affecting the metabolic activities of fungi. Since this fact is o-en- 
erally appreciated, manv workers have concerned themselves with 
problems involving the influence of temperature upon selected 
species of fungi. These studies have dealt with temperature as a 
factor in spore germination, mycelial growth, and reproduction 
of the chosen organisms; with determinations of their cardinal 
temperatures, temperature coefficients, and lethal temperatures; 
with attempts to correlate temperatures that are favorable or in- 
hibitorv to infection and the subsequent development of disease 
or decav with those that are favorable or inhibitory to the Growth 
of the pathogens; and with attempts to establish a rational basis 
to account for the geographical distribution and seasonal incidence 
of fungi. In the aggregate the reports of these studies contain a 
large volume of data together with varied interpretations of them. 
In the account that follows an attempt has been made to select 
from these numerous reports representative materials that will 
aid in evaluating the effects of temperature on fungi. 

It does not seem to be possible completely to isolate tempera- 
ture as an environmental factor in studies with fungi. Such non- 
temperature factors as relative humidity, rate of accumulation and 
concentration of staling products and other by-products, char- 
acter of the substrate, initial reaction and rate of change of re- 
action of the medium, and aeration of the medium, as well as 
factors internal to the fungus, such as strain differences and a^e 
of the mycelium, exert an influence, whether the fungus is being 
grown on artificial media or on the natural substrate. 

Furthermore in experimental conditions temperatures are either 
maintained continuously or else fluctuate to only a small decree, 
whereas in nature they vary continually. Whether all metabolic 
activities can be maintained at a constant optimal level over indefi- 

96 



CARDINAL TEMPERATURES 91 

nite periods or whether one activity is favored by a given tempera- 
ture whereas another is adversely affected by the same tempera- 
ture is none too well known at present. The investigator is led 
to suspect, however, that physiologic unbalance results if tem- 
peratures are maintained, because at a constant level a single 
temperature may not necessarily be optimum for the germination, 
the mycelial development, and the reproduction of all species. 

The duration of exposure of a fungus to a given temperature 
should also be taken into consideration. This factor becomes im- 
portant in a study of the rate of growth, which varies within the 
culture period, there being a lag at the initiation of growth, fol- 
lowed by a period of acceleration and eventually terminated by a 
period of deceleration. These facts are expressed in the well- 
known sigmoid growth curve, characteristic of all organisms. 

Another difficulty that presents itself, as has been indicated in 
Chapter 1, is the inadequacy of methods for measuring growth. 
In nearly all reports use is made of the diameter of colonies or of 
the amount of surface area of colonies, when as a matter of fact 
growth is three-dimensional. These two criteria are of value 
in comparing the growth of an organism at different constant tem- 
peratures on the same medium, but they largely lose their value 
when a comparison is made of the same or different organisms 
grown on different media. In studying the rate of growth of 
Verticillium albo-atrum, Chaudhuri (1923) was led to conclude 
that the "rate of spread" on different media may be associated 
with extremely different rates of mycelial production. 

CARDINAL TEMPERATURES 

Each fungus may be presumed to possess a minimum, an opti- 
mum, and a maximum temperature. The minimum and the maxi- 
mum limit growth at low and high temperatures, respectivelv. 
These values are difficult to fix absolutely and usually are oniv 
closely approximated. The optimum temperature is that which 
permits greatest metabolic activity; it is usually based upon meas- 
urement of the greatest increment of growth during some defi- 
nite time interval. Respiratory activity and mycelial extension, 
however, may be correlated with one optimum, whereas, as has 
been indicated and will be discussed subsequentlv, conidial pro- 
duction may occur at a different optimum. Observations by 



98 EFFECTS OF TEMPERATURE ON FUNGI 

Fawcett (1921) indicate that the optimum cannot be considered 
apart from the time factor. 

The temperature range within which fungi are active is rather 
limited in comparison to that of bacteria. At 0° C their growth 
is completely checked, and relatively few are active at 42° C. 
The optimum temperature is not median in any instance between 
the minimum and the maximum temperatures. In other words, 
temperature does not increase the rate of fungus activity uni- 
formly from the minimum to the optimum, and decrease it uni- 
formly from the optimum to the maximum. 

In connection with the rate of reaction (physiological proc- 
esses) in fungi, the {generalized rule of van't Hoff, which states 
that for every rise of 10° C the reaction rate is doubled or trebled, 
holds true, within the range approximating 10° C to 30° C. At 
high temperatures, however, as Blackman (1905) has indicated, 
this rule is modified by a time factor, for ". . . when the process 
is conditioned as to its rapidity by a number of separate factors, 
the rate of the process is limited by the pace of the 'slowest 
factor.' Such controlling factors are sometimes spoken of as 
"pace-makers." Their influence is universally demonstrated in 
graphs showing cardinal temperatures in all fungi studied. The 
growth curve is observed to decline sharply and precipitously 
from the optimum to the maximum. In Table 8 are assembled 
the cardinal temperatures for a few representative species grown 
on semisolid media. 

The most extensive study to date involving temperature in rela- 
tion to the growth of fungi capable of producing decay of wood 
is that of Cartwright and Findlay (1934). They measured the 
diameter of colonies grown on 2% malt agar, using the average 
daily increment of growth of five colonies as an index. Their ob- 
servations are summarized in Table 9. 

From these data it is apparent, first of all, that the temperature 
requirements of species within the families Thelephoraceae, Polv- 
poraceae, and Agaricaceae are variable between species, even 
within the same genus. 

Cartwright and Findlay (1934) indicate that comparative 
growth rates on malt agar may not necessarily indicate the growth 
rates on timber in the forest. Stereum purpureum, for example, 
grows rapidly on malt agar but slowly on wood. Nevertheless 
a given wood-destroying species, such as S. frtistitloswn on oak, 



CARDINAL TEMPERATURES 



99 



TABLE 8 

Cardinal Temperatures of Various Fungi 



Fungus 
Fusarium coeruleum 

Fusarium eumartii 

Fusarium discolor var. sulphureum 

Fusarium oxysporum 

Fusarium radicicola 

Fusarium trichothecioides 

Verticillium albo-atrum 

Verticillium albo-atrum 
Rhizoctonia solani 
Merulius sihestris 
Merulius domesticus 
Merulius sclerotiorum 
Polyporus vaporarius spumarius 
Verpa bohemica 
Lenzites saepiaria 
Polyporus versicolor 
Lenzites tigrinus 
Gloeosporium musarum 
Glomerella cingulata 
Glomerella gossypii 
Gloeosporium jructigenum 
Colletotrichum lagenarium 
Colletotrichum lindemuthianum 
Magnusia nit i da 
Magnusia brae hy trie hi a 
Pythiacystis citrophthora 
Phytophthora terrestris 
Phomopsis citri 
Diplodia natalensis 
Ceratostomella pili/era 
Ceratostomella coerulea 
Ceratostomella pluriannulata 
Ceratostomella ips 



Source of Data 

Edson and Shapovalov 

(1920) 
Edson and Shapovalov 

(1920) 
Edson and Shapovalov 

(1920) 
Edson and Shapovalov 

(1920) 
Edson and Shapovalov 

(1920) 
Edson and Shapovalov 

(1920) 
Edson and Shapovalov 

(1920) 
Chaudhuri (1923) 
Lauritzen (1929) 
Falck (1907) 
Falck (1907) 
Falck (1907) 
Falck (1907) 
Falck (1907) 
Lindgren (1933) 
Lindgren (1933) 
Lindgren (1933) 
Edgerton (1915) 
Edgerton (1915) 
Edgerton (1915) 
Edgerton (1915) 
Edgerton (1915) 
Edgerton (1915) 
Sweet (1941) 
Sweet (1941) 
Fawcett (1921) 
Fawcett (1921) 
Fawcett (1921) 
Fawcett (1921) 
Lindgren (1942) 
Lindgren (1942) 
Lindgren (1942) 
Lindgren (1942) 



Temperatures 
{degrees C) 



Mini- 
mum 

5 



Opti- Maxi- 
mum mum 

25 35 



25 



35 



5 25 35 

5 30 

5 30 

5 25 35 

5 25 35 

10 22.5 30 

2 23 34.5 

3 25 30 
3 22 30 
3 25 30 
3 25 30 
3 22 30 
5 32-35 45 
27-32 40 
7 32-35 43 

29-30 37.5 
27-29 37.5 
27-29 37.5 

24-25 34-35 
24 35 

21-23 30-31 
5 32 43 

5 32 43 

8.7 26.5 31.9 

12.0 31.5 36.1 

9.1 27.0 31.4 

8.4 28.0 36.0 

4 28-29 34-35 

3 25-27 32-34 

4 28-29 34-35 
6-8 30-32 37-39 



100 



EFFECTS OF TEMPERATURE ON FUNGI 



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102 EFFECTS OF TEMPERATURE ON FUNGI 

may completely invade the wood to the exclusion of all other 
Thelephoraceae and Polvporaceae. Conceivably the temperature 
differential may be an important factor when two or more species 
are competing for occupancy of a given piece of wood, but it may 
not necessarily constitute the controlling factor. 

Another inference from the data of Cartwright and Findlay 
(1934) involves temperature as an ecological factor affecting the 
geographical distribution of fungi. It is well known that certain 
species, just as is true also of seed plants, are quite sharply re- 
stricted in their natural habitat to Arctic regions, to temperate 
regions, or to the tropics. In pathogens this distribution might be 
anticipated to be coextensive with that of the suscepts and there- 
fore not necessarily governed primarily by temperature. In sapro- 
phytic species, temperature might not be expected to be as potent 
a factor as the kind of substrate, and saprophytes might be antici- 
pated to be cosmopolitan in distribution. Nevertheless, many 
saprophytes are restricted in distribution, but evidence indicates 
that with them temperature is a major factor. This conclusion 
finds support in Weimer and Harter's (1923) studies on the tem- 
perature relations among species of Rhizopus. They found that 
R. chinensis is distinctly more tolerant of high temperature than 
any of the other ten species tested. 

Humphrey and Siggers (1933) made an extensive study of tem- 
peratures favorable to the growth of wood-rotting fungi in cul- 
ture, and on this basis were able to arrange them into three groups: 
(1) a low-temperature group (20° to 24° C), (2) an intermediate- 
temperature group (24° to 32° C), and (3) a high-temperature 
group (above 32° C). 

In the first group are included Coniophora cerebella, Stereum 
gausapatuni, Merulius lacrymans, Phlebia merismoides, Folypoms 
abietiiuts, P. schnveinitzii, Tomes amiosus, F. officinalis, F. mgroli- 
neatns, Trametes pini, and Colly bia velutipes. In the second group 
Humphrey and Siggers placed Merulius sihestris, At. tremellosus, 
Corticium chrysocreas, C. effuscatum, Peniophora gigantea, 
Stereiim jrustulosum, S. fasciatum, S. rameale, Porta incrassata, P. 
subacida, P. xantha, Polyporus radiatus, P. robinophilus, P. sinuo- 
sus y P. sulphireus, P. versicolor, Daedalea ambigua, D. quercina, 
D. unicolor, Trametes serialis, Tomes everhartii, F. igniarius, F. 
marmoratus, F . pijiicola, F. rimosus, F. subroseus, Ganoderma 
applanatum, Lenzites berkeleyi, Irpex mollis, Hydnum ochracewn, 



RESISTANCE TO TEMPERATURES 103 

H. pulcherrimwm, Lentimis lepidens, Schizophyllum commune, 
and Pleurotus o streams. The third group comprises Phlebia stri- 
gosazonata, Stereum fuscum, Poly poms hirsutus, Ganoderma luci- 
dum, Lenzites saepiaria, L. trabea, and Pamis riidis. 

Studies to date on the temperature relations of wood-destroy- 
ing fungi have involved their Growth on artificial media rather 
than on wood [Herrick (1939)]. It is conceivable that there may 
be little, if anv, correlation of growth rates on such different sub- 
strates. Lindgren (1933) indicated that two reasons may be as- 
signed for a lack of correlation: (1) the chemical and phvsical 
differences between nutrient agar and wood; and (2) the time 
factor, cultivation on agar being confined to periods of short dura- 
tion and on wood in nature to long periods. These reasons appear 
to be sufficient to render unreliable anv predictions of the rate of 
decay of timber on the basis of the rate of growth on agar of the 
causal fungus. 

RESISTANCE TO LOW TEMPERATURES AND 
HIGH TEMPERATURES 

Experiments to determine the ability of fungi to survive when 
subjected to temperatures in excess of those known to inhibit 
growth and reproduction are meager. The results of these experi- 
ments, however, show that fungi are much more tolerant of low 
than of high temperatures. Evidently, as the temperature is 
elevated above the maximum for growth, desiccation and coagu- 
lation of proteins occur, and these reactions become the proximate 
cause of death. At low temperatures, on the other hand, these 
profound changes in proteins may not be accomplished, and 
other explanations are needed to account for the death of the 
fungus. 

Low temperatures. The temperatures employed in ordinary 
refrigeration and cold storage are^very effective in inhibiting the 
growth of such fungi as those causing decay of meats, fruits, vege- 
tables, and other foodstuffs. 

Lauritzen (1929) found that a storage temperature of less than 
2° C is required to prevent decay of turnips, induced by Rhizoc- 
tonia solam. At a maintained temperature of 8° to 10° C this 
fungus caused 62 to 87% decay within a period of 2 years. 

Brooks and Cooley (1917) stored apples inoculated with vari- 
ous decay-producing fungi at 0° C with the result that the rots 



104 



EFFECTS OF TEMPERATURE OX FUXG1 



developed. The organisms involved included Alternaria sp., Bo- 
try t is cinerea, Cephalothechmi roseum, Neofabrea malicorticiSj 
Fenicillhmi expansum, Sclerotinia cinerea, Sphaeropsis malonmi. 




-1 -2 -3 -4 

Temperature (degrees Centigrade) 

Fig. 4. Effect of cold upon survival of Aethalium septicum. A. Slow cool- 
ing, followed bv exposure for 10 minutes at the given temperatures. B. 
Rapid cooling, followed bv exposure for 5 seconds. C. Rapid cooling, 
followed by exposure for 10 minutes. (After Gehenio and Luyet.) 

and Vohitella fn/cti. Storage at 10° C inhibited Glomerella cin- 
gulata; at 15 3 C, Fusarium radicicola. These results support the 
observations of Schneidcr-Orelli (1912), who grew on gelatin the 
following species at maintained temperatures of 0° C, 4.5° C, and 
9.5° C: Botrytis cinerea, Fusarium putrefaciens, Gloeosporhtm 
album, G. her barum, Monilia fructigena, Mi/ cor piriformis, and 



RESISTANCE TO TEMPERATURES 



10$ 



Fenicillium glaucum. Neither Gloeosporiwn fructigenum nor 

Rhizopus nigricans, however, grew at 0° C, although meager colo- 
nies of these organisms developed at 4.5° C. Since so many decay- 
inducing fungi are able to grow at or near 0° C, it is essential that 
subzero conditions be provided for many readily perishable foods 
that must be kept for months before they normally reach the con- 
sumer. Consequently storage in dry ice has been employed effec- 
tively to meet these conditions. 





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Fig. 5. Comparison under controlled conditions of temperatures favorable 

for the development of tobacco-root rot with those favorable for the growth 

of the causal fungus. Tobacco-root rot develops best at temperatures below 

those optimum for the pathogen. (After Jones.) 

Numerous species of fungi are able to survive subzero weather, 
as their going into dormancy in fall and their reappearance in 
spring indicate. The cold of winter may decimate the fungus 
population, but it does not cause the extinction within a given area 
of any considerable number of species. 

Buller and Cameron (1913) exposed the fructifications of 
Schizophyllnm commune for several winter months to tempera- 
tures that ranged between — 15° C and —40° C. After the fructi- 
fications had been brought inside for a few hours, they resumed 
casting their spores. Moreover, when fructifications that were 
actively discharging spores were quickly frozen at — 3 1 ° C, they 
still retained their viability. 

Bennett ( 193 1 ) subjected the vegetative and perithecial stages of 
Gibberella saitbinettii to —20° C every third day for 45 days, per- 
mitting the temperature at no time to reach zero. Afterwards the 



106 EFFECTS OF TEMPERATURE ON FUNGI 

cultures were more vigorous and produced perithecia more abun- 
dantly than similar cultures that had been kept at normal tem- 
peratures. 

The ability of fungi to tolerate extreme cold is illustrated by 
Buller's (1913) findings with Schizophyllum commune and by 
Faull's (1930) findings with Neurospora crass a. Buller exposed 
the fructifications of 5. commune to — 190° C for 3 weeks without 
apparent injury, and Faull subjected the ascospores of N. crassa 
to temperatures from — 170° to — 190° C for 24 to 48 hours with- 
out delaying their germination. When wet, the conidia of this 
species were unimpaired by exposure to —80° C for 1 hour; when 
dry, to —170° to —190° C for an equal period. Toleration of 
these extremely low temperatures leads the investigator to antici- 
pate that the spores of certain species will be found to survive 
at absolute zero ( — 273° C), the point at which all reactions and 
hence all biological processes are theoretically supposed to be in- 
hibited, provided, of course, that the period of exposure is not 
too protracted. 

Becquerel (1910) dried the conidia of Aiucor, Rhizopus, Asper- 
gillus, and Sterigmatocvstis, sealed them in tubes under vacuum in 
which the pressure was reduced to 10 -4 cm of mercury, and ex- 
posed them at — 190° C for 77 hours; after 2 years' storage, they 
germinated normally. 

In the experiments of Kadisch (1931) with several dermato- 
phytes, Achorion gypseum survived 3 hours 1 exposure to —252° C 
in one instance, and in another withstood 2 hours at —268° C, 
followed by 4 hours at —268.8° C and then 1 )<> hours at —272° C. 

It would be anticipated that mycelia cannot tolerate as extreme 
temperatures as can spores. Evidence in support of this supposi- 
tion has been presented by Bartetzko (1910), Lindner (1915), 
and Lipman (1937). Bartetzko (1910) subjected germinating 
spores of Aspergillus, Penicillium, Botrytis, and Phycomyces in 
liquid nutrient media to — 14° for 2 hours without injury. When 
the young hyphae of Aspergillus in 1% glucose solution were ex- 
posed to —12° for 2 hours, they were killed, whereas in 5% glu- 
cose solution there was no apparent injury at —26° for an equal 
period. The other species exhibited similar differences in glucose 
solutions of different concentrations. 

Lindner ( 1915) exposed Aspergillus niger and Penicillium glau- 
cum y growing on 3% gelatin, to —10° to —13° C. Age of the 



RESISTANCE TO TEMPERATURES 101 

hyphae and duration of exposure were found to be important 
factors. Twenty-four-hour-old cultures were more easily killed 
than 48-hour-old cultures. Aerial hyphae were more easily killed 
than submerged hyphae. 

Lipman (1937) employed 12 species of fungi, cultured for 24 
hours on synthetic agar or on potato agar. After gradual cooling 
he immersed them in sealed tubes for 48 hours in liquid air; he 
then gradually warmed them. Of the 12 species, belonging in 
Aspergillus, Penicillium, Rhizopus, Mucor, Absidia, Mortierella, 
Rhizoctonia, Armillaria, Trichoderma, Pythium, and Fusarium, 
8 survived. As an explanation Lipman hypothesizes that this ex- 
traordinary tolerance may be causally related to the tiny spaces 
that exist between the colloidal micelles, which because of their 
small size prevent dehydration through ice formation. 

Not all fungi are capable of tolerating the extremes of tempera- 
ture which have been mentioned. Gehenio and Luyet (1939) ex- 
posed the plasmodium of Aethalhun septimm so as to study the 
effect of cold on vitality and the influence of the duration of ex- 
posure, as well as to determine whether cold per se or the sud- 
denness of the temperature change is responsible for injury. They 
found, first of all, that there may be marked injury at tempera- 
tures of freezing or slightly above if the plasmodia are cooled 
abruptly, whereas with slow cooling the injury may not be ap- 
preciable until the temperature descends to about —2.5° C. They 
also noted that the plasmodia may be killed after exposure of only 
5 seconds to temperatures of —1° or — 2° C. This sensitivity to 
cold finds support in the observations that some tropical species 
of seed plants are killed if exposed to temperatures above 0° C. In 
these cases death cannot be attributed to the formation of ice 
crystals. Here the mechanism of death, as postulated by Gehenio 
and Luyet, consists of gelation of the protoplasmic sol under the 
action of cold, the gelation being accompanied by syneresis. The 
squeezing out of the dispersion medium, if gelation is complete, is 
not a reversible process and hence is lethal. 

The problem of the causes of death by low temperatures in 
fungi, in other plants, and in animals is summarized in the mono- 
graph by Luyet and Gehenio (1940). Their summary indicates 
that death from cold has been attributed to the following causes: 
(1) bursting of the cells by expansion in ice formation, with con- 
sequent mechanical injury; (2) destruction of the fine structure 



108 EFFECTS OF TEMPERATURE ON FUNGI 

of the protoplasm bv ice crystals; (3) crushing between the ice 
masses as freezing progresses; (4) thawing at too rapid a rate; (5) 
dehydration of protoplasm, resulting in increased permeability, 
increased viscosity, coagulation of proteins, ionic dissociation, loss 
of water-binding properties of cytoplasm, and or svneretic re- 
lease of water. 

High temperatures. Fungi, it has been pointed out, generally 
are unable to tolerate exposure to high temperatures. The de- 
cline in ability to germinate or to grow is normally very sharp in 
the zone beyond the optimal. 

The lethal effects of temperature on germination of spores is 
considered in Chapter 9 and therefore need not be discussed here. 
In many instances such temperatures as inhibit germination and 
growth or are lethal are not excessive. For example, Wolf et al. 
(1934) found that sporangia of Peronospora tabacina exposed to 
85° F for 1 hour are incapable of germination. 

Fawcett and Barger (1927) observed that oranges kept at 90.5° 
F, which is above the maximal limit for Pemcillhmi italicum and 
P. digitatimi, are not decayed during 28 days' exposure. On the 
other hand, Faull (1930) noted that the ascospores of Neurospora 
crassa, when heated for more than 1 hour at 50° C, retain their 
ability to grow. 

In the fermentation of cigar tobaccos, temperatures of 140° to 
150° F are not unusual. Aspergillus niger, commonly present on 
the cured leaves, is unable to develop at these temperatures but 
may induce spoilage if too much time elapses for the bulk to be- 
come hot or to cool after fermentation. Temperatures near 100° F 
approximate the optimum for this mold. 

Treatment with hot water has been employed to free seed oats 
from loose smut, caused by Ustilago avenae, and wheat from 
naked smut, caused by U. tritici. Such treatment is practicable 
because the temperature lethal for the smut fungi is lower than 
that which kills the cereal embryos. Similarly, cotton seed, if 
slowly desiccated, can be rendered free from viable external 
conidia and internal mycelium of the anthracnose fungus, Glomc- 
rella gossypii. Lehman (1925) predried cotton seeds at 50° C, 
for 36 hours or at 60° C for 18 to 24 hours and then heated their; 
to 95° C for 10 to 12 hours, without reducing their percentage 
germination and with complete elimination of the anthracnose 
fungus. 



INFLUENCE OF TEMPERATURE ON INFECTION 109 

In some cases seed disinfection does not require exposure to ex- 
cessive temperatures. Edgerton (1915) found that 30° to 31° C 
is maximum for the growth in culture of Colletotrichinn linde- 
muthianum and has been able to produce in Louisiana, during 
summer, anthracnose-free bean seed from a crop planted with 
infected seed. 



INFLUENCE OF TEMPERATURE ON INFECTION 

The severity of certain soil-borne diseases, especially those 
caused by Fusarium, Verticillium, Rhizoctonia, Sclerotinia, and 
Thielaviopsis, is known to be correlated with temperature. Data 
bearing on this matter have been amassed from the use of soil- 
temperature tanks equipped with thermostatic controls. Plans 
for the construction and operation of this type of apparatus are 
described by Jones, Johnson, and Dickson (1926). Their account 
should be carefully read to obtain an appreciation of the problems 
relating to the influence of temperature in the development of 
plant diseases and to the construction and operation of ecostats. 
These workers conclude that disease is the resultant of the "inter- 
action of the plastic host and a plastic parasite under the play of 
variable environment." Temperature, as a variable, modifies the 
metabolic activity not only of the host but also of the parasite, 
and it may happen that such temperatures as approximate opti- 
mum for the one may exercise an adverse influence upon the other. 

By means of soil-temperature tanks Gilman (1916) determined 
that symptoms of cabbage yellows, caused by Fusarium conglu- 
tinans, are absent at maintained soil temperatures between 12° and 
16° C, but that characteristic symptoms appear within the range 
17° to 22° C. When this organism is grown in culture, its opti- 
mum, indicated by a daily increase in the diameter of colonies, 
approximates 25° C. 

Johnson and Hartman (1919), also using soil-temperature tanks, 
found that soil temperatures of 17° to 23° C are most favorable for 
the development of tobacco-root rot. The disease gradually di- 
minished in severity above 26° C and was absent at 29° to 30° C. 
As they indicate, account must be taken in experimentation of 
such other factors as soil moisture, soil reaction, supplv of nu- 
trients in the soil, and amount of infestation, none of which can be 
isolated and evaluated completely. The sum total of all these 



110 



EFFECTS OF TEMPERATURE ON FUNGI 



factors, whether favorable or unfavorable to the development 
of the disease, determines the severity of the attack. 

The destruction of stem tissues of potato and injury to the 
growing points by Corticium vagum are limited within the range 
9° to 27° C, [Richards (1921) |, with greatest damage between 
15° and 21° C. The severity of attack decreases very rapidly 
above 21° C, and damage is minor at 24° C and above. 

7 C 



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Fig. 6. Relation of the growth rate of Fusarium conglutinans and the de- 
velopment of cabbage yellows at various controlled soil temperatures. 
Both have quite the same optima. (After Jones, Johnson, and Dickson.) 

Infection of onions by Urocystis cepulae is governed by soil 
temperature [Walker and Wellman ( 1926) ] . Abundant infection 
by this smut fungus occurs at temperatures as low as 10° to 12° C, 
which is about the minimum permitting germination and growth 
of onions. Temperatures extending up to 25° C favor infection, 
but above this point the amount of infection is rapidly decreased. 
At 29° C and above, the onion seedlings remain free from in- 
fection. 

Observations of the foregoing type afford a basis in accounting 
for the seasonal incidence of certain plant diseases and for their 
geographical distribution. Jones (1924) pointed out that onion 
smut does not occur in southern Texas, although the pathogen 
has been repeatedly introduced into this region. The soil temper- 



TEMPERATURE AND REPRODUCTION 111 

ature is above that lethal to the smut fungus during the period 
when the seedlings are being grown in seed beds and are being 
transplanted. The prevalence of peach-leaf curl and apple scab is 
correlated with cold, wet spring weather. Late blight of potatoes 
is entirely absent, or at least never epiphvtotic, in the Coastal 
Plains area of the southeastern United States if the crop matures 
in late May or in June, when summer temperatures prevail. The 
fundus which causes downy mildew of tobacco disappears rather 
quickly after a few warm days with temperatures in excess of 85° 
F [Dixon, McLean, and Wolf (1936)]. The observations of 
Stevens (1917) led him to conclude that temperature is the chief 
climatic influence in the growth of the chestnut-blight fungus, 
Endothia parasitica. Sclerotium rolfsii is limited to warm regions 
and becomes of importance only during hot weather. 

TEMPERATURE AND REPRODUCTION 

There is abundant evidence that temperatures favorable for 
germination or for growth of fungi may be slightly lower than 
those favorable for reproduction. In some instances mycelial 
growth occurs at high temperatures that are inhibitory to repro- 
duction. Ames (1915) determined that the spores of Thielaviop- 
sis paradoxa germinate at 5° to 6° C, and, although there is slight 
growth at 10° C, this organism must be provided with tempera- 
tures in excess of 10° C to induce fruiting. If the temperature 
is elevated to 36° C, however, the mycelium develops, but conidia 
are not produced. Similar differences were noted at both the 
upper and lower limits for Glomerella rufomacirians, which ger- 
minates at 4° C, but requires a minimum of 12° C to produce 
spores. Pemcillhnn digitatinn is able to germinate and grow at 
30° C, but no conidia are formed at this temperature. 

Sweet (1941) recorded that the formation of cleistothecia by 
Magnnsia nitida and M. brachytrichia occurs throughout the 
range 16° to 38° C, although conidial germination is secured 
throughout the range 1.5° to 43° C. Production of conidia, 
however, is limited to the range 10° to 38° C in M . nitida, and 16° 
to 40.5° C in M. brachytrichia. 

Sporulation by Peronospora tabacina occurs within a range of 
temperature from 42° to 63° F and is most abundant at 56° F 



112 



EFFECTS OF TEMPERATURE OX FUNGI 



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and temperature. (After Scheffer and Livingston.) 



TEMPERATURE AND REPRODUCTION 



113 



[Dixon, McLean, and Wolf (1936)]. Mycelial growth, however, 
may occur at temperatures either below or above this range. 

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thora sphaerosperma. Although growth occurs at 12° C, conidia 
are not formed. 

Temperatures within the range 21° to 25° C were observed by 
Longree (1939) to be optimum for sporulation of Sphaerotheca 
pannosa var. rosae, but mycelial growth occurs well beyond both 
of these limits. Crosier (1933) reported 21° C as optimum for 
sporangial production by Phytophthora infestans. Krause (1930) 
concluded that perithecial formation by Neocosmospora vasin- 
fecta and Nectria coccinea is markedly influenced by temperature. 
At 7° C Nectria coccinea requires 45 days to produce perithecia; 



114 EFFECTS OF TEMPERATURE ON FUNGI 

at 22.5° C, 30 days; and at 30° C, 17 days. At 22.5° C Neocos- 
vwspora vasinfecta requires 45 days; at 28.5° C, 30 davs; and at 
31.5° C, 17 davs. Undoubtedly low temperature is a primary 
factor in the formation of sporophores by many Thelephoraceae 
and Polyporaceae, but not all species. This statement is substan- 
tiated by the occurrence in North Carolina of fresh sporophores 
of Fomes annosus, Poly poms abietinus, P. sanguineus, Sterenm 
lobatum, and S. jasciatinn at any time in the interval from Oc- 
tober to March. 



TEMPERATURE AND ZONATIOX 

Alternation of light and darkness is known to stimulate the pro- 
duction of daily bands of conidia and hence of zonation in various 
fungi grown in Petri dishes, as described in Chapter 6. Tempera- 
ture may also play an important role in zonation. Bisby (1925) 
made the observation that Fnsariimi discolor sulphureum, which 
forms zones in response to alternating light and darkness, can be 
induced to form zones in constant darkness provided that tem- 
perature is favorable. At a temperature of 16° to 18° C zonation 
does not occur, even though the cultures are exposed to alternate 
light and darkness. At 21° C zones can be formed under the 
stimulus of lisrht, but similar cultures in constant darkness are 
without zones. At 30° C, however, rings were formed when the 
cultures were maintained in constant darkness. 

TEMPERATURE COEFFICIENTS 

By temperature coefficient is meant the ratio of the rate of a 
given physiological process, for example, respiration, at any given 
temperature to the rate at which this process proceeds at another 
temperature. Temperature coefficient is frequently represented 
by the symbol Q ut , meaning that the interval is 10° and that the 
rate at the given higher temperature is divided by the rate at the 
temperature 10° lower. Biologists well appreciate the fact that 
within a range which approximates the minimal and maximal 
temperature limits for the given organism, the reaction-velocity 
changes follow van't HoiTs rule. According to this rule, Q u > for 
the physiological orocess in question should lie between 2 and 3 
as a minimum. 



TEMPERATURE COEFFICIENTS 



115 




116 



EFFECTS OF TEMPERATURE OX FUNGI 



Temperature coefficients have been abundantly determined and 
interpreted. Fawcett (1921) measured the growth-temperature 
coefficients of Pythiacystis citrophthora and Fhytophthora terres- 
tris within the range 8° to 36° C, of Fhovwpsis citri within the 
rans;e 8° to 32° C, and of Diplodia natalensis within the range 8° 
to 45° C. He found that for each 24-hour observation period, the 



320 



2 
I 



E 160 



.as 

£ 

C3 








10 15 20 25 

Temperature ( degrees Centigrade ) 



30 



oo 



Fk.. 10. The extent of decav, measured in terms of diameter of lesions, 
induced in turnips bv Rhizoctonia solani at various maintained temperatures. 

(After Lauritzen.) 

Qiu for mycelial growth is greatest for the lowest temperature 
shown and becomes smallest for the highest temperatures. At 8° 
to 18° C Fhytophthora terrestris showed a coefficient of 30, and 
at 26° to 36° C a coefficient of 0.4". lor the 8° to 18° C range 
the Oio of Fhowopsis citri was 4.0; for the 21° to 31° C range, 0.5. 
Within the range investigated, the coefficient of Pythiacystis 
citrophthora was 12.3 for the lowest temperature and 0.05 for the 
highest; that of Diplodia natalensis was 16.7 for the lowest tem- 
perature and 0.05 for the highest. 

ScherTer (1936) determined the rate of carbon dioxide produc- 
tion bv Polystictus versicolor. He found that production was 



TEMPERATURE AND OXYGEN TENSION 111 

greater as temperature was increased, and at 29.5° C was critical. 
The loss of carbon (as CO;.) was least within the range 25.5° to 
29.5° C; it was relatively great at 17.5° and at 33.5° C. Moreover 
the rate of C0 2 production per unit of mycelial area and the rate 
of growth were quite alike within the range 17.5° to 29.5° C. 

TEMPERATURE AND OXYGEN TENSION 

In the light of findings that the respiratory quotient is highest 
at the lowest temperatures and, conversely, lowest at the highest 
temperature, it would be expected that oxygen tension would also 
modify physiological processes. Evidence of such modifying 
effect has been presented by Scheffer and Livingston (1937). 
They grew Polystictus versicolor on malt agar in special tubes, 
by means of which they could modify the oxygen tensions and 
then keep them at constant levels. At the same time they main- 
tained constant temperatures by means of thermostatically con- 
trolled incubators. By these procedures they found that C0 2 
production per unit area of mycelial mat was always most rapid 
as 2 pressure became greater. At 33.5° C with 745-mm pres- 
sure of 2 , C0 2 production was most rapid; it was least rapid at 
17.5° C with zero 2 pressure. Mycelial growth, however, was 
most rapid at the optimum temperature for P. versicolor, that is, 
at 29.5° C, at all 2 pressures from 16 mm to 745 mm. When 
C0 2 production in atmospheres of pure 2 was compared with 
that in pure N 2 , Scheffer and Livingston noted that the rate per 
unit of mycelial area was two to five times as rapid in oxygen as 
in nitrogen. 

The availability of 2 is known to operate in another manner, 
as has been demonstrated by Chaudhuri (1923). He aerated 
liquid nutrient media on which Verticillhim albo-atram was being 
grown at different temperatures, employing rate of spread as a 
measure of yield of fungus material. His data show that aeration 
markedly increases both the rate of growth and the total amount 
of growth in a given volume of liquid media. Since V . albo- 
atnnn is known to produce staling products, Chaudhuri postu- 
lates that these increases are to be attributed to the oxidation of 
w r aste products. 



118 



EFFECTS OF TEMPERATURE ON FUNGI 



90 



85 



80 



75 



70 



65 



60 



B 55 
1 

I 50 



8 45 

I 

"o 
o 

i 

s 

1 



40 



35 - 



30 



25 



20 



15 



10 



Fusarium discolor 
var. sulphureum 



Fusarium 
oxysporum 



Fusarium 
radicicola 




15 20 25 
Degrees Centigrade 

Fig. 11. The growth, measured in terms of diameter of colonies, of soil- 
inhabiting pathogens as influenced by temperature. Verticillhtm albo-atrwn 
has the slowest growth rate; 25° C is the optimum temperature. (After 

Edson and Shapovalov.) 






IMPLICATIONS U9 



IMPLICATIONS 



Experiments involving the maintenance of fungi in culture at 
a given constant temperature for considerable periods have a 
limited usefulness. This conclusion finds support in the fact that 
in nature fungi do not encounter constant temperature. Experi- 
ments with controlled temperature have demonstrated, it appears, 
that optimal temperature requirements exist for each metabolic 
activity of a given fungus and for each phase in its developmental 
cycle. It is desirable therefore that a much larger body of data 
showing these facts be accumulated, for from such experiments 
would certainly come increased understanding of temperature as 
an environmental factor in fungus activities. 

Some persons are inclined to make light of the popular idea that 
"diseases are caused by weather." Indeed, such persons may with 
fairness be accused of overemphasizing the "germ theory." They 
are content to stress the primary cause of disease and to overlook 
secondary or attendant causes. Since temperature is one of the 
components of weather, it cannot be ignored in its influence, 
among pathogenic fungi, upon such sequential phenomena as 
spore dispersal, spore germination, incubation and severity of the 
resultant disease, and, finally, the development of reproductive 
elements by the pathogen. 

LITERATURE CITED 

Ames, A., "The temperature relations of some fungi causing storage rots," 

Phytopathology, 5: 11-19, 1915. 
Bartetzko, H., "Untersuchungen iiber das Erfrieren von Schimmelpilzen," 

Jahrb. iviss. Botan., 41: 57-98, 1910. 
Becquerel, P., "Recherches experimentales sur la vie latente des spores des 

Mucorinees et des Ascomycetes," Conip. rend., 150: 1437-1439, 1910. 
Bennett, F. T., "Gibberella saubinettii (Mont.) Sacc. on British cereals. II. 

Physiological and pathological studies," Ann. Applied Biol., 18: 158-177, 

1931. 
Bisby, G. R., "Zonation in cultures of Fusarhmi discolor sulphureum," 

My col., 11: 89-97, 1925. 
Blackman, F. F., "Optima and limiting factors," Ann. Botany, 19: 281-295, 

1905. 
Brooks, Charles, and J. S. Cooley, "Temperature relations of apple-rot 

fungi," /. Agr. Research, 8: 139-164, 1917. 



120 EFFECTS OF TEMPERATURE ON FUNGI 

Brooks, Charles, and J. S. Cooley, "Temperature relations of stone-fruit 

fungi," /. Agr. Research, 22:451-465, 1922. 
"Time-temperature relations in different types of peach-rot infection," 

/. Agr. Research, 31: 507-543, 1928. 
Buller, A. H. R., "Upon the retention of vitality by dried fruit bodies of 

certain Hvmenomvcetes, including an account of an experiment with 

liquid air," Trims. Brit. Mycol. Soc, 4: 106-112, 1913. 
Buller, A. H. R., and A. T. Cameron, "On the temporary suspension of 

vitality in the fruit bodies of certain Hvmenomvcetes," Proc. Trans. 

Roy. Soc. Canada, 6:73-78, 1913. 
Cartwright, K. St. G., and W. P. K. Findlay, "Studies in the physiology 

of wood-destroving fungi. II. Temperature and the rate of growth," 

Ann. Botany, 48: 481-495, 1934. 
Chaudhuri, H., "A studv of the growth in culture of Verticillium albo- 

atrum B. and Br.," Ann. Botany, 31: 519-539, 1923. 
Crosier, YVillard, "Studies in the biology of Phytophthora infestans (Mont.) 

de Barv," Cornell Agr. Expt. Sta. Menu, 755:40 pp. 1933. 
Dickson, J. G., "Influence of soil temperature and moisture on the develop- 
ment of the seedling blight of wheat and corn caused by Gibberella 

saubinettii," J. Agr. ^Research, 25:830-870, 1923. 
Dixon, L. F., Ruth A. McLean, and F. A. Wolf, "Relation of climatological 

conditions to tobacco downy mildew," Phytopathology, 26: 735-759, 

1936. 
Edgerton, C. W., "Effect of temperature on Glomerella," Phytopathology, 

5: 247-259, 1915. 
Edson, H. A., and M. Shapovalov, "Temperature relations of certain potato- 
rot and wilt-producing fungi," /. Agr. Research, 18: 511-524, 1920. 
Falck, R., "Wachstumsgesetze, Wachstumsfaktoren, und Temperaturwerte 

der holzzerstorenden Mycelien," Moller's Hausschivammforschungen, 

Hefte 7:53-152, 1907. 
Faull, J. H., "On the resistance of Neurospora crassa," My col., 22:288-303, 

1930. 
Fawcett, H. S., "The temperature relations of growth in certain parasitic 

fungi," Univ. Calif. Pub. Agr. Sci., 4: 183-232, 1921. 
Fawcett, H. S., and W. R. Barger, "Relation of temperature to growth of 

Penicillhim italicum and P. digitatnm and to citrus-fruit decay produced 

by these fungi," /. Agr. Research, 55:925-931, 1927. 
Gehenio, P. M., and B. F. Luyet, "A study of the mechanism of death in 

the plasmodium of Mvxomycetes," Biodynaviica, no. 55: 1-22, 1939. 
Gilman, J. C, "Cabbage yellows and the relation of temperature to its 

occurrence," Ann. Mo. Botan. Garden, 3: 25-82, 1916. 
Herrick, J. A., "The growth of Sterenm gausapatum Fries in relation to 

temperature and acidity," Ohio J. Sci., 39: 254-258, 1939. 
Humphrey, C. J., and P. V. Siggers, "Temperature relations of wood-de- 
stroying fungi," /. Agr. Research, 41: 997-1008, 1933. 
Johnson, James, and R. E. H\rtman, "Influence of soil environment on the 
root rot of tobacco," /. Agr. Research, 11: 41-86, 1919. 



LITERATURE CITED 121 

Jones, L. R., "The relation of environment to disease in plants," Am. J. 

Botany, 11: 605-609, 1924. 
Jones, L. R., James Johnson, and J. G. Dickson, "Wisconsin studies upon 

the relation of soil temperature to plant disease," Wis. Agr. Expt. Sta. 

Bull, 11: 144 pp. 1926. 
Kadisch, E., "Beitrage zur Wirkung der Kalte auf pathogene Fadenpilze, 

Hefen, und Bakterien. Ausdehnung dieser Versuche bis in die Nahe 

des absoluten Nullpunktes (bis — 272° C)," Med. Klin., 27th year: 

1074-1078, 1109-1112, 1931. 
Krause, A. W., "Untersuchungen uber den Einfluss der Ernahrung, Belich- 

tung, und Temperatur auf die Perithecienproduktion einiger Hypo- 

creaceen. Beitrag zur Kulturmethodik einiger parasitischer und sapro- 

phytischer Pilze," Z. Parasitenk., 2: 419^176, 1930. 
Lauritzen, J. I., "Rhizoctonia rot of turnips in storage," /. Agr. Research, 

3£: 93-108, 1929. 
Lehman, S. G., "Studies on treatment of cotton seed," N. C. Agr. Expt. Sta. 

Tech. Bull., 26:71 pp. 1925. 
Lindgren, R. M., "Decay of wood and growth of some Hymenomycetes as 

affected by temperature," Phytopathology, 25:73-81, 1933. 
"Temperature, moisture, and penetration studies of wood-staining Cera- 

tostomellae in relation to their control," U. S. Dept. Agr. Tech. Bull., 

801: 35 pp. 1942. 
Lindner, J., "Uber den Einfluss giinstiger Temperaturen auf gefrorene 

Schimmelpilze. Zur Kenntnis der Kaltresistenz von Aspergillus niger" 

Jahrb. iviss. Botan., 55: 1-52, 1915. 
Lipman, C. B., "Tolerance of liquid air temperatures by spore-free and very 

young cultures of fungi and bacteria growing on agar media," Bull. 

Torrey Botan. Club, 64:531-546, 1937. 
Longree, K., "The effect of temperature and relative humidity on the 

powdery mildew of roses," Cornell Agr. Expt. Sta. Mem., 223: 43 pp. 

1939. 
Luyet, B. F., and P. M. Gehenio, Life and death at low temperatures. 341 

pp. Biodynamica, Normandy, Mo. 1940. 
Richards, B. L., "Pathogenicity of Corticium vagum on the potato as af- 
fected by soil temperatures," /. Agr. Research, 27:459-482, 1921. 
Sawyer, W. H., "Observations on some entomogenous members of the 

Entomophthoraceae in artificial culture," Am. J. Botany, 16:81-121, 

1929. 
Scheffer, T. C, "Relation of temperature and time to carbon dioxide pro- 
duction and growth in continuously aerated malt-agar cultures of Poly- 

st ictus versicolor,''' Plant Physiol., 11: 535-564, 1936. 
Scheffer, T. C, and B. E. Livingston, "Relation of oxvgen pressure and tem- 
perature to growth and carbon dioxide production in the fungus Poly- 

stictus versicolor" Am. ]. Botany, 24: 109-119, 1937. 
Schneider-Orelli, Otto, "Versuche liber die Wachstumbedingungen und 

Verbreitung der Faulnispilze des Lagerobstes," Xentr. Bakt., Parasitenk., 

II Abt., 32: 161-169, 1912. 



m EFFECTS OF TEMPERATURE ON FUNGI 

Stevens N E., "The influence of temperature on the growth of Endothia 
parasitica? Am. J. Botany, 4: 112-118, 1917. 

Sweet H R. "Studies on the biology of two spec.es of Magnusia. I. Effect 
of' temperature on germination of spores and on growth and repro- 
duction" Am. J. Botany, 28: 150-161, 1941. 

™ T r axo F L Wellm^x, "Relation of temperature to spore ger- 

"^^'^o^Uroc^s **** h 4* *—*> *»*- 

wiTt and L. L. Harter, 'Temperature relations of eleven species 

of RhizoDus " /. Aer. Research, 24: 1-40, 1923. 
W oi SuC R»™ McLean, and F. R. D.kk.s, "Downy m.l- 

dew of tobacco," Phytopathology, 24: 337-363, 1934. 



Chapter 6 

EFFECTS OF RADIATION ON FUNGI 

Although students of fungi have long been interested in the 
reactions to light of this group of organisms, little progress in this 
field was made until after the beginning of the present century. 
The primary reason for this state of affairs is that the existence of 
radiations other than visible light was unknown until approxi- 
mately 1900. From physical researches it is now known that 
radiations of the following groups exist, some of them possessing 
wavelengths in excess of those of visible light and others being 
shorter. 

1. Hertzian rays, the wavelengths of which range from 1 X 10 6 
to 3 X 10 14 Angstrom units, an Angstrom unit (A) being 1/10,- 
000,000 of a millimeter. Those waves in the upper portion of the 
range between 1 X 10 11 and 3 X 10 14 A are used in radio com- 
munication. 

2. Infrared or heat rays, the wavelengths of which range from 
8000 to 4 X 10 6 A, thus overlapping the lower end of the range 
of Hertzian waves. 

3. Visible light rays, the wavelengths of which range from ap- 
proximately 4000 to 8000 A. 

4. Ultraviolet rays, the wavelengths of which range from 136 
to 4000 A. 

5. X-rays, the wavelengths of which range from 0.06 to 1000 A, 
thus overlapping the lower end of the ultraviolet range. 

6. Gamma-rays, the wavelengths of which range from 0.01 to 
1.4 A. 

7. Cosmic rays, the wavelengths of which range down to 
1/10,000 A. 

Of these groups, infrared rays, visible light rays, ultraviolet 
rays, and X-rays have been used in experimentation with fungi. 
Such studies have been concerned mainly with the morphogenic 
effects of radiation, the fungicidal effects, and the modifying 

123 



124 



EFFECTS OF RADIATION ON FUNGI 



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MORPHOGENY REACTIONS 125 

effects upon reproduction. Unfortunately many of the results, 
especially those with visible light, are not reproducible because 
account has not been taken of three correlated factors: (a) quan- 
tity or intensity of light, (b) quality of light or wavelength of 
radiations, and (c) duration of exposure or total radiation. Even 
in some studies employing monochromators intensity was not 
kept constant or was not measured when quality was changed. 
It is all too apparent, furthermore, that such other factors as age 
of the culture, hydrogen-ion concentration, temperature, and 
screening effects from the culture media and from the massing 
of hyphae must be considered in experimentation on the reactiv- 
ity of fungi to radiation. Since this has not always been done, 
a barrage of criticisms may be levelled against the experimental 
procedures and consequently against the conclusions. In the 
account that follows some of the extensive publications in this 
field are brought together, but many meritorious ones have been 
excluded from the discussion. Although certain generalizations 
may be drawn from these publications, none appears to rest on 
too secure a basis, and the only broad statement warranted ap- 
pears to be that much further work is needed. Such studies 
should be attempted, however, only by mycologists well grounded 
in the physical principles of light and other radiations. Much of 
value on the effects of radiation will be found in the summary 
by Smith (1936). 

MORPHOGENIC REACTIONS 

A survey of studies to determine whether light is required for 
the development of fructifications by fungi reveals that light 
exerts a profound influence upon some species but that others 
appear to be completely indifferent to it. Several early mycolo- 
gists noted that in the complete absence of light the stipes of 
some species of Hymenomycetes are longer than normal, that 
other species, normally sessile, develop stipes, and that other 
species, normally pileate, branch in clavarioid fashion. Coprinus 
stercorarius, in darkness, has been observed to grow stipes 2 to 
3 ft long. Buller (1906) noted that Poly poms squamosw, kept 
in the dark, developed coal-black stag-horn-shaped sterile stro- 
mata, 15 cm tall and having white tips, in place of normal stalked 
pilei. The formation of the pileus is entirely conditioned by the 



126 



EFFECTS OF RADIATION ON FUNGI 



presence of daylight, exposure for a single hour, even if followed 
by return to the darkroom, being sufficient to result in the pro- 
duction of pilei. Likewise in Lentinus lepideus [Buller (1905)] 
the stimulus of light is necessary for formation of the pileus. 
Fruiting bodies grown in weak light have grotesque shapes. Al- 
though stipes are at first positively heliotropic and indifferent to 




Fig. 13. Petri-dish culture of Aspergillus clavatus in ordinary diffuse light. 

geotropism, by the time pilei begin to form they lose this reactiv- 
ity to light, and negative geotropism dominates pilear develop- 
ment. Aspergillus clavatus forms short conidiophores when 
grown in total darkness, whereas conidiophores of two lengths, 
one short and the other an inch or more long, are produced on 
exposure to diffuse daylight [Wolf (1938)]. The colonies pro- 
duced under these considerations might be suspected to belong 
to two distinct species of Aspergillus. 

Psalliota campestr'is, the cultivated mushroom, when grown in 
caves or cellars that are illuminated only to permit gathering the 
crop, is completely indifferent to light. Many subterranean 
fungi, as would be anticipated, are unaffected by light. Evidence 
has accumulated, moreover, that a considerable number of slime 



MORPHOGENY REACTIONS 



121 



molds, Hyphomycetes, Pyrenomycetes, and Basidiomycetes can 
develop in the absence of light. Long and Harsch (1918), for 
example, found that sporophores of Polyporus cinnabar inns, P. 
farloivii, and Trametes serialis develop in culture in absolute dark- 
ness. 

Zonation. A variety of morphogenic effects may be expressed 
by those species that respond to light. Perhaps the most striking 




Fig. 14. Culture of Aspergillus clavatus grown in blue light. 

is zonation, resulting from alternation of day and night, for it has 
been encountered by all who have cultivated fungi. Moreau 
(1912) observed the zone of conidia, produced daily, in Penicil- 
lium glaucum, Hedgcock (1906) in Cephalothecinm roseum, and 
Bisby (1925) in Fusarium discolor sulphur eum. Bisby reported 
that exposure of the cultures to bright daylight for a period of 
% to y 2 second was sufficient to produce a ring of conidia. As a 
more accurate measure, he noted that exposure to a 2 5 -candle- 
power tungsten light for 2 to 2% minutes was sufficient. 

In order to relate zonation to radiation of certain wavelengths, 
Hedgcock (1906) subjected Cephalothecinm roseum and Reide- 



128 



EFFECTS OF RADIATION ON FUNGI 



meister (1909) exposed Botrytis cinerea to illumination in which 
portions of the spectrum were screened out. The results of their 
studies and those of others are contradictory. In red lisrht and 
in darkness few conidia of B. cinerea were noted by Reidemeister 
(1909), but they formed abundantly in blue light. Colonies of 
Aspergillus clavatus grown in blue light produced tall conidio- 




Fig. 15. Culture of Aspergillus clavatus grown in total darkness or in red 

light. 

phores with a few short ones, whereas in red light and in dark- 
ness all were short [Wolf (1938)]. 

Since some fungi produce concentric zones in total darkness, 
the alternation of day and night must be regarded as only one of 
the complex factors involved in this phenomenon. Bisbv (1925) 
induced zonation in Fusarium discolor sulphur eum in total dark- 
ness if the temperature was alternated. The effect of temperature 
has been substantiated by more recent studies with other species. 
In an analysis of his studies on Fusarium and Monilia jructigena 
Brown (1925) showed that zonation in response to light changes 
is correlated with the following factors: (1) the capacity of the 



PHOTOTROPISM 129 

species to react to light in the matter of sporulation, (2) staling 
produced by mycelial growth, provided that the amount of stal- 
ing does not in any way impede the extension of the mycelium, 
and (3) the concentration of nutrients available, which should 
not permit of sporulation so intense as to interfere with the pro- 
duction of successive daily zones. 

PHOTOTROPISM 

The fact that fungi lack chlorophyll and that certain species 
are capable of completing their entire developmental cycle in 
the absence of light might at first thought incline the student to 
the belief that they would not react phototropically. On the 
other hand, it might be anticipated that radiant energy would 
influence rate of growth, and that consequently species in which 
growth or elongation is localized might respond to differences in 
the intensity of light. Species with long sporangiophores, such 
as Pilobolus or Phy corny ces nitens, or with long conidiophores, 
such as Aspergillus clavatus, should be especially suited for studies 
of this sort, since they respond to unilateral illumination. It 
should be possible with such species to determine the minimal 
amount of light required to stimulate a phototactic response and 
to establish that quality, quantity, and duration of exposure are 
functions of each other. In P. nitens it has been found that 
response follows a change of % to % candlepower per meter 
per second. Response may not occur immediately, so that re- 
action time may be said to consist of an exposure and a latent 
period. The duration of this latent period is constant for any 
particular intensity unless the exposure time is reduced below the 
minimum threshold; below this minimum the reaction time in- 
creases progressively as the duration of exposure decreases. 

Among Phycomycetes. In response to light, Phy corny ces 
nitens has long been known to bend in a zone just beneath the 
sporangium. Blaauw (1914), who investigated the phototropic 
response of this fungus by use of physical methods, regarded the 
sporangiophore as a cylindrical lens which concentrates the light 
on the cell wall opposite the source, causing greater photochemi- 
cal activity in this area. The net result of this photochemical 
action is bending of the sporangiophore, a response to unequal 



130 



EFFECTS OF RADIATION OX FUNGI 



rates of growth on opposite sides of the growing zone. This ex- 
planation is not entirely satisfactory if it is borne in mind that 
the sporangiophores are radially symmetrical and that the regions 
of sensitivity and of growth coincide. 



4 





Fig. 16. Response of Phycomyces to incident light. In A, grown in air, 
the sporangiophores bend to direct the sporangia toward the light. In B, 
the sporangia are surrounded with paraffin oil and have turned away from 
the source of light. Density of the surrounding medium conditions the 

(After Buder.) 



direction of refraction of light. 



Further evidence to clarify this problem was presented by 
Buder (1918). He grew P. nit ens, with the sporangiophores di- 
rected vertically, in chambers whose vertical sides were parallel. 
He then immersed the sporangiophores in one chamber in paraf- 
fine oil; in the control chamber the sporangiophores were sur- 
rounded by air. Illuminating both unilaterally, he found that 
those in oil were negatively phototropic, whereas those in air 
were positively phototropic. In explanation Buder pointed out 






PHOTOTROPISM 131 

that, since air is a less dense medium than is the content of the 
sporangiophore, the rays of light refracted from the front half of 
the cylindrical cell converge on the side opposite the source of 
light. Similarly, the oil is a more dense medium than is the con- 
tent of the sporangiophore, and hence the rays of light after 
refraction diverge from one another. When air is the medium, 
the back half of the growing zone is lighted the more intensely; 
when oil is the medium, the front half. The growth response is 
therefore in opposite directions in the two cases. 

Castle (1933) has also contributed to an understanding of the 
response of P. nitens to light. His solution of the problem is 
based upon three assumptions: (1) bending is a resultant of un- 
equal absorption of light by the two halves of the cell (the half 
toward the source of light and the half most distant from the 
source); (2) the primary action of light is upon the protoplasm; 
and (3) the absorption of light is brought about by a substance 
or substances (pigment) equally distributed within the cell. From 
these reasonable assumptions he deduces that the factors which 
govern the unequal action of light in the two halves of the cell 
are the following: (1) the refractive index of the cell, (2) the 
size of the cell, more specifically its radius, and (3) the coefficient 
of absorption possessed by the intracellular pigment. 

All known species of Pilobolus, which commonly occur on 
the fresh dung of herbivors when it is kept in a moist chamber, 
exhibit photic reactions. Among those who have investigated 
the response of these species to light are Allen and Jolivette 
(1914), Parr (1918), Pringsheim and Czurda (1927), van der 
Wey (1929), and Buller (1934). Allen and Jolivette admitted 
light through a pinhole and found that Pilobolus aimed point- 
blank at the light. The accuracy of the aiming was remarkable, 
for when the culture was 20 cm distant from the opening, 95% 
of the sporangia struck within a ring 4 cm in diameter, the remain- 
ing 5% being within the next 1 or 2 cm. A greater degree of 
precision was obtained with white or blue light and less accuracy 
with yellow light; the aiming was very inaccurate with red light. 
Of course the distance that the sporangia needed to travel modified 
the precision. Allen and Jolivette also made the interesting obser- 
vation that, when Pilobolus was exposed to two equal beams of 
white light with the angle between them greater than 10° of arc. 



132 



EFFECTS OF RADIATION ON FUNGI 



the aiming was as accurate as if one source of lioht was nonexist- 
ent. The sporangia were aimed at one or the other source of light, 
not midway between the two. Allen and Jolivette were unable to 
explain how this result was achieved, but Buller (1934) later 
found the mechanical basis to reside in the ocellar structure of the 
subsporangial swelling. 




L 



J L 



1 



J L 



J L 



J 



12 

Millimeters 

Fig. 17. Median longitudinal section of Pilobohis kleinii. The fructification 
is directed toward the source of light. The basal sporangial wall has gelat- 
inized, and the broken line indicates where the sporangium has separated 
from its attachment. Certain ravs cannot penetrate the black sporangium, 
which fits as a cap at the apex of the subsporangium. The ravs which pene- 
trate the upper sporangial wall converge at the basal perforated septum, 
which is red. The photochemical changes induced bv converged light 
within the subsporangium induce swelling and eventual bursting at the tip. 
The sporangium is carried away in toto with the squirt. (After Buller.) 

Later Parr (1918) concerned herself with precise measurements 
of the responses of Pilobohis to wavelengths of the different re- 
gions of the spectrum, to the presentation time, and to the energy 
values involved. Her important conclusions include the follow- 
ing: (1) Pilobohis responds phototropicallv to light in all re- 
gions of the spectrum. ( 2 ) The presentation time required to 
react phototropicallv increases gradually from the red rays to 
the violet; that is, Pilobohis is more sensitive to violet than to red. 
(3) The presentation time varies in inverse ratio to the square 
root of the wave frequency. (4) For any given light source the 
total energy value may be expressed as the product of the square 
root of the wave frequency multiplied bv the presentation time. 



PHOTOTROP1SM 



133 



This value decreases with a decrease in energy value of the spec- 
tral regions. 

The method bv which Pilobolus aims and discharges its spo- 
rangia toward the source of light was elucidated by Buller in a 
series of observations that began in 1919. He discovered that 



c 

& 



750* 


» 




\ 


\ 
























\ ° 




c 


»\ 
\ 






















700 








\ 


\ 




























\ 

o 


^ 






























\ 

\ 

\ 


















650 






*\ 






< 


k 






















o \ 


\^- Tungsten 


> 


^-Nernst 


























V 














600 








°\ 








\ 

\ 
°\ 






























\ 
\ 

\ 






















c\ 








t 


\ 










550 










< 


> \ 








\ 

\ 
































\ 

\ 

\ 








500 














c\ 








\ 
































S 




















°\ 










\o 

\ 
\ 




450 


























> 


\ 






























\ 
















> 
















400 































50 



55 60 65 70 

Presentation time (minutes) 



75 



Fig. 18. Response of Pilobolus to light from two sources to show relation 
of presentation time to frequency of light. Presentation time is regarded as 
the time required for one-half of the sporangiophores to respond. The time 
required for heliotropic response is seen to decrease progressively from red 
light waves (those with low frequency) through yellow, green, blue, violet, 
and indigo (those with high frequency). (After Parr.) 



134 EFFECTS OF RADIATION ON FUNGI 

the subsporangial swelling is an ocellus which acts as a lens. 
When the fruiting structures are in heliotropic equilibrium, the 
light is focussed on a red perforate septum at the base of each 
subsporangium. The red color is imparted by a carotinoid pig- 
ment. The sporangium itself is black and hence casts a shadow 
on some part of the subsporangium beneath. According to Buller 
(1934), the sequence of events is somewhat as follows: Light 
strikes the upper end of the fruiting body, and the incident rays 
enter the part of the upper wall of the subsporangium that bulges 
around the sporangium. They are focussed on the wall below, 
where a region of greater photochemical activity is thereby pro- 
duced. The stimulus is thus transmitted to the motor region of 
the stipe (stalk of sporangiophore), and in response differential 
growth occurs. .Most rapid growth, as has been discussed in 
Phy corny ces mteiis, occurs on the side of the stipe nearest the 
area where the light is focussed. As a consequence the ocellar 
mechanism is tilted until the rays fall symmetrically upon the 
red perforate ring at the base of the subsporangium. In this posi- 
tion a state of physiological equilibrium becomes established, and 
the sporangium is directed head on toward the light. Some ap- 
preciation of the rate of response may be gained from Buller's 
observation on P. longipes, in which he found the stipe capable of 
turning through an angle of 90° and of completely orienting the 
sporangium in about an hour. 

The discharge of the sporangium is also the result of photic 
effects. When the rays are properly centered, the photochemical 
reactions on the protoplasmic content of the subsporangium re- 
sult in increased osmotic pressure. Eventually the pressure is 
sufficient to separate the subsporangial and sporangial walls, the 
rupture beginning as a collar around the periphery of their zone 
of contact. When this release of tension occurs, the subsporangial 
wall, being weakest beneath the sporangium, bursts. The spo- 
rangium is thus carried away by a squirting process. 

Among the striking observations on Pilobolus made by Allen 
and Jolivette (1914), as has been mentioned, was that, if two 
equal beams of white light are converged upon the fruiting bodies, 
with an angle of convergence greater than 10°, the sporangio- 
phores direct the sporangia toward one or the other of the two 
sources of light. Contrary to expectations, the aim of the spo- 
rangiophores is therefore not in the direction of the resultant of 



PHOTOTROPISM 13$ 

these two forces. The explanation for this response occurs in 
the accounts of Pringsheim and Czurda (1927) and van der Wey 
(1929) and has been confirmed by Buller (1934). All are in 
accord that this response may be explained by these assumptions: 
(1) the subsporangium acts as an ocellus, and (2) the red annular 
area in the base of the subsporangium is the region for light per- 
ception. Then, when two spots of light become focussed along 
the basal wall, the one nearest the annular area gives the greater 
stimulus to the motor region of the stipe. In consequence the 
stipe bends, and when the nearest spot comes to rest directly on 
the annular area, heliotropic equilibrium becomes established. 

Among Ascomycetes. The position of the fruiting bodies of 
many Discomycetes and Pyrenomycetes within or on the sub- 
stratum and the orientation of their asci may be presumed to be 
governed by phototropism or by geotropism. Some of these 
fungi, for example, subterranean species, may be wholly unaf- 
fected. In fact, little is known about the specific effect of either 
of these tropic forces on Ascomycetes. This subject constitutes 
a fertile field for study, especially in connection with leaf-inhabit- 
ing species. 

As long ago as 1890 the tips of the asci of Ascobohis demidatits 
were known to bend phototropically. The meager studies sub- 
sequently made on phototropic responses among disk fungi are 
assembled and interpreted by Buller (1934). Discomycetes pos- 
sess hymenia that are plane, concave, or convex. If the hymenia 
are plane, no structural adaptations are required to enable the 
ascospores to be discharged without striking some part of the 
hymenial surface. In certain species with concave or convex 
hymenia it has been shown that the ends of the asci may be 
curved phototropically, or else the opercula of the asci may be 
asymmetrically situated near the apices. By this means ascospores 
are ejected into the environment and do not lodge on the oppos- 
ing walls of the fruit bodies. 

In Ascobohis magnificiis and A. stercorarius [Buller (1934)] 
the tip of the ascus protrudes above the hymenium. This tip, 
containing the ascospores, curves toward the source of light, and 
eventually the ascospores are discharged toward the light. In 
C Maria (Lachnea) scutellata, which normally is plane and nor- 
mally possesses straight asci, curvature may be induced by uni- 
lateral illumination. In Aleuria vesiculosa both asci and pa- 



136 



EFFECTS OF RADIATION ON FUNGI 



raphyses are positively phototropic. Since its hymenial surface is 
hemispherical, the amount of bending of the ascus tip is related 
to the position of the ascus. Asci near the center are straight, 
whereas those near the periphery may be bent through an anp;le 
of 45°. In Morchella cornea, M. crassipes, and Ptyc hover pa 
(Verpa) bohem'ica the fertile portions of the fruit bodies may be 
regarded as compound disks. The asci are phototropic and be- 
have as though each alveolus were a disk. The stipes of some of 




12 M. 6 P.M. 



12 P.M. 



6 A.M. 



12 M. 



6 P.M. 



Fig. 19. Diurnal cycle of development of asci by Tctphrina deformans. 

(After Yarwood.) 

these stalked species bend in response to light, thus carrying the 
fertile tissues into the position most favorable for ascospore dis- 
charge and dissemination. 

A diurnal rhythm in the discharge of ascospores is known to 
exist in certain species. Ingold (1939) observed that Hy poxy Ion 
jusciim discharges its spores nightly during the approximate pe- 
riod between 9 p.m. and 5 a.m. In Nectria cimiabarina and 
Podospora curvula, however, ascospore discharge occurs in the 
daytime. 

By direct microscopic examination and by use of spore traps, 
Yarwood (1941) found that in Taphrina deformans the ascoge- 
nous cells give rise to asci in the evening and that nuclear division 
and increase in size of asci occur throughout the night. During 
the following daylight period the ascospores become morpho- 
logically mature, and maximum discharge occurs during the early 
portion of the succeeding night. This rhythm is attributed to 
alternating light and darkness, but the significant effect of light 



LUMINESCENCE 



131 



is not understood. In T. deformans discharge at night appears to 
be an adaptation favoring infection. 

The perithecial beaks of Neurospora sitophila have been shown 
[Backus (1937)] to be positively phototropic and to discharge 
their spores toward the light. Observations indicate also that the 
beaks of such rostrate fungi as Gnomonia, Ceratostomella, and 
Diaporthe are positively phototropic. The phototropic responses 
of such genera as Linospora and Ophiodothella, whose perithecia 



May 18 



May 19 



May 20 



May 21 




s 


3 


3 


3 


3 


3 3 3 


3 


3 


3 


3 


3 


co 

r-i 


Pu 


Ph 

9 


< 

to 


CO 
r-i 


CU Ph < 

CO CO <£, 

Intervals of time 


r-i 


Ph 


Ph 
CO 

T-i 


< 

CO 


CO 

T-I 



Fig. 20. Period of ascospore output from a stroma of Hypoxylon jus cum 
at a temperature within the range 17 to 19.5° C. (After Ingold.) 

and asci lie horizontally and whose beaks stand vertically, should 
constitute an interesting subject of study. 



LUMINESCENCE 

Nearly everyone has noted that old stumps, decaying logs, and 
leaf mold may emit a weird glow at night. This phenomenon, 
which has been called "fox fire," is usually caused by luminous 
fungi, most of which are Hymenomycetes. Various other or- 
ganisms, including species of bacteria, flagellates, sponges, jelly- 
fish, hydroids, bryozoans, marine worms, earthworms, Crustacea, 
myriapods, insects, molluscs, squids, and fish, are known to emit 
light. An informative treatment of this general subject is given 
in Harvey's (1940) Living Light. Buller (1924, 1934) studied 
luminescence in Paints stipticns and Omphaiia flavida, and his 
account of this phenomenon among fungi will be found very 
stimulating;. 

According to Buller's list (1924), the pilei of the following 
species are luminous: Clitocybe illndens, Panns incctndescens, P. 



138 EFFECTS OF RADIATION ON FUNGI 

stipticus, Pleurotus incandescens y P. facifer, P. gardneri, P. igneus, 
P. noctilucens, P. olearius, P. phosphor eus, P. prometheus. The 
rhizomorphs of Armillaria mellea and the sclerotia of Colly bia 
tuberosa and C. cirrhata are luminescent. Among other species 
claimed to be luminous are Fomes annosus, Poly poms sulphur ens, 
Pomes piui, Colly bia longipes, Corticium coerideum, Dictyophora 
phalloides, and Xylaria hypoxylon. 

The mycelium and pilei of Pamts stipticus in North America 
are luminous, whereas those in England are non-luminous [Buller 
(1924)]. Dried fruit bodies do not glow, but, if revived by 
moistening, they again emit light. Not only moisture but also 
favorable temperature relations are necessary for luminescence. 
The minimum temperature for P. stipticus is —2° to — 4° C, the 
maximum 35° to 37° C, and the optimum 10° to 25° C. 

Oviphalia flavida is of special interest because it has long been 
known to be the cause of a serious leaf disease of the coffee tree 
in the American tropics. Buller (1934) discovered that the lesions 
induced by O. flavida are luminous and reported that they may 
be seen in the dark at a distance of 6 to 10 ft. In culture the 
mycelium too is luminous. This organism is capable of forming 
peculiar structures called gemmifers and gemmae if the stimulus 
of alternating day and night is provided. 

Luminosity in these fungi, as in other luminous organisms, is 
the result of an oxidative change within the cells, luciferin bein^ 
acted upon by the enzyme luciferase in the presence of oxygen. 
Apparently this enzyme has not been extracted from fungi, nor 
has it been demonstrated to be capable of functioning apart from 
living cells. 

INHIBITORY EFFECTS 

Exposure of fungi to sunlight has long been known to modify 
their rate of growth. If the total illumination is small, growth 
may be retarded, but with larger amounts death may ensue. This 
matter appears to have been given rather extensive study, as Smith 
(1936) has indicated. 

It would be anticipated that not all species of fungi are equally 
sensitive to sunlight. Abundant evidence in support of this con- 
clusion from comparative studies is not available. Fromme (1915) 
noted that the germ tubes of urediniospores of Puccima rhamm 
are negatively phototropic and mentioned that a similar reaction 



INHIBITORY EFFECTS 



139 



for those of P. dispersa had earlier been recorded. The germ 
tubes of several other fungi, cited in his report, are indifferent in 
their reaction to light. It would seem that the restriction of some 
pathogens to the lower leaf surface instead of both leaf surfaces 
may be, in part at least, a light response. For example, the downy 



35 



30 



25 



o 
to 

-2 
c 

<D 

a 

CO 

c 

-2 



20 



15 



10 



High intensity- 




o 


o 


O 


o 


o 


8 


o 


o 


O 


(i 


o 


05 


o 


U3 


^J 


iS 


in 


"t 


CT> 


CO 


00 


OJ 


1-H 






1-H 


T-H 


CM 


CO 



Dosage (roentgen units) 

Fig. 21. Effect of dosage on mutation rate at low intensity (240 r per 
minute) and at high intensity (5400 r per minute). (From Sansome, Deme- 

rec, and Hollaender.) 

mildews fructify quite commonly on unexposed surfaces, whereas 
powdery mildews behave in this respect as though quite indiffer- 
ent to sunlight. 

Numerous studies have been made to determine which portions 
of the spectrum are most injurious or possibly lethal. As might 
be anticipated, it has been found that radiations with shortest 
wavelengths are most effective in retarding growth. It has been 
noted furthermore that the ultraviolet regions are more effective 
than the blue, but in many of these studies intensities are not 
measured, and consequently the results cannot be satisfactorily 



140 EFFECTS OF RADIATION ON FUNGI 

evaluated. The methods employed by Oster (1934) in his study 
of Sac char omyces cerevisiae appear to be well suited to similar 
studies of other fungi. He used monochromatic light and found 
that inhibition of colony size could be obtained at a low energy 
level. Under such conditions few new buds were formed, giant 
cells were sometimes produced, and the metabolic functions were 
retarded, as was shown by lowered 2 consumption. At a wave- 
length of 2652 A, 457 ergs/mm 2 were required to kill 50% of the 
cells, but at 3022 A, 23,500 ergs/mm 2 were necessarv. The shape 
of the curves for lethal action at different wavelengths suggests 
that more factors than single quantum hits on a sensitive region, 
that is, adsorption of energy by the nucleoproteins, are responsible 
for these effects. Oster suggests that the age of the cells at the 
time of exposure is also a factor in the energy relations involved. 
The effect of temperature must always be taken into considera- 
tion in experiments of this type. 

Ultraviolet radiations of wavelengths between 2537 and 2650 A 
were found most effective in killing Trichophyton mentagroph- 
ytes [Hollaender and Emmons (1939)]. They suspended the 
spores in physiological salt solution, using wavelengths in the 
range 2280 to 2950 A in measured quantities. In a subsequent 
report these investigators [Emmons and Hollaender (1939) | cor- 
related time of exposure of spores of this same species to mono- 
chromatic light of 2650 A with energy required to cause death 
and with percentage survival. Their experimental data on these 
points are contained in Table 10. 





TABLE 10 




ect of Ultraviolet Light on Trichoph 


tton tnentagrof 


Duration of 


Energy {ergs per 


Survival 


Exposure 


spore in ten 


Ratio 


{minutes) 


thousandths) 


{percentage) 


5 


7.25 


81.0 


15.5 


22.7 


53.0 


34.0 


50 '.2 


42.5 


53 


78.7 


16.4 


78 


116.7 


7.7 


101 


151.7 


3.93 


132 


200.0 


1.03 


162 


247.4 


0.61 


198 


304.4 


0.24 


280 


436.4 


0.153 



INHIBITORY EFFECTS 



141 



Dimond and Duggar (1941) determined the lethal effects of 
monochromatic ultraviolet radiations 2650 A in wavelength on 
Aspergillus melleus, Rhizopas minus, and Mncor disperses. They 
correlated ergs of energy required with volume of the spores, 
using the volume of A. melleus as unity. Their data, which are 
presented in Table 11, indicate that resistance is not directly cor- 

TABLE 11 
Lethal Effect of Ultraviolet Radiations on Three Species of Fungi 



Species 


Ergs per Spore for 
50% Killing 


Ratio 


Mean Volume 
of Spore, /x 3 


Ratio 


Aspergillus melleus 
Rhizopus suinus 
Mucor dispersus 


0.0064 

0.088 

0.12 


1.0 

13.7 

17.5 


8 

28 

113 


1.0 

3.4 

14.1 



related with the volume of the spore. Pigmentation of spores and 
differences in the number of spore nuclei are employed as addi- 
tional factors in accounting for differences in the action of radia- 
tions. 

Recently Sharp (1938) made certain refinements in methods of 
studying the effect of ultraviolet light on bacteria that would 
appear to be adaptable for use with fungi. In attempts to elimi- 
nate the shielding or screening effects of masses of bacteria and 
of the medium he atomized broth cultures into the air, passed the 
bacteria-laden air through a tube where thev were exposed to 
monochromatic ultraviolet light, and then captured the treated 
bacteria at the exit on culture media. It should be possible to 
substitute suspensions of spores in water for broth cultures of 
bacteria in such an apparatus. 

Several investigators have been concerned with the use of ultra- 
violet rays as a potential fungicide. Fulton and Coblentz (1929) 
tested a group of pathogens by use of a 1 10-volt quartz lamp with 
a mercury cathode and a tungsten anode operated on 320 watts 
(80 volts, 4 amp). The spores of all organisms were at the sur- 
face of the agar plates. The investigators eliminated temperature 
effects and found that the following survived exposure for one 
minute: Helminthosporium sp., Alternaria sp., Cladosporium sp., 




142 



STIMULATORY EFFECTS 143 

Pestalozzia sp., Chaetomella sp. (all from cranberry), Diplodia sp. 
(from lime fruit), Aspergillus niger, Penicillium italicum, Col- 
letotrichum gloeosporioides, and Ceuthospora limitata. The fol- 
lowing species, however, were killed by this treatment: Rhizopus 
nigricans, Penicillium digitatum, P. expansum, Phomopsis citri, 
Glomerella rufo-maculans, Gloeosporium limetticolum, Antho- 
stromella destruens, Ac author hynchus vaccinii, Gloeosporium 
minus, Alelanconium sp. (from grape), Fusarium sp. (from 
orange), Botrytis sp. (from apple), Phytophthora sp. (from 
orange), and Guignardia sp. (from cranberry). Similar studies 
by Landen (1939) were concerned primarily with attempts to 
destroy the viability of chlamydospores and sporidia of Ustilago 
zeae. Employing a large crystal-quartz monochromator, he found 
that sporidia are more sensitive than chlamydospores. Long ul- 
traviolet rays between 3022 and 3 130 A required a dosage of 
1.5 X 10° ergs/mm 2 to be lethal. Dillon-Weston and Hainan 
(1930) irradiated cultures of several species, including Rhizopus 
nigricans, Dematium pulhdans, Neurospora sitophila, and Sclero- 
tinia trifoliorum. They employed daily exposures with low in- 
tensities and therewith merely modified the rate of growth. 

Germination of the urediniospores of Puccinia graminis tritici 
was inhibited if they were floated on the surface of water during 
exposure to sunlight, but if they were placed in the dark under 
otherwise similar conditions, they germinated readily [Dillon- 
Weston (1931)]. Similar results followed if he exposed them 
to a mercury-vapor lamp for ultraviolet radiations. 

STIMULATORY EFFECTS 

The evidence on stimulation of fungi by ultraviolet light is 
contradictory. The results of studies by a number of workers 
indicate that exposure to such light is followed by an increased 
growth rate. Using Fusarium eumartii, Smith (1935) found that 
irradiated cultures were at first retarded, but that the rate of 
growth was increased after the period of retardation. In such 
cultures the total growth was never greater than that in the con- 
trols. Since temperature and the accumulation of labile nutritive 
products also favored an increased rate of growth, Smith regarded 
stimulation as an indirect effect of radiation. This interpretation 
is not in accord with the results of Hutchinson and Newton 



144 EFFECTS OF RADIATION ON FUNGI 

(1930), who obtained most stimulation in slow-growing cultures 
of yeast. They also conclude that some wavelengths result in 
stimulation, others in retardation, of growth. Since they did not 
take into consideration differences in total energy in the different 
wavelengths that they used, however, their conclusions cannot be 
accepted with finality. 

EFFECT ON SPORULATION 

Among other effects of ultraviolet irradiation is modification in 
spore production. Ramsey and Bailey (1930), using a quartz- 
mercury-vapor arc with filters to screen out radiations below a 
certain wavelength, and exposing for 15 to 30 minutes at a dis- 
tance of 60 cm, found the greatest production of conidia by 
Macrosporhmi tomato and Fusarium cepae within the range 2535 
to 2800 A. Radiations within this range could also be used to 
inhibit the growth of these organisms or to kill them. Their evi- 
dence indicates that increased sporulation was not the result of 
increased temperature nor modification of the medium, as was 
suggested by Smith ( 1935) from her studies of F. eumart'u. When 
the filters employed by Ramsey and Bailey permitted the trans- 
mission only of radiations of wavelengths greater than 3120 A, 
there was slight stimulation in spore production. Radiations of 
wavelengths greater than 3334 A, however, were without appre- 
ciable effect in this respect. They also noted that the minimum 
duration of exposure for stimulation was 30 seconds and that sev- 
eral exposures at short intervals were more effective than a single 
exposure equal in duration to the sum of the several short ex- 
posures. When irradiated, their cultures of F. coeruleum formed 
conidia, whereas this strain was never observed to do so in non- 
irradiated cultures. On the other hand, their cultures of F. argil- 
laceum, when irradiated, failed to form conidia, producing only 
chlamydospores. 

Stevens (1928, 1930, 1931) exposed Glomerella crngulata and a 
species of Coniothvrium to a Cooper-Hewctt quartz-mercury- 
vapor arc operated at 4.5 amp and 66 volts. The agar plates were 
uncovered during exposure at a distance of 21 cm from the source 
of light. With exposures at less than 1 min, perithecia were 
formed in abundance by G. cingulata and pvcnidia by Coniothv- 
rium. In both species these structures normally appeared on the 



INDUCTION OF SALTATIONS 145 

same medium but were always sparse. Stevens (1931) induced 
Colletotrichum lagenarium to form the perithecial stage in cul- 
ture, whereas this stage had never been observed previously under 
any conditions. Although he did not regard temperature as a 
significant factor, he noted that the presence in the medium of 
such sugars as favor growth also favors increased spore produc- 
tion after irradiation. 

There is evidence that ultraviolet radiation may hasten sporula- 
tion [Hutchinson and Ashton (1930)]. Short exposures induced 
Colletotrichum phomoides to sporulate earlier, and long exposures 
delayed sporulation. Hutchinson and Ashton concluded that 
within certain limits the time of sporulation is an inverse expres- 
sion of the rate of growth. 

EFFECT OF X-RAYS 

Both ultraviolet and X-rays have been used as therapeutic 
agents, especially in dermatomycosis and actinomycosis. The 
medical aspects of their use appear to be better known than their 
general effects on fungi. The evidence concerning X-rays indi- 
cates that fungi are rather insensitive to their action but that large 
dosages are lethal. Haskins and Moore (1934) found that soft 
X-rays were 2.1 times as potent in killing conidia of Penicillium 
as were hard X-rays. The soft X-rays used by them had a wave- 
length of 1.3 to 1.5 A; the hard ones, from 0.18 to 0.21 A. Lethal 
action of X-rays against plant pathogens was earlier reported by 
Pichler and Wober (1922), who successfully freed wheat seed 
from Ustilago tritici, barley seed from U. mida, and potato 
tubers from Synchytrhnn (Chrysophlyctis) endobioticam. 

Nadson and Philippov (1925) suppressed the formation of 
zygotes in Mucor genevensis and Zygorhyn chits moelleri by 
exposure of cultures to X-rays. Marked changes in protoplasmic 
structure resulted in Sac char omyces cerevisiae and Nadsonia ful- 
vescens after exposure to X-rays [Nadson (1937)]. 

INDUCTION OF SALTATIONS 

None of the effects of radiation which have thus far been 
given consideration in the present account can be regarded as 
mutations, for evidence is lacking that they are heritable. Radia- 



146 EFFECTS OF RADIATION ON FUNGI 

tions of short wavelengths, however, have been used to produce 
heritable mutations with many biological materials, as is well 
known, and a voluminous literature on this subject exists. Rela- 
tively few studies have been made of induction of heritable mu- 
tations in fungi. Dickson (1932, 1933) exposed malt-agar cul- 
tures of Mucor gevevensis, Phy corny ces blakesleeamts, and the 
ascospores of seven species of Chaetomium to X-rays for 50 min- 
utes at a distance of 26 cm. Changes in color and amount of 
mycelium were induced in colonies arising as subcultures from the 
irradiated materials, and these changes were manifest by sector- 
ing. Stevens (1930) obtained sectoring in cultures of Glomerella 
c'nigulata exposed to ultraviolet radiation. Greaney and Machacek 
(1933) exposed cultures of Hehninthosporhnn sativum to a mer- 
cury-arc lamp (110 volts, 60 cycles) for 4 minutes on each of 3 
successive days. During exposure the cultures were placed at a 
distance of 35 cm from the arc. As a result of this treatment a 
saltant having hyaline mycelium and almost colorless conidia ap- 
peared. In all these cases the saltants remained constant in sub- 
cultures through succeeding- generations. 

Lockwood and associates (1945) irradiated 217 isolates of Asper- 
gillus t err ens with, ultraviolet rays, and as a consequence 141 were 
changed morphologically. Among the 76 that were unchanged 
morphologically, 59 were found capable of producing more ita- 
conic acid than the parent strains. None of the 141 strains that 
were altered morphologically, on the other hand, was found cap- 
able of this increased production of itaconic acid. 

Emmons and Hollaender (1939) irradiated the dermatophyte, 
Trichophyton mentagrophytes, and thereby induced the produc- 
tion of mutants. This organism lacks a sexual stage, and conse- 
quently the investigators were unable to make a genetical analy- 
sis of the mutants. By use of Neurospora cr asset [Sansome et al. 
(1945)], however, it was found that two types of mutants could 
be induced, one of which was caused by chromosomal aberration. 
They varied dosage and intensity and noted that increase in in- 
tensity resulted in increase in mutation rate. At low dosage there 
was a straight-line relationship between increase in mutation rate 
and increase in energy. 



. IMPLICATIONS 141 

MODE OF ACTION OF SHORT RADIATIONS 

When the percentage of survivors is plotted against the total 
energy used to kill yeast, typical S-shaped curves are secured from 
the data of WyckofT and Luyet (1931) and Oster (1934). Several 
different explanations of why curves of this type should be ob- 
tained have been offered. Some workers regard them merely as 
expressions of normal probability of survival of the individuals. 
Others attribute the form of the curve to multiple quantum hits 
on a sensitive region of the cell, presumably on the nucleus or cer- 
tain of its constituent elements. A single hit is regarded as the 
adsorption, by the sensitive region, of 1 quantum. The adherents 
of the multiple-quantum theory, knowing the amount of energy 
and the survival percentage, calculate the number of hits required 
to kill. Needless to say there is little accord in observations on the 
number of quanta required. The significance of this fact is not 
clear, but the situation might be clarified if the influence of age, 
nutrition, acidity, temperature, and such factors was taken into 
consideration. In conclusion, it is apparent that many phenomena 
attributed to the action of radiation are not caused by light alone 
but are correlated in a causal relationship with other factors. 

IMPLICATIONS 

It appears that the present-day mycologist and physicist, each 
in his own field, can do little more to extend knowledge of the ef- 
fects of radiations on fungi. Conceivably they might achieve re- 
sults were they to collaborate. In lieu of such collaboration, ad- 
vances in knowledge will be conditional upon the presence of 
workers who may properly be termed bio-physicists. This name 
connotes possession of basic training in both biology and physics 
and, what is more important, a consuming zeal to apply this 
training to explorations leading to the furtherance and dissemina- 
tion of knowledge in mycology. Such "myco-physicists" should 
be able to correct or clarify many of the contradictory conclu- 
sions and concepts now extant. 



148 EFFECTS OF RADIATION ON FUNGI 



LITERATURE CITED 

Allen, Ruth F., and H. D. Jolivette, "A study of the light reactions of 

Philobolus," Trans. Wis. Acad. Aci., 77:533-598, 1914.^ 
Backus, M. P., "Phototxopic response of perithecial necks in Neurospora," 

My col., 29: 383-386, 1937. 
Bisby, G. R., "Zonation in cultures of Fusariwn discolor snlphureimi," 

Mycol., 11: 89-97, 1925. 
Blaauw, A. H., "Licht und Wachstum," Z. Botan., 6: 641-703, 1914. 
Brown, W., "Studies in the genus Fusarium. II. An analysis of factors which 

determine the growth forms of certain strains," Ann. Botany, 39: 375- 

408, 1925. 
Buder, J., "Die Inversion des Phototropism bei Phvcomyces," Ber. dent. 

botan. Ges., 36: 104-105, 1918. 
Buller, A. H. R., "The reactions of the fruit bodies of Lentinus lepideus to 

external stimuli," Ann. Botany, 19: 427^38, 1905. 
"The biologv of Polyporus squamosus, a timber-destroving fungus," 

/. Econ. Biol, 1: 101-138, 1906. 
Researches on fungi. Vol. 1: pp. 47-78, 120-121, 1909; vol. 3: 357-411, 

1924; Vol. 6: 36-45, 90-130, 264-324, 397-454, 1934. Longmans, Green, 

London. 
Castle, E. S., "The phvsical basis of the positive phototropism of Phy- 

comyces," /. Gen. Physiol., 17:49-62, 1933. 
Dickson, Hugh, "The effect of x-rays, ultra-violet light, and heat in pro- 
ducing saltants in Chaetomiwn cochliodes and other fungi," Ann. 

Botany, 46: 389-404, 1932. 
"Saltation induced bv x-ravs in seven species of Chaetomium," Ann. 

Botany, 41: 735-754," 1933. ' 
Dillon-Weston, W. A. R., "Effect of light on urediniospores of the black- 
stem rust of wheat, Pnccina graminis tritici" Nature, 128:67-68, 1931. 
Dillon-Weston, W. A. R., and E. T. Halnan, "The fungicidal action of 

ultra-violet radiation," Phytopathology, 20: 959-965, 1930. 
Dimond, Albert, and B. M. Duggar, "Some lethal effects of ultra-violet 

radiation on fungus spores," Proc. Nat. Acad. Sci., 27:459-468, 1941. 
Emmons, C. W., and Hollaender, A., "The action of ultraviolet radiation 

on dermatophytes. II. Mutations induced in cultures of dermatophytes 

by exposure of spores to monochromatic ultraviolet radiation," Am. 

]'. Botany., 26:467-475, 1939. 
Fro.mme, F. D., "Negative heliotropism of urediniospore germ tubes," 

Am. J. Botany, 2:82-85, 1915. 
Fulton, H. R., and W. W. Coblentz, "The fungicidal action of ultra-violet 

radiation," /. Agr. Research, 38: 159-168, 1929. 
Gri \m y, F. J., and J. E. Machacek, "The production of a white fertile 

saltant of H ehnimthosporiwn sativum by means of ultra-violet radia- 
tion," Phytopathology, 25:379-383, 1933. 
Harvey, E. Newton, Living light. 328 pp. Princeton University Press. 

1940. (Cf. pp. 37-42.) 



LITERATURE CITED 149 

Haskins, C. P., and C. N. Moore, "The inhibition of growth in pollen and 
mold under x-ray and cathode-ray exposure," Radiology, 25:710-719, 
1934. 

Hedgcock, G. G., "Zonation in artificial culture of Cephalothecium and 
other fungi," Mo. Botan. Garden, Ann. Rept., 1906: 115-117, 1906. 

Hollaender, A., and C. W. Emmons, "The action of ultraviolet radiation 
on dermatophytes. I. The fungicidal effect of monochromatic ultra- 
violet radiation on the spores of. Trichophyton mentagrophytes" J. 
Cellular Comp. Physiol., IS: 391-402, 1939. 

Hutchinson, A. H., and M. R. Ashton, "The effect of radiant energy in 
growth and sporulation in Colletotrichinn phomoides" Can. J. Re- 
search, 3: 187-198, 1930. 

Hutchinson, A. H., and D. Newton, "The specific effects of monochro- 
matic light on the growth of yeast," Can. J. Research, 2: 249-263, 1930. 

Ingold, C. T., Spore discharge in land plants. 178 pp. Clarendon Press, 
Oxford. 1939. 

Landen, E. W., "Spectral sensitivity of spores and sporidia of Ustilago 
zeae to monochromatic ultra-violet light," /. Cellular Comp. Physiol., 
14:217-226, 1939. 

Lockwood, L. B., K. B. Raper, A. J. Mover, and R. D. Coghill, "The 
production and characterization of ultraviolet-induced mutations in 
Aspergillus terreus. III. Biochemical characteristics of the mutations," 
Am. J. Botany, 52:214-217, 1945. 

Long, W. H., and R. M. Harsch, "Cultures of wood-rotting fungi on arti- 
ficial media," /. Agr. Research, 72:33-82, 1918. 

Moreau, F., "Sur les zones concentriques que forment dans la cultures les 
spores de Penicillium glaucum" Bull. soc. hot. Trance, 59: 491-495, 1912. 

Nadson, G. A., "De certaines irregularities des changements de la 'matiere 
vivante' sans l'influence des facteurs externes, principalement des ravons 
X et du radium," Actualites Sci. hid., 513: 1-26, 1937. 

Nadson, G. A., and G. Philippov, "Influence des rayons X sur la sexualite 
et la formation des mutantes chez les champignons inferieurs (Muco- 
rinees)," Compt. rend. soc. biol., 93:Ml-M5, 1925. 

Oster, R. H., "Results of irradiating Saccharomyces with monochromatic 
ultra-violet light," /. Gen. Physiol., 18: 71-88, 1934. 

Parr, Rosalie, "The response of Pilobolus to light," Ann. Botany, 32: 177- 
205, 1918. 

Pichler, F., and A. Wober, "Bestrahlungsversuche mit ultraviolettem Licht, 
Rontgenstrahlen, und Radium zur Bekampfung von Pflanzenkrankhei- 
ten," Zentr. Bakt., Parasitenk., II Abt., 57: 319-327, 1922. 

Pringsheim, E. G., and V. Czurda, "Phototropische und ballistiche Prob- 
leme bei Pilobolus," Jahrb. iviss. Botan., 66: 869-872, 1927. 

Ramsey, G. B., and A. A. Bailey, "Effects of ultra-violet radiation on 
sporulation in Macrosporium and Fusarium," Botan. Gaz., 89: 113-136, 
1930. 

Reideaieister, W., "Die Bedingungen der Sklerotien und Sklerotienringbil- 
dung von Botrytis cinerea auf kunstlichen Nahrboden," Ann. Mycol., 
7: 19-44, 1909. 



150 EFFECTS OF RADIATION ON FUNGI 

San some, E. R., A I. Demerec, and A. Hollaender, "Quantitative irradia- 
tion experiments with Neurospora crassa. I. Experiments with X-ravs," 
Am. J. Botany, 32:218-226, 1945. 

Sharp, D. G., "A quantitative method of determining the lethal effect of 
ultra-violet light on bacteria suspended in air," /. Bact., 35: 589-599, 1938. 

Smith, Elizabeth C, "Effects of ultra-violet radiation and temperature on 
Fusarium. II. Stimulation," Bull. Torrey Botan. Club, 62: 151-164, 1935. 
"Effects of radiation on fungi." In Biological effects of radiation, II: 
889-918, 1936. 

Stevens, F. L., "Effect of ultra-violet radiation on various fungi," Botan. 
Gaz., 86:210-225, 1928. 
"The response to ultra-violet irradiation shown bv various races of 

Glomerella cingulata" Am. J. Botany, 77:870-881, 1930. 
"The ascigerous stage of Colletotrichum lagenarium induced by ultra- 
violet irradiation," My col., 23: 134-139, 1931. 

\Ye\, H. G. van der, "Uber die phototropische Reaction von Pilobolus," 
Proc. konink. Akad. Wetenschappen Amsterdam, 32:4-13, 1929. 

Wolf, Frederick A., "Fungal flora of Yucatan caves," Carnegie Inst. Wash- 
ington Pub., 491: 19-2 1" 1938. 

AYyckoff, R. W. G., and B. F. Luyet, "The effects of x-ravs, cathode, and 
ultra-violet rays on yeast," Radiology, 11: 1171-1175, 1931. 

Yarwood, C. E., "Diurnal cycle of ascus maturation of Taphrina deformans," 
Am. J. Botany, 2^:355-357, 1941. 



Chapter 7 
EFFECTS OF REACTION OF SUBSTRATE ON FUNGI 

Amono- the chemical environmental influences to which fungn 
are generally known to respond is the reaction of the substrate. 
Long ago the theory was advanced that the chemical activities of 
acids, bases, and salts may be attributed chiefly to the ionized 
portions. Abundant experience has shown that fungi are more 
tolerant of acid ions [H + ] than of basic ions [OH - ]. If, for 
example, it becomes necessary to separate mixed cultures of fungi 
and bacteria, the growth of bacteria may be inhibited by the 
addition of lactic acid in the proportion of 1 drop of 50% lactic 
acid to 10 ml of agar in making the poured plates that are to be 
planted with the mixed cultures. 

Many fundamental facts regarding the effects of the ionized 
portions were established before the differences between total 
acidity (titratable acidity) and active acidity (hydrogen-ion-con- 
centration) were appreciated. The work of Clark (1899) is not- 
able in this connection. He studied the effects of the concentra- 
tion of a variety of mineral and organic acids upon the germina- 
tion of spores and mycelial development of Sterigmatocystis 
nigra, Oedocephahim albidzmi, Pemcillium glaiicwn, and Botrytis 
cinerea. As a result he found that the OH~ group is rather 
more toxic to all species than the H+ ions and that molds differ 
specifically in tolerance. Furthermore, to inhibit the germina- 
tion of these molds, a concentration of the mineral acids 200 to 
400 times that fatal to higher plants is required. 

Subsequently the classical studies of Michaelis and S0rensen on 
the theory of the hydrogen ion and its measurement laid the 
foundations of present-day knowledge. These matters, an under- 
standing of which is essential to all biologists regardless of their 
special field of interests, are summarized and elucidated in a vol- 
ume by Clark (1928). With the help of this book the student can 
learn the fundamentals of ionization, conductivity, and use of in- 
dicators, at least to a sufficient degree to be able to measure hvdro- 

151 



152 EFFECTS OF REACTION OF SUBSTRATE ON FUNGI 

gen-ion concentration, without mastering the underlying theories 
and principles. 

Meaning of hydrogen-ion concentration. Substances are con- 
ceived to be aggregates of molecules; a molecule is the smallest 
unit of mass that possesses the characteristics of the given sub- 
stance. A molecule is, however, conceived to be an aggregate 
of atoms, which, if all alike, constitute an element. 

Included in atomic structure are positive electric units or 
charges (protons) and negative units (electrons). In each ele- 
ment protons and electrons are arranged according to a definite 
configuration, which is concerned with the behavior of the atoms. 
A complete atom may be deprived of one or more positively 
charged electrons, whereupon the remaining particle is a posi- 
tively charged ion and is designated a cation. Some atoms, how- 
ever, are able to acquire extra electrons, thereby becoming nega- 
tively charged ions which are designated anions. 

The behavior of acids, bases, and salts in water solutions is at- 
tributed to the activities of their constituent ions. If, for example, 
an acid (base or salt) is dissolved in water, its molecules become 
dissociated to a certain amount. The radical of the molecule ac- 
quires an electron from the acid hydrogen atom, becoming a 
negatively charged ion and leaving the nucleus of the hydrogen 
atom positively charged. This hydrogen ion is designated as H + 
to distinguish it from the complete atom H; the remainder of the 
molecule may be symbolized by A - . Dissociation of an acid may 
therefore be formally expressed as follows: [HA] ^± [H + ] + 
[A~ ]. This reaction is shown to be reversible, but if temperature 
is kept constant, equilibrium will eventually become established. 

ThCn [H+] X [A-] = „ 

[HA] ai 

meaning that the product of the number of positively charged 
ions and negatively charged ions, divided by the number of un- 
dissociated molecules, is a constant for each given acid. It follows 
from this concept that if the value of K is large, the numerator 
must be large in proportion to the denominator. Such acids arc 
strong acids. If, on the other hand, the numerator is small in 
proportion to the denominator, the acid is a weak acid. The in- 
tensity of reaction of an acid therefore depends upon the hydro- 
gen-ion concentration. 



SIGNIFICANCE OF THE SYMBOL pH 153 

Measurement of hydrogen-ion concentration. The concen- 
tration of hydrogen ions is expressed numerically in terms of a 
normal solution. A normal hydrogen-ion solution contains 1 
gram of hydrogen ions or the equivalent per liter. Normal solu- 
tions are therefore made up on the basis of molecular weight to 
secure a solution containing 1 gram of hydrogen or the equivalent 
per liter. The dissociation constant of a IN solution of the 
strongest acid, HC1, at 25° C is essentially 1. The dissociation 
constant of the weakest acid, pure water, has been determined to 
be 1/10,000,000 N, which constitutes neutrality. It follows there- 
fore that the dissociation constants of all other acids are fractions 
that range between these extremes. 

The dissociation of pure water at 25° C, if expressed formally, 
would be written 

[H+] X [OH-] _ 
[HOH] ~ Au " 

If*the concentration of hydrogen ions in water is 1/10,000,000 
gram (or 10 ~ 7 ), if expressed logarithmically) and water is neutral, 
then the concentration of hydroxy 1 ions is also 1/10,000,000 gram 
(or 10~ 7 ). The number of molecules of water dissociated is 
so small in comparison with the total number that [HOH] may 
be considered unity and omitted, making the formal equation 
[H+] x [OH-] = K w , or [H+] (10" 7 ) X [OH~] (10~ 7 ) = 
K w (lO- 1 ^). 

Significance of the symbol pH. Since the hydrogen-ion con- 
centration of a solution is, with few exceptions, a fraction of the 

normal, it may be expressed as jtt+i, that is, the reciprocal of 

[H+ ] . By use of the reciprocal the negative exponent is avoided. 
The symbol pH is therefore used to designate the logarithm of the 
reciprocal of the hydrogen-ion concentration. The hydrogen-ion 
concentration of pure water, for example, is 1/1*0,000,000 N. 
Expressed otherwise, 

[H+] = 1 X 10- 7 , 
or 

r+ 



or 



log [H+] = -7, 
- log [H+] = +7, 



or j 

log jjpj = 7, 

or 

pH = 7. 



154 EFFECTS OF REACTION OF SUBSTRATE ON FUNGI 

The pH of a solution is rarely an even decimal fraction of nor- 
mal. For this reason the quantity between two succeeding frac- 
tions may be indicated by a multiplying factor. If, for instance, 
the concentration of ions is 0.000273 N, it may be written 2.73 X 
10 ~ 4 . By the use of the logarithm table, it will be found that the 
logarithm of 2.73 = +0.434 and that of lO" 4 = -4.000. Since 
the logs are added when multiplying, +0.434+ (—4.000) = 

— 3.566. Therefore losr rTT , , = 3.566, or pH = 3.566. 

& [H + J r 

If the actual figure for the hydrogen-ion concentration is sought 
when the pH value is given, it can be determined by the reverse 
of the procedure of calculation just described. Suppose that the 
given pH value is 9.63, which may be expressed thus: [H + = 
1 X 10~ 963 . The exponent —9.63 = — 10 plus + 0.37; or, other- 
wise stated, it equals 10 -10 X 10 + - 37 . The logarithm table shows 
that +0.37 corresponds with the number 2.34. Substitution of 
this number in the original equation, [H+] = 1 X 10 _9G3 , gives 
the identity [H+] = 2.34 X-IO" 10 . 

In acids dissociated in water, the concentration of hydrogen ions 
must be greater than that of the water itself and therefore must 
range between pH 7.0 (the hydrogen-ion concentration of water) 
and pH 0. Likewise, bases dissociated in water have a hydroxyl- 
ion concentration greater than that of water itself, so that this 
concentration can range between pOH 7.0 (the hvdroxvl-ion 
concentration of water) and pOH (the hvdroxvl-ion concen- 
tration of a normal basic solution). Since the acid or base was dis- 
sociated in water, [OH - ] ions are always present in acid solutions 
and [H + ] ions in basic solutions. The concentration of hydrogen 
ions therefore varies inversely as the concentration of hydroxvl 
ions, and vice versa, the product of the concentration of both kinds 
being always 1 X 10 -14 . From this relation it is evident that, if 
the concentration of either ion is known, that of the other can be 
readily computed. Thus if the pH of a solution is 10 ~ 4 , the pOH 
is 10 -10 . From the foregoing discussion it is apparent that any 
pH value between and 7 indicates an acid solution, with decreas- 
ing acidity as the number increases. Similarly, any pH value be- 
t\\ een 7.0 and 14.0 indicates a basic solution with increasing basic- 
ity (decreasing acidity) as the number increases. 

.Measurement of pH. Two methods are employed in measur- 
ing hydrogen-ion concentration, one electrometric, the other 
colorimetric. The electrometric method is the more accurate and 



pH AND GROWTH 



155 



requires the more expensive and elaborate apparatus. The colori- 
metric method yields approximate measurements that can be 
simply and quickly achieved. The electrometric method depends 
upon the ability of solutions of acids, bases, and salts to conduct 
an electrical current, differences being attributable to concentra- 
tion; the colorimetric method involves changes in the colors of 
indicators, each within a given range of pH, and the matchino- 
of colors with standards. 

pH and growth. Hydrogen-ion concentration does not equally 
influence all the vital processes or activities of fungi, as might be 
anticipated. Much of what has been learned about the effects 
of pH and pOH on fungi has come from studies on the influence 
of reaction upon growth rather than upon individual processes, 



TABLE 12 

Range of Hydrogen-Ion Concentration Permitting Growth of Various 

Fungi 



Authority 

Sherwood (1923) 
Herrick (1939) 
Johnson (1923) 
Johnson (1923) 
Johnson (1923) 
Johnson (1923) 
Johnson (1923) 
Johnson (1923) 
Meacham (1918) 
Meacham (1918) 
Meacham (1918) 
Meacham (1918) 
Webb (1919) 
Webb (1919) 
Webb (1919) 
Webb (1919) 
Jackson (1940) 
Jackson (1940) 
Wolpert (1924) 
Wolpert (1924) 
Wolpert (1924) 
Wolpert (1924) 
Wolpert (1924) 
Wolpert (1924) 
Wolpert (1924) 
Wolpert (1924) 



Organism 

Fusarium lycopersici 
St ere um gausapatum 
Mucor glomerula 
Fusarium bullatum 
Aspergillus oryzae 
Aspergillus terricola 
Penicillium i tali cum 
Penicillium var labile 
Lenzites saepiaria 
Fomes roseus 
Merulius lacrymans 
Coniophora cerebella 
Lenzites saepiaria 
Aspergillus niger 
Penicillium cyclopium 
Botrytis cinerea 
Pythium sp. 
Rhizoctonia solani 
Lenzites saepiaria 
Daedalea confragosa 
Polystictus versicolor 
Ar miliaria me lie a 
Pholiota adiposa 
Polyporus adustus 
Pleurotus ostreatus 
Schizophyllum commune 



Range within Which Growth 
Occurred 

2.8 and 8.4 
2.0 and 8.2 
3.2-3.4 and 8.7-9.2 
2.3-2.2 and 9.2-11.2 
1.6-1.8 and 9.0-9.3 
1.6-1.8 and 9.0-9.3 
1.9-2.2 and 9. 1-9.3 
1.6-1.8 and 10.1-11.1 

1.9 (optimum, 3.0) 
1.9 (optimum, 3.0) 
1.0 (optimum, 3.0) 
1.9 (optimum, 3.0) 
Below 2.8 and 7.4 

2.8 and 7.4-8.8 

Below 2.8 and approximately 9.6 
Below 2.8 and 7.4 
2.5-3.5 and 8.5 
2.5 and 7.5-8.5 

3.4 and 7.3 

3.5 and 7.2 
2.5 and 7.6 

2.9 and 7.4 
2.8 and 7.0 
3 . 5 and 7 . 6 
3.0 and 7.5 
3.4 and 7.0 



156 EFFECTS OF REACTION OF SUBSTRATE ON FUNGI 



such as reproduction, respiration, and enzyme production. In 
general, these studies have been concerned with establishing the 
range of pH within which growth can be accomplished. All 
show that optimum growth occurs if the media are acid, and there 
is abundant evidence to indicate that the range of pH that will 
permit growth varies with the species, with the composition and 

100 



75 - 



£ 50 
m 
S 

>> 

S 






£ 25 - 







1 1 1 1 1 1 1 1 1 


1 




Polystictus versicolor^ // """V* yPleurotus ostreatus 




/ 7"-^ \ '■ 

/ ' * N \ • 






— / / * \\ 

Armillaria mellea^J / *\* 




— 


A / / V 






/V '' \ ^ 






/ / / x^^ 




— 


/ / , \ » 












/.' ' "~-/ X >. , » 






— / #' ^ >» « ; 












/ // / ^Schizophyllum commune '\ 
I'll • 






/ // ' ^ 






/ •'/ / v. 


! l\ 




/ /;/ /! i i i l i it 







5 6 

Reaction of medium (pH ) 



8 



Fig. 23. Growth of certain Basidiomycetes in Richards' solution adjusted 
to different initial degrees of aciditv. Cultures maintained at 25° C. (After 

Wolpert.) 

the initial reaction of the culture medium, and with temperature. 
Representative results of the influence of reaction upon growth 
of fungi are assembled in Table 12. 

The most significant feature indicated by Table 1 2 is that essen- 
tially all the region of pH permitting growth lies within the acid 
range. As a secondary feature it is apparent that each species 
may differ in the limits of this range. It should be indicated that 
the optimum pH does not occur at the midpoint. The under- 
lying reasons for these facts seem to rest upon the isoelectric points 
of the constituent proteins of the different species, as shown by 
Robbins (1924). In Rhizopus nigricans and Fiisarinm ly coper sici 



VARIATION OF pH RANGE WITH MEDIA 151 

the isoelectric points of the proteins were shown to be in the 
vicinity of pH 5.0 and 5.5, respectively. When Robbins stained 
the mycelial mat of R. nigricans with an acid dye, it retained the 
dye on the acid side of the isoelectric point, but the mycelium 
was unstained on the basic side. This result was determined by 
washing mycelia stained with eosin, for example, with solutions 
of pH 3.5, 3.9, 4.5, 5.7, 5.8 and 6.9. These solutions were made 
with appropriate mixtures of 0.1 M phosphoric acid and of 0.1 M 
sodium hydroxide. After thorough washing with a buffer solu- 
tion of pH 3.5 or 3.9, the mycelia were bright red; with a solution 
of pH 4.5, intermediate red; with a solution of pH 5.7 or 5.8, 
pink; with a solution of pH 6.9, hyaline. 

Variation of pH range with media. Failure of various work- 
ers to agree on the pH range that will support growth in a ^iven 
fungus may be attributed to differences in culture media. These 
differences involve kind and proportion of nutrients, buffering, 
and initial reaction. The influence of the composite of these 
factors is illustrated bv the work of Wolpert (1924). He em- 
ployed a modified Richards' solution, on the one hand, and a 
2.5% peptone-mineral nutrient, on the other, with the results 
shown in Table 13. 

TABLE 13 

Comparison of pH Range of Certain Basidiomycetes on Two Different 

Media 

pH Range That Inhibits Growth 



Organisms Richards' solution Peptone solution 

Lenzites saepiaria 3 . 4 and 7.3 2.8 and 7 . 5 

Daedalea confragosa 3 . 5 and 7.2 2.8 and 7 . 6 

Polystictus versicolor 2 . 5 and 7.6 2.5 and 7 . 5 

Armillaria mellea 2.9 and 7.4 2.0 and 7.8 

Pholiota adiposa 2 . 8 and 7.0 2.8 and 7 . 8 

Pleurotus ostreatus 3-. and 7.5 3.0 and 8 . 5 

Schizophyllum commune 3.4 and 7.0 2.8 and 8.5 

During growth each species increased the acidity in Richards' 
solution, Lenzites saepiaria being the most active acid-producer. 
All of them except L. saepiaria and Pleiirotns ostreatus decreased 
the acidity in peptone solution when the initial reaction was less 
than pH 6.0 and increased it when the initial reaction was greater 
than pH 6.0. 



158 EFFECTS OF REACTION OF SUBSTRATE ON FUNGI 

As is well-known, temperature modifies the rate of growth, but 
it is also an important factor in modifying the pH range. Tem- 
peratures favorable for optimal development tend to be corre- 
lated with the widest pH range. Wolpert (1924) grew each of 
the species listed in Table 13 at 15° C, 25° C, and 35° C, all other 
factors being identical. Lenzites saepiaria, Pleurotits ostreatus, 
and Arnnllaria mellea have high optimal temperatures, Schizo- 
phyllum commune has a low one, and the remainder have inter- 
mediate optimal temperature requirements. 

The comparative growth of Ophiobohts graminis on Czapek's 
nutrient fortified with cane sugar and on corn-meal decoction led 
Webb and Fellows (1936) to conclude that the nutritional and 
physical nature of the media, irrespective of all other factors, 
greatly modifies the influence exerted by free hydrogen or hy- 
droxvl ions on the growth of fungi. 

pH of fungus tissues. Essentially nothing is known about the 
hydrogen-ion concentration of fungus tissues. Armstrong (1929) 
measured the reaction of the juice of crushed stipes and pilei of 
certain fleshy fungi, with the results shown in Table 14. 

TABLE 14 
Hydrogen-Ion Concentration of Fungus Tissues 



Fungus 


pH 


Agaric us campestris 


Ca. 5.9 


Amanita muse aria 


6.2 


Ar miliaria mellea 


5.6-5.9 


Clavaria rugosa 


Ca. 6.2 


Clavaria corniculatus 


Ca. 6.2 


Clitocybe laccata 


6.2 


Collybia radicata 


5.9 


Coprinus atramentarius 


6.2-6.8 


Coprinus micaceus 


5.6-5.9 


Cortinarius violaceus 


6.2 


Hekella crispa 


6.2 


Hypholoma fasciculare 


Ca. 5.9 


Lactarius blennius 


Ca.S.6 


Leotia chlorocephala 


5.6-6.2 


Mycena pura 


Ca. 5.9 


Mycena vulgare 


Ca. 5.9 


Panus torulosis 


5.6-5.9 


Polystictus versicolor 


5.9 


Typhula incarnata 


Ca. 5.9 



CORRELATION OF REACTION OF THE SOIL 159 

These determinations of pH may not necessarily be those of 
the vacuolar sap, just as the pH of the crushed tissues of green 
plants may not be that of the cell sap. They appear of interest 
in indicating that fungus tissues are acid. Recognition of their 
significance, however, awaits the development of methods for 
determining true pH values of fungus-cell sap. 

pH and pigmentation. The pigments in many species of 
fungi may function as natural indicators that change color with 
a change of reaction. One factor that controls the" development 
of pigment, moreover, is the reaction of the medium. The pres- 
ence of appropriate carbohydrates may also constitute a control- 
ling factor. These relationships with species of Fusarium were 
studied by Sideris (1925). If he employed dextrose solutions and 
made no attempt to control the changes in reaction during growth, 
pigment was produced within the range pH 3.0 to 7.5. If the pH 
was kept constant, pigment was produced only within the rano-e 
3.5 to 5.5. 

pH and enzymic activity. The effect of pH on enzymic ac- 
tivity was mentioned in Chapter 2. Evidently the effect of re- 
action upon individual metabolic processes in fungi has not been 
the subject of much study. Karrer ( 192 1 ) recorded that Fusarium 
sp. from cotton, when grown in nutrient solution, yield the great- 
est total amount of amylase if the initial reaction is pH 4.5 and the 
final reaction is pH 7.8; Collet otrichum gossypii, if the initial re- 
action is pH 7.0 and the final is pH 7.9. For Fenicilliinn italicum 
pH 3.0 and pH 4.5 are equally favorable. Amylase accumulation 
is completely inhibited within the range pH 9.0 to 1 1.0 in Fusarium 
sp. and in C. gossypii, and at pH 9.0 in P. italicum. Further evi- 
dence of the influence of hydrogen-ion concentration upon the 
activity of amylase was presented by Sherman, Thomas, and Bald- 
win (1919). They showed that pancreatic amylase is active within 
the range pH 4.6 to 10, pH 7.0 being optimum; malt amylase 
within the range pH 2.5 to 9.0, pH 4.4 to 4.5 being optimum'; and 
amylase from Aspergillus oryzae within the range pH 2.6 to 8.0, 
pH 4.8 being optimum. 

Correlation of reaction of the soil, optimum pH of the 
pathogen, and incidence of disease. Experimentation involving 
these matters in connection with soil-borne pathogens has en- 
gaged the attention of certain plant pathologists, notably Chupp 



160 EFFECTS OF REACTION OF SUBSTRATE ON FUNGI 

(1928), Maclnnes (1922), Schaffnit and Meyer-Hermann (1930), 
Sherwood (1923), and Sideris (1929). 

Schaffnit and Meyer-Hermann (1930) determined the pH 
optima for growth of a group of soil-borne fungi as a basis for 




5.8 



6.0 



6.2 6.4 6.6 6.8 

Hydrogen -ion concentration 



.0 



7.2 



7.4 



Fig. 24. Relation of hydrogen-ion concentration to infection of cabbage 
by Plasmodiophora brassicae. Infection is inhibited as alkalinity is ap- 
proached. (After Chupp.) 

their field trials. From these results thev concluded that such 
organisms may be grouped as follows: 

1 . Litrophilic: those that prefer alkaline soils, including Pythhnn 
de baryanum, Momlwpsis aderholdii, Fusarium nivale, F. equiseti, 
Ophiobohts graminis, and Typhula grarmneum. If the soils are 



CORRELATION OF REACTION OF THE SOIL 161 

made acid, F. nivale, O. gramhiis, and T. gramineum may dis- 
appear. 

2. MesanthrophiUc: those that thrive best in neutral soils, in- 
cluding Fusariimi aurantiacum, F. avenaceum, F. herbarum, 
Phoma betae, and Thielavia basicola. 

3. Oxyphilic: those that thrive best in acid soils, including 
Plasmodiophora brassicae and Rhizoctonia violacea. If the soil 
reaction reaches pH 7.5, P. brassicae is checked. Synchytrium 
endobioticnm may also be placed within the oxyphilic group, but 
it is actually intermediate between this group and the aestatic 
group. 

4. Aestatic: those that possess the ability to thrive in a wide 
range of soil reactions, including Fusarium cidmorum, F. poly- 
morphum, Helminthosporium sativum, Ophiobolus herpotrichus, 
and Rhizoctonia solani. 

SchafTnit and Meyer-Hermann (1930) indicate that not only is 
the reaction changed by the addition of acid or basic materials 
to soil but also that such changes are always accompanied by 
changes in the physical properties of the soil. Furthermore it has 
become a matter of common knowledge that changes in reaction 
may not be permanent and that changed availability of minerals 
to growing crops accompanies changes in soil reactions. 

Chupp (1928) noted that pH 7.2 to 7.4 is the upper limit at 
which Plasmodiophora brassicae causes club root of crucifers and 
that in the range more acid than pH 6.0 all the plants may be 
affected. 

In black-root rot of tobacco, soil reaction and soil tempera- 
ture are correlated factors. Doran (1929) observed that this dis- 
ease does not develop at any temperature provided that the pH of 
the soil is 5.6 or lower. Marked injury is apparent, however, at 
15° C with pH 5.7; at 18° C with pH 5.7 to 5.8; at 21° C with pH 
5.8; at 27° C with pH 5.8 to 5.9. M 30° C there was little, if any, 
injury with pH values of 6.0 to 6.9. 

That acid soils yield a scab-free crop of potatoes, unless lime is 
applied, is the common experience of potato growers. Studies by 
Gillispie (1918) on the scab organism, Actinomyces cloromo genus, 
showed that it is inhibited at pH 4.8 to 5.2, varying with the iso- 
lates. He correlated these results with the fact that the acidity 
of Caribou loam of Maine ranges from 4.9 to 5.5 and hence may 
restrain the growth of the scab organism. 



162 EFFECTS OF REACTION OF SUBSTRATE ON FUNGI 

Alkaline fungicides. In the lio-ht of observations that fungi 
generally grow better in acid than in basic media, various attempts 
have been made to utilize this fact in wood preservation and the 

800 



700 - 



600 - 



CO 

§> 500 



s 

I 400 

0) 

s 

CtH 

o 

£ 300 

.SP 



200 - 



100 - 



I j I | 1 , , | ! | . , 1 

A 




l\ 




1 \ 


_ 


*+ 1 \ 




' \ ' ! 




/ v 1^- Schizophyllum commune 




\ 


— 


t I 




/ ^~--^ 




/ 










— Ik \ 




in \ 




. V^^Polystictus versicolor 


- 


/ \ ' 




7 1 /^v » 




V \ y' \ I 


- 


// ^^^/ \ ' 




Ma f *^ 1 





1 1 / K \ 

J' S *^, 4 K^Armillaria mellea \ 




// "^--/V^ "xX I 


■ - 


1/ ! \ N \ l 




,// / 1 |x » 1 








/// / --—' » ; \ \\ \ 






- 


•Mi >' « «' m - « 1 




'Mi » I x 




/ / ,U — Pleurotus ostreatus \\ Xs 1 


_ 


' J 1 \ \ ^ 








^ / /L-" 1 , 1 , 1 1 \ n \i 





5 6 

Reaction of medium 



8 



9 



Fig. 25. Growth of certain Basidiomvcetes at 25° C in peptone nutrient solu- 
tions with varying initial reactions. Comparison with Fig. 23 shows that 
nutrition is also a factor in growth. (After Wolpert.) 

prevention of decay in fruits. Stains of sapwood can be largely 
prevented if the lumber is dipped into an alkaline bath immedi- 
ately after it is sawn [Scheffer and Lindgren ( 1 940 ) | . Such 
treatment gives protection until the water content of the lumber 
can be reduced to 20% or less, at which point lack of moisture 
inhibits the growth of sapwood-staining species. 



IMPLICATIONS 163 

Attempts to prevent the decay of apples and oranges by use of 
alkaline dips have been less successful. Marloth (1931) recorded 
that sodium tetraborate is more toxic to Penicillium dlgitatwn 
than to P. italicum, but that sodium bicarbonate is more toxic to 
P. it ah cam. Attempts to prevent decay of citrus fruits by these 
molds and by Pbomopsis citri and Diplodia natalensis have not 
been uniformly successful, presumably because the mycelia early 
become internal to the "buttons" or cut pedicels and because the 
spores of Penicillium are difficult to wet. 

Implications. The totality of evidence from studies of the ef- 
fects of reaction on fungi shows convincingly that pH is an en- 
vironmental factor of enormous consequence in modifying; their 
metabolic activities. There is specificity of minimal, optimal, and 
maximal pH requirements, but it must not be overlooked that the 
effects of the concentration of hydrogen ions cannot be isolated 
completely from those induced by other ions. These effects are 
always intricately correlated. For this reason it is not unlikely 
that in the past too much consideration has been given to the 
influence of the hydrogen ion and too little to that of other ions. 
No doubt this situation prompted the supercilious suggestion 
that doctoral dissertations dealing with these problems may be 
regarded as fulfilling requirements for the "pH D." 

So long as growth continues in a fungus culture, the reaction 
of the medium continues to change, although the changes may be 
masked by relatively large amounts of buffer substances present 
in the medium. In other words, the analytic and synthetic proc- 
esses that occur during utilization of organic substrates result in 
the production of many kinds of organic acids and such other 
products as pigments, polysaccharides, sterols, and vitamins (see 
Chapter 4). Among the acids known to be producted by fungi 
are aconitic, carlic, carlosic, carolic, dimethylpyruvic, fulvic, fu- 
maric, gallic, glycolic, glycuronic, itaconic, kojic, malic, cf-man- 
nonic, mycophenolic, oxalic, penicillic, puberulic, pyruvic, spiculi- 
sporic, succinic, and terrestric. It would seem that determination 
of the kinds of acids produced in fungus metabolism and the con- 
ditions influencing their production should be given increasing 
consideration, rather than devoting so much attention to accumu- 
lation of data on pH changes during growth. 



164 EFFECTS OF REACTION OF SUBSTRATE ON FUNGI 



LITERATURE CITED 

Armstrong, J. I., "Hydrogen-ion phenomena in plants. I. Hvdrion con- 
centration and buffers in fungi," Proto plasma, 8: 222-260, 1929. 

Chupp, C, "Club root in relation to soil alkalinity," Phytopathology, 18: 301- 
306, 1928. 

Clark, W. M., The determination of hydrogen ions, 3rd ed. xvi -f- 717 pp. 
Williams and \\ llkins Co., Baltimore. 1928. 

Clark, J. F., "On the toxic effect of deleterious agents on the germination 
and development of certain filamentous fungi," Botan. Gaz., 28: 289- 
327, 378-404, 1899. 

Dorax, W. L., "Effects of soil temperature and reaction on growth of to- 
bacco infected and uninfected with black-root rot," /. Agr. Research, 
39: 853-872, 1929. 

Gillispie, J. L., "The growth of the potato-scab organism at various hv- 
drogen-ion concentrations as related to the comparative freedom of 
acid soils from potato scab," Phytopathology, 8:257-269, 1918. 

Herrick, J. A., "Growth and variability of Stereum gausapatam in culture," 
Phytopathology, 7^:504-511, 1939.' 

Jackson, L. W. R., "Effects of H-ion and Al-ion concentrations on damp- 
ing-off of conifers and certain causative fungi," Phytopathology, 
SO: 563-579, 1940. 

Johnson, H. W., "Relationships between hvdrogen-ion, hvdroxvl-ion, and 
salt concentrations, and the growth of seven soil moulds," Iowa Agr. 
Expt. Sta. Research Bull, 16: 307-344, 1923. 

Karrf.r, Joanne L., "Studies in the phvsiologv of the fungi. XIII. The ef- 
fect of hvdrogen-ion concentration on amvlase produced by certain 
fungi," Ann. Mo. Botan. Garden, 8: 63-96, 1921. 

MacInnes, Jean, "The growth of the wheat-scab organism in relation to 
hvdrogen-ion concentration," Phytopathology, 12: 290-294, 1922. 

Marloth, R. H., "The influence of hvdrogen-ion concentration and of 
sodium bicarbonate and related substances on Penicillium italicum and 
P. digitatwn" Phytopathology, 21: 169-198, 1931. 

Meacham, A I. R., "Note upon the hvdrogen-ion concentration nccessarv to 
inhibit the growth of four wood-destroving fungi," Science, 48: 499-500, 
1918. 

Robbins, W. J., "Isoelectric points for the mvcelium of fungi," /. Gen. 
Physiol., 5:259-271, 1924. 

Schaffntt, E., and K. Mf.ver-Her.m ann, "Ubcr den Einfluss der Bodenreak* 
tion auf der Lebenweise von Pilzparasiten und das Ycrhalten ihrer Wirt- 
pflanzen," Phytopath. Z., 2:99-166, 1930. • 

Scheffer, T. C, and R. M. Lindgren, "Stains of sapwood and sapwood 
products and their control," U. S. Dept. Agr. Tech. Bull., 114: 124 pp. 
1940. 

Sherman, H. C, A. C. Thomas, and M. E. Baldwin, "Influence of hydrogen- 
ion concentration upon enzvmic activity of three typical amylases," 
Am. Chem. Soc. ]., 41: 181-239, 1919. 



LITERATURE CITED 165 

Sherwood, E., "Hvdrogen-ion concentration as related to the Fusarium wilt 
of tomato seedlings," Am. J. Botany, 10: 537-553, 1923. 

Sideris, C. P., "The role of the hvdrogen-ion concentration on the develop- 
ment of pigment in Fusaria," /. Agr. Research, 30: 1011-1019, 1925. 
"The effect of the H-ion concentration of the culture solution on the 
behavior of Fusarium chromophythoron and Allium cepa and the de- 
velopment of pink-rot-disease symptoms," Phytopathology, 19: 233-268, 
1929. 

Webb, R. W., "Studies in the physiology of the fungi. X. Germination of 
the spores of certain fungi in relation to hydrogen-ion concentration," 
Ann. Mo. Botan. Garden, 6: 201-222, 1919. 

Webb, R. W., and H. Fellows, "The growth of Ophiobohis gramims Sacc. 
in relation to hydrogen-ion concentration," /. Agr. Research, 33: 845- 
872, 1936. 

"Wol^ert, F. S., "Studies in the phvsiologv of the fungi. XVII. The growth 
of certain wood-destroving fungi in relation to the H-ion concentration 
of the media," Aim. Mo. Botan. Garden, 11:48-96, 1924. 



Chapter 8 
SPORE DISSEMINATION 

All students of fungi are impressed with the seemingly limitless 
profligacy of these organisms in the production of spores. Arthur 
(1929) records that more than 2 billion sporidia may be formed 
by a single gall of Gymnosporangimn juniperi-virginianae. 
Fovies applanatiis, which may attain a size of 0.75 X 0.5 meters, 
may have an annual production of 5 million million spores. The 
crop of aeciospores from a single barberry bush was found by 
careful computation to be 64,512,000,000. The pileus of Psalliota 
ccnnpestris may produce 1,800,000,000 basidiospores, that of 
Coprimts comatus 5 billion, and that of Poly poms squamosus 1 1 
billion. 

Meyer (1936) reported that a sporophore of Tomes fomentarius 
shed 1115 grams of spores in a period of 20 days. Each spore had 
a computed weight of 0.000,000,000,146 gram. The calculated 
number of spores produced by this sporophore, therefore, was 
7,636,986,301,369. 

Moss ( 1940) estimated the number of spores formed by Calvatia 
gigantea as 20 million million. This ability to produce spores in 
abundance is made possible among many Basidiomycetes by the 
lar^e size of the fructifications and by such structural modifica- 
tions as gills and pores that increase the spore-bearing surface. 
Certain leathery and woody polypores have been found to be 
capable of shedding spores continuously for 6 months or longer. 
Organisms in other groups may shed spores in abundance only 
under special conditions. Monilia sitophila, for example, may 
cover burned sugar-cane stubbles to the extent that acres of land- 
scape look pink. The metal lic-lustered Blakeslca trispora may be 
equally widely prevalent on mowed, withered Sida spinosa and 
other weeds in orange groves. Heald (1937) states that Tilletia 
tritici may be so abundant during the threshing season in eastern 

166 



DISTRIBUTION OF SPORES 161 

Washington that over 5 million smut spores lodge on each square 
foot of soil. 

Of much more interest than the ability of fungi to produce 
spores in abundance is the development of mechanisms or devices 
that serve to provide maximum distribution of these spores. 
Survival of given species mav in large part be conditioned by dis- 
persal into habitats where food is available. In most species of 
fungi special mechanisms are lacking, and hence distribution, 
among both aquatic and terrestrial forms, appears to be largely 
fortuitous. 

DISTRIBUTION OF SPORES 

Aquatic fungi. The environment in which aquatic species ex- 
ist is more constant than the habitat of terrestrial species, and cor- 
related with this fact is the possibility that a larger proportion of 
their spores may germinate and develop into new individuals. For 
these reasons problems of dissemination of aquatic fungi might not 
be expected to stimulate as much interest as similar problems in- 
volving terrestrial fungi. Nearly all aquatic fungi are among the 
Phycomycetes, the spores of many of which are motile (planetic). 
The most primitive of these are holocarpic. Each such plant pro- 
duces 20 to 30 spores, each of which possesses a single flagellum. 
After a brief period of motility, which rather closely restricts the 
distance that the spore may migrate from the parent plant, the 
spore initiates the assimilatory phase of the cycle of development. 
After a few days the sporangium is again mature, and conditions 
for dispersal have once more been prepared. 

Other more highly specialized species possess differentiated 
sporangia or other sporiferous cells, from which cells having two 
flagella are liberated. Evidence is lacking that biflagellate species 
are significantly better able to disseminate themselves and to com- 
pete to greater advantage than monoflagellate ones. Undoubtedly 
diplanetism, that is, two morphologically distinct motile stages 
that always occur sequentially, so highly developed among the 
Saprolegniales, must be regarded as an evolutionary advance over 
monoplanetism. In the Saprolegniales diplanetism is accompanied 
by certain morphological differences in spores whose significances 
are wholly unknown. Typically, on first escaping from the spo- 
rangium the swarm spores are pear-shaped and terminally biflagel- 
late. After swarming for a brief period, they encyst and then 



168 SPORE DISSEMINATION 

escape from the cvst as reniform, laterally biflagellate swarmers. 
Encvstment follows; after this stage they give rise to germ tubes. 
This pattern of behavior varies in the different genera. Sometimes 
polvplanetism occurs as reported bv Weston (1919) in Dic- 
tvuchus, bv Hohnk (1933) in Saprolegnia torulosa and Achlya 
racemosa, and bv Salvin ( 1940) in Achlva, the number of swarm- 
ings beimj controlled by reserve food in the swarm spore and by 
unknown environmental factors. 

Terrestrial fungi. Various adaptations occur among terres- 
trial fungi to aid in their geographic distribution. The spores of 
many are pulverulent, so that dissemination bv air currents is 
favored. Others accumulate in a mucous matrix that is water sol- 
uble, the occurrence of dews and rains being required for spore 
dissemination. Some become wet with ease, others with difficulty; 
some have thin walls, others very thick, resistant walls; some are 
smooth, others are armed with spines, tubercles, or echinulations. 
The fructifications of some species are malodorous, encouraging 
visitation by flies, bees, ants, and other insects, whereas others 
are attractive to mvcophagous animals, such as nematodes, beetles, 
snails, slugs, and rodents. 

For convenience of discussion the dissemination or dispersal of 
fungi may be considered to be accomplished by (a) agencies re- 
lated to the environment of the species and (b) the fungus itself 
through structural adaptations. 

The environmental agencies include movement of air as con- 
vection currents and winds, movement of water, occurring as 
dew, rains, and streams, and transport by insects and other animals, 
including man. Many species are dispersed on seed, fruits, cut- 
tings, seedlings, and transplants. 

Air currents as a factor in dispersal. For nearly 150 years 
it has been taken for granted from observational evidence that the 
spores of fungi are wind-borne. Proof that wind is an important 
agency in the spread of pathogenic fungi has been forthcoming 
only in recent years. It arose from attempts to explain the occur- 
rence of epidemics, especially of rusts. According to Arthur 
(1929), Marshall reported the following observations made in 
1782 upon the results of planting a barberry bush in a field of 
wheat: "About the barberry bush there appeared a long but 
somewhat oval-shaped stripe of a dark livid color, obvious to a 
person riding on the road at a considerable distance. The part 



DISTRIBUTION OF SPORES 169 

affected resembled the tail of a comet, the host itself representing 
the nucleus, on one side of which the sensible effect reached about 
twelve yards, the tail pointing toward the southwest, so that prob- 
ably the effect took place during a northeast wind. ... As the 
distance from the bush increased, the effect was less discernible, 
until it vanished imperceptibly." Ward (1882), in connection 
with studies on Hemileia vastatrix in Ceylon, was among the first 
to demonstrate that rusts are wind-borne; he trapped the uredinio- 
spores on slides coated with glycerin. Klebahn (1904) believed 
wind responsible for bringing grain-rust spores to Germany, be- 
cause during a dust storm which swept from northern Africa to 
northern Europe, he caught 3800 spores of Puccinia gramijjis at 
Hamburg and 5600 at Thiiringen in cotton-batting spore traps, 
4 in. in diameter. 

Within the United States a volume of evidence has been ac- 
cumulated to show that the grain rusts are unable to survive the 
winter in the cold climates of the central part of the Cereal Belt, 
where the alternate host is absent, and that urediniospores are car- 
ried northward from Mexico and Texas toward Canada. By 
means of aeroplanes, Stakman and his associates (1923) entrapped 
viable rust spores during April over Waco, Texas, at various alti- 
tudes ranging from 1000 to 16,500 ft. In late summer in Manitoba 
at an altitude of 5000 feet, 259 urediniospores were entrapped on 
2 sq in. of surface in one instance, and 116 urediniospores in an- 
other. 

The later work of Stakman et al. (1940) showed that the telio- 
spores of Puccinia gramims are of no consequence in the annual 
cycle of this rust in the South. The uredinial stage does not sur- 
vive the winters north of Texas or the summers in Texas and 
areas southward. In the North rust is dependent on barberry and 
on urediniospores blown in from farther south. Toward the end 
of summer and in the fall urediniospores are blown southward, 
and the rust survives the winter in fields of early-sown wheat in 
Texas and northern Mexico. 

Observations by Pennington (1924) indicate that aeciospores of 
Cronartium ribicola, while usually carried only a few hundred 
feet, may under exceptional conditions be transported 150 to 200 
miles and then cause infection. Gyimiosporanghnn juniperi-vir- 
ginianae was found by Schneiderhan (1926) to have produced 



110 



SPORE D1SSEMIXAT10\ 



11.5 spots per leaf on apple trees 1% miles away from infected 
cedars and 0.32 spot per leaf on trees 3 miles distant. 

The foregoing evidence regarding dispersal of rusts bv air cur- 
rents is representative for this group of fungi but is inadequate in 
indicating the importance of this agencv for other groups of fungi. 
Stakman and his coworkers (1923) identified other genera, such 
as Alternaria, Helminthosporium, Cladosporium, Cephalothecium, 
and Ustilago, on their spore traps. Heald et al, (1915) found that 




^& 





Fig. 26. Spore trap of weather-vane type. A. The dish whose inner surface 
is coated with glvcerin remains directed toward the wind. B. Dishes may 

be stacked during transport. 

ascospores of Endothia parasitica may be entrapped in the air in 
considerable numbers 300 to 400 ft. from diseased chestnuts, sub- 
stantiating the observations of others. Many similar observations 
have been recorded for Venturia hiaequalis. Burrill and Barrett 
(1909) showed that Diplodia zeae is distributed bv winds; Wolf 
(1916) made the same observation for Cercospora personata. 
Peronospora tabacina is verv quickly dispersed from infected to- 
bacco seed beds to healthy ones several miles distant [Wolf et al. 
(1934)], and it is reasonable to assume that its introduction into 
New England and Canada was the result of the carriage of spo- 
rangia several hundred miles through the air. 

The aerial dissemination of plant pathogens is briefly treated 
in a recent report by Craigie (1939). His studies show that the 
funp-i causing stem rust of cereals, leaf rust of wheat, and crown 
rust of oats are air-borne in western Canada, being carried several 



DISTRIBUTION OF SPORES 



111 



Open 



>f Cover- 



hundred miles from their place of origin. In conclusion it may 
be said that ample evidence has shown that fungus spores "fly 
through the air with the great- 
est of ease" [Keitt (1942), Chris- closed 
tiansen (1942), Durham (1942)]. 

Spore-trapping devices. Vari- 
ous devices for determining the 
presence and movement of wind- 
borne spores have been em- 
ployed. These techniques were 
described and illustrated in a re- 
port by the Committee on Appa- 
ratus in Aerobiology ( 1 941 ) . The 
simplest method consists of expos- 
ing a surface coated with vaseline, 
glvcerin, gelatin, or agar. 

Rittenberg (1939) exposed agar 
plates on shipboard during cruises 
in the Pacific in the area from 
Monterey to the Cedros Islands 




and extending seaward 400 miles. 
He entrapped such soil-borne or- 
ganisms as Alternaria, Catenularia, 
Cephalosporium, Cladosporium, 
Penicillium, Spicaria, Sporotri- 
chum, Stemphylium, and Tri- 
choderma. . 

Some workers have employed 
aspirators, by means of which a 
definite volume of spore-laden air 
is drawn through a filter of steri- 
lized cotton or sugar crystals. 
Others expose dishes containing 
water or cotton batting. All de- 



Coated 
glass 
slide- 



Air 
current 



i>\ 



I 



Hook or 
handle 



Fig. 27. Schematic representa- 
tion of "sky-hook" type of spore 
trap. (Adapted from Meier and 
Lindberg.) 



vices are serviceable. In order 

to keep the sticky surface directed toward the wind, the exposure 
plates may be fastened to a weather vane or may be inclined from 
the vertical for protection from rains. The most ingenious ap- 
paratus used is the "sky hook," as employed by A4eier and Lind- 



112 SPORE DISSEMINATION 

bersr (1935) in their aerobiologncal studies in the Arctic. Proctor 
(1934) at the Massachusetts Institute of Technology used an 
ingenious automatic device. 

Rate of fall of spores. Rate of fall of spores in still air has 
been given consideration by a number of investigators, including 
Buller (1909), Ukkleberg (1933), Stephanov ( 1935), and Gregory 
(1945). In general the terminal velocity has been found to be of 
the order expected from Stokes' law. According to this law, 




where V = terminal velocity, p = density of the spore, a = den- 
sity of the medium, g = acceleration due to gravity, r = radius of 
the spore, and /x = viscosity of the medium. Deviations from the 
expected rate may be ascribed to the following factors: ( 1 ) shape 
of spore; that is, they are seldom ideal spheres; (2) irregularities 
in outer surface of spore wall; (3) rapid desiccation during 
falling; and (4) inaccuracy in determining the density of the 
spores. 

Buller (1909) found that the rate of fall of basidiospores of 
Colly bia dryophila is 0.49 mm per second and of Coprinus plica- 
tilis 4.29 mm per second. Ukkleberg (1933) determined that the 
rate of fall of urediniospores of Pnccinia graminis tritici is 11.57 
mm, of P. graminis secalis 10.58 mm, of P. coronata avenue 10.00 
mm, and of P. triticina 12.62 mm per second. He found that the 
rate of fall of aeciospores of P. graminis tritici is 10.56 mm, and 
of P. graminis secalis 10.20 mm per second. 

Insects as vectors of fungi. It is well known that such in- 
sects and arachnids as ticks, fleas, flies, mosquitoes, lice, bees, wasps, 
beetles, and mites are capable of transmitting microorganisms, 
especially species responsible for important diseases of man and 
various animals. Much less is known, however, regarding the role 
that animals play in the transmission of species pathogenic to 
plants. Although the presence of certain virus diseases of plants 
appears to require the presence of specific insects as vectors, for 
instance, aster yellows, carried by Cicadula sexnotata, curly top 
of beets, carried by Eutetix tenella, and kroepoek of tobacco, car- 
ried by Bemesia gossipiperda, no portion of the life cycle of the 
pathogen, whether virus, bacterium, or fungus, appears to develop 
within the body of the vector. Instead the infective agent is 



DISTRIBUTION OF SPORES 113 

merely taken into the body of the insect and passes unharmed 
through the alimentary tract, or is regurgitated or accidentally ad- 
heres at the surface, or is mechanically transferred to the host 
plant. The relation of insects to disease in plants is therefore less 
spectacular than in animals but is none the less quite as important. 

Rand and Pierce (1920) are among the first to bring together 
from widely scattered sources the information extant on insects as 
agents in the transmission of fungi. The later accounts of Rand, 
Ball, Caesar, and Gardner (1922) and the comprehensive works 
of Leach (1935, 1940) describe the present status of this topic. In 
the appendix to the volume by Leach (1940) is a long list of in- 
sect-transmitted fungi. 

Abundant evidence is at hand to show that the brown-rot fungus 
of stone fruits, Sclerotinia fructicola, is transported by bees, wasps, 
May beetles, and squash bugs at the season when the fruit is ripen- 
ing. Heald [Arthur (1929)] demonstrated that mites are carriers 
of Sporotrichum anthophihim, the cause of bud rot of carnations. 
Punctures made by the cabbage maggot, Pegomya brassicae, af- 
ford portals of entry for Phoma oleracea, the cause of cabbage 
blackleg. The woolly aphis, Schizoneura lanigera, is associated 
with the spread of the apple-canker fungus, Nectria ditissima. 
Similarly Ehrlich (1934) showed that N. coccinea infects beech, 
but only if the bark is infested with Cryptococcus fagi. Initial 
infection is possible provided that the living tissues of the bark 
have been injured by the insect while feeding. The fungus then 
grows parasitically and kills the beeches within 2 or 3 years. 

Certain species of Orthoptera, Lepidoptera, Coleoptera, and 
Hemiptera were found by Wolf (1916) to distribute Cercospora 
personata on peanuts. Among these orders grasshoppers, because 
of their powers of flight, were regarded as especially important 
vectors of this peanut-leaf-spot fungus. A single longicorn 
beetle, Leptostyhis maculata, was found to transport 320,000 
spores of Endothia parasitica and, according to Studhalter and 
Ruggles (1915), 19 other insect species also act as carriers of this 
fungus. 

The larval forms of many species find rust spores to be suitable 
food, and they effectively aid in distributing them. Arthur (1929) 
records that the larvae of Smyrithurus sp., a neuropterous insect, 
carries Puccinia rubigo-vera iritici, and the larvae of Diplosis sp., 



114 SPORE DISSEMINATION 

a cecidomyid, transports Uroviyces bid enti cola. Honeybees dis- 
tribute the urediniospores of rust on Populus and the aeciospores 
of Caeovia nit ens. The scarabeid beetle, Serica sericea, is among 
many species that transport Cronarticum ribicola. . Basidiobohis 
ranarum is carried to froo-s and salamanders within the bodies of 
various beetles. Gypsy-moth larvae, Porthetria dispar, have been 
found by actual count to bear from 1120 to 23,320 aecio- 
spores of Cronartium ribicola. Arthur (1929) records that Rat- 
hay noted 135 species of Coleoptera, Hymenoptera, Hemiptera, 
and Diptera as carriers of rust spores, Diptera being especially at- 
tracted to the saccharine exudate of pycnia. Doubtless they are 
important agents in the spermatization of rusts. 

The Dutch elm pathogen, Ceratostomella ulmi, is transported 
by bark beetles, Scolytus scolytus and 5. multistriatus. Several 
species of Ips and Dendroctonus are known to be capable of dis- 
seminating spores of fungi (various species of Ceratostomella) 
associated with blue stain of loos and lumber. 

The spore dispersion of Phallales appears to be dependent upon 
sarcophagid and muscid flies. The sporiferous tissue of Phallales 
becomes slimy at maturity and is nauseatingly putrid. This pene- 
trating odor is attractive to flies, and in consequence they carry 
the spores externally and also void them intact in their excreta. 
Ithyphallus coralloides, suspected of causing root rot of sugar 
cane, is so attractive to flies that they can be driven away from the 
fructifications only with difficulty. Various ants and beetles are 
also attracted to this species and no doubt carry the spores under- 
ground to situations favorable for germination and development. 
Various flies are also attracted to the saccharine exudate contain- 
ing conidia of the sphacelial (conidial) stage of Claviceps, espe- 
cially C. purpurea and C. paspali. 

Brodie (1931) has shown that flies transport the conidia of 
Coprimts lagop7is, as a result of which the mvcelia become diploi- 
dized. Similarly the transfer of pycniospores of Puccima graminis 
and P. helianthi by flies and other insects attracted to the sugary 
exudate has been demonstrated [Craigie (1931)]. 

The tree cricket, Oecanthns niveus, actively transports the 
spores of Leptosphaeria comothyrhivu the cause of canker on 
apple trees. 

The flea beetle, Epitrix cucimieris, the Colorado potato beetle, 
Leptinotarsa decemlineata, and the horn worm, Protoparce caro- 



DISTRIBUTION OF SPORES 115 

Una, have been found to have conidia of Alternaria solani and of 
Septoria ly coper sici on their bodies. 

Hendree (1933) isolated from the fecal pellets of termites and 
from the frass and wood enclosing their burrows 33 genera of 
fungi, among them Trichoderma and Penicillium. In her opinion 
these fungi are a common dietary element of the termites Reticuli- 
termes hesperus, Zootermopsis angiisticollis, and Kalotermes 
minor. 

Such insect visitors as honeybees, bumblebees, carpenter bees, 
thrips, and ants were found [Smith and Weiss (1942)] to be 
capable of transporting spores of Ovulinia azaleae, causing flower 
spot on cultivated azaleas. 

It has been noted that the females of certain woodwasps, Sir ex 
gigas and S. cy aliens, always have elements of the wood-rotting 
fundus, Sterenm sanguine olentum, in the pouches at the anterior 
end of the ovipositor. Whether this association is symbiotic is not 
known. 

The only conclusion warranted from the foregoing discussion, 
which is representative of a large volume of reports of insects as 
disseminating agents, is that many species of insects are concerned. 
Furthermore, many fungi, both pathogenic and saprogenic, are 
insect-borne. It remains to be determined whether virulence in 
fungi is modified by passage through the alimentary tract. It is 
known that some species have already germinated when the fecal 
pellets are voided, although essentially nothing is known about the 
effects of digestive enzymes on germination. 

More attention should be given also to the necessity of host in- 
juries by the insect for inoculation and infection. In this connec- 
tion there is evidence that sugar cane injured by the sugar-cane 
borer, Spenophorns obscnrus, is more subject to attack by Col- 
letotrichiim jalcatnm. Moreover, onions infested with thrips are 
predisposed to infection by Feronospora destrnctior, and grasses 
punctured by aphids are more susceptible to Erysiphe graminis. 

Leach (1935) has expressed the opinion that insects are not 
merely disseminators of inoculum in the case of pathogenic fungi, 
but that the insect-fungi relationship is highly organized and has 
broad biologic and evolutionary significance. 

Other animals as vectors of fungi. Besides insects, many 
other animals transport fungi, but usually dissemination by them 
is entirely fortuitous. Among the animals known to be or sus- 



116 SPORE DISSEMINATION 

pected of being carriers are slugs, snails, sow bugs, various rodents, 
birds, and domestic animals. Slugs and snails feed upon a large 
variety of fungi, especially powdery mildews, discomycetes, rusts, 
mushrooms, polypores, and leathery fungi [Buller (1922), Wolf 
and Wolf (1940)]. The spores either are voided or are dragged 
along and scattered by the migrations of these animals in search 
of food. Fleshy Hymenomvcetes appear to be especially attrac- 
tive. Poisonous species are devoured with impunity. The pos- 
session of a highly developed olfactory sense guides the animals 
in the location of the fruit bodies of these species. Gravatt and 
.Marshall (1917) made the observation that slugs (Agriolimax 
agrestris), snails (Sabulina octona), and sow bugs (Armadillidium 
-j nl gave) eat and distribute spores of Cronartium ribicola. Heald 
and Studhalter (1914) found that birds, especially woodpeckers, 
are of importance in the dissemination of Endothia parasitica. An 
estimate of the numbers of spores of this fungus carried by two 
downy woodpeckers {Dryobates piibescens medianus) was 757, 
074 and 624,341 and by a brown creeper (Certhia jamiliaris ameri- 
cana), 254,019. 

A number of fungi, notably species of Pilobolus, Sordaria, 
Panaeolus, Anellaria, and Coprinus, normally occur on dung and 
are regarded as coprophilous. Their spores are distributed by 
such herbivorous animals as horses, cattle, sheep, goats, rabbits, 
and geese. These animals swallow the spores and herbage to- 
gether, and either the spores pass undamaged through the ali- 
mentary tract or else their germination is favored by the digestive 
enzymes which they encounter en route. After having been 
eaten, the spores of these coprophilous species may remain for 
hours within the alimentary tract before being voided in the feces. 
Meanwhile the animal may transport them for miles. Soon after 
discharge from the animal's body the spores will develop into new 
plants, and their fruiting bodies will mature. Some dung-fungi 
are especially adapted to such habitats. Buller (1934) has shown, 
for example, that the sporangia of Pilobolus, on being shot away, 
adhere to herbage 3 to 8 ft. distant from the dung heap. The 
sporangia cling by virtue of the gelatinous material that arose by 
dissolution when the sporangium separated from the swollen 
subsporangium. Since these sporangia cannot be wet, they are 
not affected by rains and in consequence may adhere intact to the 



DISTRIBUTION OF SPORES 111 

vegetation for several weeks. Moreover the sporangial wall is 
black, so that injurious radiations are screened out. 

There is no evidence that certain other coprophilous species, 
for example, Lachnea stercorea and Humaria gramdata, have any 
structural adaptations for such habitats. Undoubtedly many 
species grow in dung quite by accident. At any rate, mycologists 
have come to recognize that herbivorous animals are excellent 
collectors of fungi. 

The human agency. lYian, unwittingly and wittingly in the 
distribution of seed, seedlings, cuttings, nursery stock, bulbs, and 
roots, has spread and will continue to spread fungi of economic 
importance throughout the world. Many of these fungi have 
caused him enormous financial losses. To relieve and prevent this 
situation, both state and federal inspection services have been 
instituted and quarantines established. 

In North America alien or exotic species appear to be much 
more destructive than indigenous ones, as is evident from the 
ravages of chestnut blight (Endothia parasitica), blister rust of 
white pines (Cronarthnn ribicola), Dutch elm disease (Ceratosto- 
mella idmi), late blight of potato (Phytophthora infestans), 
downy mildew of tobacco (Feronospora tabacina), and willow- 
scab (Fusicladhnn saliciperdum) . Furthermore, there is evidence 
that pathogens introduced from one continent into another may 
find conditions in the new land more favorable for development 
in epidemic proportions, as did grape mildew (Plasviopara viti- 
cola) and late blight of potato (Phytophthora infestans), intro- 
duced into Europe from the New World, and coffee rust (Hemi- 
leia vastatrix), introduced into Ceylon, presumably from Africa. 
A list of rusts [Arthur (1929)] in Australia in 1906, comprising 
161 species, is said to contain 30 species that are not indigenous. 
Arthur also lists 41 species of rusts that have been introduced into 
^orth America, including such important ones as Cronarthnn 
ribicola, Uromyces appendicidatus phase oli, U. appendicidatus 
vignae, U. betae, U. caryophyUimis, U. trifolii, Puccini a arachidis, 
P. asparagi, P. chrysanthemi, P. glumarum, P. graminis phlei pra- 
tensis, P. graminis tritici, and P. mahaceamm. Undoubtedly man 
distributes many fungi that cling to hands and clothing and are in- 
oculated onto healthy plants inadvertently as he passes to them 
after handling diseased plants. 



118 SPORE DISSEMINATION 

Seed-borne fungi. In 1733 Jethro Tull recorded seed disin- 
fection by the use of brine. Wheat being shipped to England 
became wet in the hold. Some of it was planted, and the resulting 
crop was observed to be free from stinking smut. From this ob- 
servation came the use of salt-water steeps to prevent seed-borne 
diseases. Moreover, before this earlv period some of the tribes 
in Asia Minor passed their seed grain through flames and thereby 
removed the highly inflammable smut spores. Thev did this, 
however, as a religious ritual, because fire has long been regarded 
as a means of purification. 

Subsequent studies have shown that many grass smuts are seed- 
borne. In addition, such other pathogenic agencies as certain 
viruses, bacteria, many fungi from nearly every important tax- 
onomic e^roup, nematodes, and insects are known to be carried 
with the seed. Orton (1931) assembled a bibliography of seed- 
borne diseases which should serve as a basis for studies by others. 
In his long list are such important pathogens as Gibberella sau- 
binettiij C oil etotri chum lindemuth'iamim, Phovia Ihigam, Septoria 
apii, Diplodia zeae, Glomerella gossypii. Collet otrichwn lagena- 
riitm, Phomopsls vexans, Sclerospora grcnninicola y Urocystis 
cepnhe, Ascochyta pisi, Tilletia tritici, and Ustilago avenae. 

Soil-borne fungi. Vascular and root-rot parasites, including 
species of Fusarium, Verticillium, Cephalosporium, Thielaviopsis, 
Sclerotium, Phytophthora, Pythium, and Rhizoctonia, commonly 
persist in the soil and are distributed by numerous agencies. 
These include movement of the infested soil by washing rains or 
its transport by streams, carriage of infested soil on seedlings, 
rooted cuttings, bulbs, corms, or roots, and transport on imple- 
ments, machinery, tools, hoofs of farm animals, and shoes of man. 

Observations in the East Indies and in the United States leave 
little doubt that fields which are flooded or overflowed after rains 
may become infested with Phytophthora nicotianae, causing to- 
bacco black-shank. The rowward spread of Fusarium wilts is a 
matter of common observation. Carriage of fungi with soil or 
on seedlings may not be an unmixed evil. Evidence assembled 
by Hatch (1936) shows that in afforestation the planting of seed 
may fail, whereas the transplanting of seedlings may succeed. 
The reason for this anomaly is the dependence of tree species 
upon certain fungi which become associated in the mycorrhizal 
relationship. 



HYGROSCOPIC MECHANISM IN MYXOMYCETES 119 

Water as a vector of fungi. Water may sometimes serve as 
an important agency for dissemination of fungi, although there 
is a dearth of direct data on this point. Rain splash is known to 
be responsible for the spread of conidia of apple bitter-rot 
(Glomerella rufo-maculans), cotton anthracnose (G. gossypii), 
bean anthracnose (Colletotrichiim lindemiithiamim) , and brown- 
spot needle disease of pines (Systremma acicola). The conidia 
of these fungi and of many others are produced in a matrix that 
is corneous when dry but that dissolves when moist. Such fungi 
are adapted for distribution at times favorable for spore germina- 
tion and infection. Others are mechanically transported by dews 
or rains and thus find lodgment on new substrata. In Colorado 
years ago Cercospora beticola was found to be present in water 
in irrigation ditches and to be spread to non-infected beets by 
irrigation. Arthur (1929) mentions an outbreak of Puccinia 
sorghi on Oxalis in a corn-field that was overflowed. 

STRUCTURAL ADAPTATIONS FOR EXPULSION OF SPORES 

At maturity or soon thereafter the spores of many species of 
fungi are forcibly discharged. Expulsion of spores from the 
structures within which they are delimited or upon which they 
are borne must be regarded as a device to further the geographical 
distribution of the particular species. 

HYGROSCOPIC MECHANISM IN MYXOMYCETES 

Within the sporangia of certain slime molds, notably Trichia 
and Hemitrichia, the capillitial threads are thickened in spiral 
bands. When the sporangial wall is ruptured as the result of dry- 
ing, the tangled capillitia may be noted to be interspersed among 
the spores. As the capillitia dry, they writhe and twist by virtue 
of the unequal thickenings of the wall. As the ends of the 
threads spring free, they fling adhering spores into the air. This 
behavior is, therefore, quite like that in the liverwort, Marchantia, 
and is very efficient in conjunction with air currents in causing 
the spores to be widely disseminated. Dissemination of other 
species, however, appears to be wholly fortuitous. 

The mechanisms involved in spore discharge are quite different 
and need not indicate phylogenetic relationships. 



180 SPORE DISSEMINATION 

SPORE EXPULSION AMONG PHYCOMYCETES 

Among some aquatic fungi, such as the Chvtridiales and Sapro- 
legniales, sporangiospores are merely ejected to the exterior of the 
sporangium, where, by virtue of their flagella, they become rather 
widely distributed. Members of these orders generally possess an 
exit tube or papilla. As the result of increased turgor after de- 
limitation of sporangiospores, the sporangium opens at the exit 
tube or papilla, and the sporangiospores are rapidly ejected, either 
en masse or singly. In Achlya and Aphanomyces they are quies- 
cent on expulsion and collect in a hollow sphere at the orifice. 
In Saprolegnia and Leptolegnia they emerge in an actively motile 
condition. In the related Aplanes they are retained within the 
sporangium. In Dictyuchus the sporangial content is cleaved into 
segments, a pore is developed from each segment, and the proto- 
plast escapes from each segment as a motile spore, leaving behind 
a reticulum of emprv cells. In Saprolegnia two planetic (motile) 
stages normally occur, a phenomenon no doubt well adapted for 
increased dissemination of the species. 

Apparently none of the Peronosporales, except species of 
Sclerospora, forcibly expels its sporangia. As observed by Wes- 
ton (1919), S. Philippine mis and 5. gram'inis, occurring on maize, 
possess a double wall separating the tip of the sterigma and the 
sporangium. At first these two walls in contact with each other 
are plane. As the sporangium grows and turgor increases, these 
membranes tend to bulge outward, and this tendency is restrained 
by adhesion of the two surfaces in contact. Eventually adhesion 
is overcome by the stress from increased turgor, and with a sud- 
den snap both membranes bulge outward, catapulting the spo- 
rangium away. It can then be caught by air currents and trans- 
ported to near-by maize plants. 

In Peronospora t abaci na and certain other species of Perono- 
spora the sporangia are effectively liberated, but by an entirely 
different mechanism. The sporangiophores grow closely 
crowded. Each sporangiophore looks like a little tree, and to- 
gether the sporangiophores constitute a miniature forest with 
interlocking branches. The entire tree, including its twig tips, 
sterigmata, is a single, inflated coenocytic cell. A slight change 
in relative humidity in the immediate environment of the sporan- 



SPORE EXPULSION AMONG PHYCOMYCETES 



181 



giophore occasioned by air currents or increased temperature 
causes the crown of the little tree to twirl and twist. In conse- 




Fig. 28. Discharges of spores by various Phycomycetes. A. Olpidium 
brassicae. B, C, D, E. Physoderma maydis. F, G. Lagenidium rabenhorstii. 
H, 1, J. Saprolegnia sp. K, L, M. Pythium de Baryamnn. O, P, Q, R. 

Albugo Candida. 

quence of these hygroscopic movements the sporangia are dis- 
lodged [Pinckard (1942)]. Long ago attention was called to 
this phenomenon by de Bary (1887), who stated from his ob- 



182 SPORE DISSEMINATION 

servations on Peronospora, Fhytophthora infestans, and Botrytis, 
"The slightest change in the humidity of the surrounding air, such 
for instance as may be caused by the breath of the observer, at 
once produces changes in their turgescence and torsion; the lat- 
ter give a twirling motion to the extremity of the gonidiophore 
and the ripe spores are thereby thrown in every direction." 

Link, in 1809, was among the earliest observers to consider the 
problem of discharge of sporangia bv Pilobolus. Since then many 
others have recorded their studies of this phenomenon, and grad- 
uallv a clear conception of the mechanism involved has evolved. 
The ingenious experimentation bv Buller is especially pertinent 
and illuminating. Members of this genus are coprophilous and 
can best be studied by cultivation on fresh dung of herbivorous 
animals, collected and placed in the laboratory in moist chambers. 
After a few davs a crop of sporangia should have formed, and 
new crops may form each day for several successive days. Each 
sporangiophore consists of a hat-shaped, black sporangium that 
surmounts a bulbous subsporangial swelling, the upper portion of 
the stipe. This subsporangial swelling functions both as an 
ocellus that causes the stipe to direct its free end toward the source 
of light and as a part of the squirting apparatus that propels the 
sporangium. 

A laver containing bright red pigment, carotene, is formed in 
the basal wall of the subsporangial swelling. This layer extends 
partly across the stipe and forms a centrally perforate, biconcave 
septum. Immediately beneath this perforate septum is the motor 
region, which responds in such fashion as to direct the sporangium 
head on toward the light, when heliotropic equilibrium is estab- 
lished. In this position the incident light is centered on the per- 
foration of the septum. Bending is a photochemical response, as 
is also the increased pressure of turgor in the subsporangium that 
follows when the sporangium faces the light. At the time of 
expulsion this pressure in Pilobolus longipes may be equivalent 
to approximately 5.5 atm. 

While these phototropic reactions are taking place, the spo- 
rangium wall splits into two layers, the inner of which remains 
intact to enclose the spores. The expansion of the columella, 
which presses upward against the sporangium, together with the 
liquefaction of the outer wall circumferentially around the base 
of the sporangium, results in Assuring of the outer wall. The 



SPORE EXPULSION AMONG PHYCOMYCETES 



183 



upper portion persists as a convex cap over the sporangium; the 
lower portion remains attached to the base of the sporangium 
with the jelly-like mass formed around the fissure. The sporan- 
gium is now ready for discharge, and this phenomenon occurs as 
soon as the swelling of the subsporangium reaches the limits of 
extensibility. Since the papil- 
lar area constitutes the weak- 
est portion of the wall, the 
subsporangium opens at this 
point, squirts away about 
one-half the fluid content of 
the subsporangium and stipe, 
and carries along the sporan- 
gium with the jet of sap. The 
gelatinous mass present around 
the base of the sporangium 
before discharge is carried 
along with the sporangium 
and sticks it to vegetation. 
When the plants are eaten, 
the spores pass through the 
alimentary tract and are 
evacuated, undigested and 
unharmed. 

The initial velocity of the 
sporangia of P. longipes and 
P. kleini approximates 20 ft. 
per second. Buller's observa- 




Fig. 29. Stages in discharge of spo- 
rangia bv Pilobolus. A. Mature spo^ 
rangium atop the subsporangial swell- 
ing. B. Circumscissile rupture of outer 
membrane of sporangium and lique- 
faction around base of sporangium. 
C. Collapsed subsporangium after 
discharge. (After Buller.) 

tions showed that the ex- 
plosive force is sufficient to carry sporangia to a vertical height 
of 72.5 in. and a horizontal distance of 91.5 in. in P. longipes 
and 90.5 in. in P. kleini. When he prepared a special drum with 
tissue paper as the membrane forming its head, the impingement 
of sporangia was audible at a distance of 21 ft. Moreover the 
sporangia are discharged with sufficient force to be felt when 
they strike the face. 

Nearly everyone has observed that flies may become attached 
to windows in attics and other little-used rooms. Upon closer 
observation a whitish halo may be noted to surround such flies. 
This halo, 2 or 3 cm in diameter, is produced by discharged 



184 



SPORE DISSEMINATION 



conidia of the entomogenous fungus, Entomophthora imiscae. 
When the fly, sluggish because of the infection, succumbs, rhi- 
zoidal hvphae grow out from crevices between the sclerites and 
anchor the fly to the pane. Expulsion of conidia by this fungus 
and most other species of Entomophthora is accomplished by the 
same mechanism. In a report Sawyer (1931) described this type 
of spore discharge in Entomophthora sphaerospenna, parasitic on 




Fig. 30. Stages in spore discharge by Entomophthora sphaerosperma. A. 
Conidiophore with papillar apex. B. Bud-like enlargement at the apex of 
conidiophore. C. The conidium has been delimited bv a septum, and 
there occur two closely opposed membranes. D. The conidiophore tip im- 
pinges into the conidium that has just been freed. E. The tip of the 
conidium becomes everted after release of pressure from conidiophore. 

(After Sawyer.) 

the larvae of Rhopobota vacciniana, attacking cranberries, Vac- 
cinium vmcrocarpon. He noted that a bud, the initial of the 
conidium, forms at the blunt apical portion of the conidiophore. 
Into this developing conidium a nucleus passes, the conidia! wall 
thickens, and a short neck becomes differentiated between coni- 
dium and conidiophore. A septum then forms across the base 
of the spore. This septum consists of two membranes in close 
apposition, one being the basal wall of the conidium, the other 
the apical wall of the conidiophore. As growth continues, the 
greater hydrostatic pressure within the conidiophore forces the 
opposed walls to bulge convexly into the conidium. Eventually 
the pressure becomes so great that the attachment between the 
conidium and the conidiophore is ruptured circumferentially. 



SPORE EXPULSION AMONG PHYCOMYCETES 



185 



The recoil of the basal wall of the conidium against the impinging 
apical wall of the conidiophore acts as a spring, and in conse- 
quence the conidium is violently pushed into space. Its passage 
through the field, when material in the humid atmosphere of a 
Van Tieghem cell is viewed with a microscope, appears like a 







Fig. 31. Schematic diagrams showing stages in sporangial (conidial) dis- 
charge in Basidiobolus ranarum. (After Ingold.) A. Zone of weakness 
apparent near base of subsporangial swelling. B. Sporangium liberated from 
upper part of subsporangium. C. Sporangium freed, but with empty, 
thimble-like subsporangium attached. D. Germination of conidium with 
secondary discharge in progress, a repetitional phenomenon. 

streak of light. The roughened ring, marking where the conidium 
was torn from its attachment of the conidiophore, can also be 
observed readily, the end of the conidium being normally everted 
on lodging. 

The accounts by Levisohn (1927) and Ingold (1934) of the 
mechanism in Basidiobolus ranarum indicate that it is quite dif- 
ferent from that in Entomophthora, being more nearly like that 
in Pilobolus. Basidiobolus ranarum occurs in frog excreta and 
can readily be made to develop and discharge its conidia on ab- 



186 SPORE DISSEMINATION 

sorbent paper in a moist chamber. This fungus possesses a sub- 
conidial bulb, and a line of dehiscence consisting of two mem- 
branes in apposition develops toward the base of this bulb. The 
upper membrane is the more elastic. When, with increased 
turgor inside, the rupture of the conidiophore takes place along 
the line of separation, the upper part of the bulb, which is least 
extensible, contracts, and the basal septum bursts. The effect is 
that the sap is squirted backward, carrying away all parts above 
the line of dehiscence on the recoil. During the rocket-like flight 
the conidium may become separated from the adhering upper 
part of the subconidial bulb or may fail to separate. The conidio- 
phore tip pushes into the conidium at the juncture to effect separa- 
tion, just as it does in Entomophthora. 

Evidently all Entomophthorales, except perhaps Massospora, are 
capable of forcibly liberating their spores. 



SPORE DISCHARGE AMONG ASCO.MYCETES 

Spore discharge among erysiphaceae. As mi^ht be antici- 
pated, cleistocarpous fungi, such as the powdery mildews, require 
a mechanism to liberate their spores that is quite unlike that of 
Pyrenomycetes and Discomycetes. Ingold (1939) has assembled 
the observations made on spore liberation among the Erysiphaceae. 
According to him, there are two types of spore liberation in this 
family. In Sphaerotheca mors-ircae, which illustrates one type, 
the cleistothecium remains dormant throughout winter, but in 
spring the single ascus swells to the extent of causing the cleisto- 
carp wall to rupture, permitting the ascus to protrude through 
the fissure. The protruded ascus continues to swell, finally burst- 
ing in a thin region at the tip and squirting out the ascospores. 

In 1884 the other type of discharge was graphically described 
for Erysiphe by W. G. Smith [Ingold (1939) ] as follows: "When 
they [the cleistocarps] burst, the contained bladders or asci often 
burst at the same time, and the living sporidia, after their six 
months' rest, fly into the air. At other times the bladders or asci 
themselves fly out of the perithecia, and sail, each with its little 
load of eight sporidia, through the air. When in the air, the asci 
burst, and the spores are set free into the atmosphere." This type 
might well be called the rocket type of discharge. The operation 



SPORE DISCHARGE AMONG ASCOMYCETES 181 

of this mechanism, as it applies to Podosphaera leucotricha, has 
been confirmed by Woodward [Ingold (1939)]. 

Discharge among other Ascomycetes. It is a matter of com- 
mon knowledge anions those who have studied Ascomycetes that 
many species of this class forcibly expel their ascospores [Ziegen- 
speck (1926)]. Even though this phenomenon has been ob- 
served in connection with a relatively small proportion of the vast 
assemblage of widely different species that constitute the Asco- 
mycetes, undoubtedly most of them will be found capable of 
such forcible discharge. Many of those who attempt to isolate 
Ascomycetes in pure culture utilize the phenomenon of expulsion. 
They have found that the simplest procedure to employ in isolat- 
ing is to place inverted agar-poured plates above mature perithecia 
at a suitable height. If favorable moisture conditions are then 
provided, an abundance of ascospores will be found to have been 
ejected onto the surface of the agar after a few hours. 

The height to which the ascospores are propelled varies with 
the species, being governed by the size of the spores or of the 
spore mass as one of the correlated factors. Hypomyces lacti- 
fliiorum has been found to shoot its spores to a height of 10 mm, 
Endothia parasitica, 22 mm, Sordaria fimicola, 60 mm, Podospora 
fimiseda, 300 mm, and P. curvicola, 450 mm. In P. curvicola 
Weimer (1917) found that the spore mass of approximately 500 
spores, held together in a gelatinous matrix, had a diameter of 
168 to 266 /x and that they were hurled up into the necks of 
2-liter culture flasks. 

Rate of ascospore discharge. The rate of ascospore discharge 
from perithecia is controlled by the external factors of moisture, 
temperature, and light. These factors are interdependent, and in 
no species does discharge occur unless the water content of peri- 
thecial tissues approximates the maximum. As may be expected, 
the output of spores is low at low temperature and increases to a 
maximum with a rise in temperature. With further increase there 
is a very rapid decline in the rate of discharge. As far as light is 
concerned, some species are stimulated by it, such as Nectria cin- 
nabar'ma and Podospora curvula, whereas others are inhibited, for 
example, Hy poxy Ion fuscum [Ingold (1939)]. 

For a few species the rate of discharge has been recorded. In- 
gold (1939) has assembled certain data on this point; they are 
shown in Table 15. 



188 



SPORE DISSEMINATION 







Fig. 32. Types of spore discharge among Ascomvcctes. A. Podospora 

curvzda (adapted from Ingold). B. Sphaerotheca mors-uvae (adapted from 

Salmon). C and D. CeratostoviclLi ampullacea (adapted from Ingold). E. 

Lecanidion atratum (adapted from Butler). 



SPORE DISCHARGE AMONG ASCOMYCETES 189 



TABLE 


15 






if Ascospore Discharge 


BY 


Several Ascomycetes 
Spore Output per 


Species 


■ 


Perithecium per Hour 


Podospora minnta 






24 


Podospora curvula 






40 


Sporormia intermedia 






184 


Hypoxylon coccineum 






1 , 800 


Diatrype disciformis 






23,000 


Endothia parasitica 






14,000 



Spore discharge among Discomycetes. Evidently the earliest 
observations of ascospore discharge were made upon the larger 
Discomycetes. Micheli (1729) described spore ejection of Peziza 
as being "like smoke." Bulliard (1791) recorded that "their seeds 
ascend like steam," if the observer shakes the fructifications or 
blows his breath upon them. De Albertini and de Schweinitz 
(1805) saw "clouds of smoke" in Rhytisma salicinum. A very 
graphic account of spore discharge by Morchella gigcis is given 
bv Plowright (1880-81), who observed the spore cloud as seen 
against a dark background with the aid of a beam of light: "When 
acted upon by a gentle current of air such as would be produced 
by gently waving the hand, it swayed to and fro without mani- 
festing any tendency to become dispersed. The component spo- 
ridia were in constant motion, rising and. falling and circling 
about, as if the law of gravity were a myth, existing only in the 
imagination of philosophers. When the cloud was quite blown 
away by a more powerful air current, it, in the course of a few 
seconds, reformed." In his chapter on the liberation or purring 
of spores by Discomycetes Buller (1934) assembled many inter- 
esting features from the accounts of early observers and added 
the results of his own observation and experimentation and those 
of his contemporaries [Falck (1916), (1923)]. 

Dickson and Fisher (1923) described a technique for photo- 
graphing discharge by Sclerotinia libertiana that is applicable to 
other large Discomycetes. Buller's (1934) observations show 
that Sarcoscypha protracta can become a miniature geyser, hurl- 
ing a column of spores to a height of about 3 in. before the spores 
lose their vertical momentum and begin to be dispersed into a 
cloud. The puffing by Urmda geaster, having ascomata which, 



190 SPORE DISSEMINATION 

before dehiscence, are brown and cigar-shaped, has earned for 
this fungus the common name, "devil's cigar." Discharge by 
these larger disk fungi creates a blast of air that carries along the 
spores, so that thev appear like a cloud. 

Among the Discomycetes spore discharge is not only visible 
but also audible. An easily perceptible hissing sound is emitted 
by many species. The noise is best heard if the fruit bodies that 
have been maintained in a moist chamber are held near the ear. 
As indicated by Buller (1934), Desmazieres noted the emission 
of sound by Helvetia epihipphnn nearly 100 years ago. De Bary 
noted it in Peziza acetabulum and Hehella crispa, Stone in H. 
elastica, Johnstone in Otidea leporina, and Buller in Aleuria 
re panda, A. vesiculosa, Asco bolus ster cor arms, Caloscy pha ful- 
gens, Ciliaria scutellata, Galactinia badia, Peziza aurantia, Pseudo- 
pie ctania mgrella, Pustidaria cat i mis, Pyronevia con flu ens, Rbizina 
in flat a, Sarcoscypba protract a, S. cor on aria, Urnula crater'unn, 
and U. geaster. The sound produced resembles most nearly the 
fizzing of a freshly drawn carbonated drink. The "effervescence" 
of some species, especially the larger ones, is protracted, lasting 
for several minutes; in others it can be heard for a few seconds 
only. 

Among the Discomycetes known to puff [Buller (1934)] are 
Arachnopeziza aurata, Ascobolus crouani, Cblorospleniuv? aeru- 
ginosinn, Dasyscypha virginea, Helotium scutula, Lachnea setosa, 
Mollisia cinerea, Orbilia xantbostigvia, and Rhytisvia acerinwn. 
The writers have noted its occurrence in Diplocarpon earliana, 
D. rosae, Peziza repanda, Sclerotinia fructicola, and S. trijolium. 

Spore discharge among Pyrenomycetes. De Bary (1887) was 
among the first to assemble the extant information regarding spore 
ejection among Pyrenomycetes. He pointed out that there are 
two types of expulsion: simultaneous and successive. In the first 
type all the spores and much of the fluid content of the ascus 
are ejected as a unit; in the second, each ascospore is discharged 
separately. De Bary augmented his account with his own obser- 
vations. Subsequently many other investigators have noted forci- 
ble spore liberation and have reported their findings with particu- 
lar species. Much of our knowledge of this phenomenon comes 
from the recent studies by Buller (1933) and Ingold (1933, 1939). 
Among Pyrenomycetes the spores of most genera, but not all, 
are forcibly liberated. These fungi may, for convenience, be 



SPORE DISCHARGE AMONG ASCOMYCETES 191 

arranged into spore-liberation types [Ingold (1933)]. In the first 
type are species of Chaetomium, Ascotricha, Daldinia, and Dia- 
trype and Ceratostoviella fimbriata. Their ascus wall is very deli- 
cate and ephemeral, and as a consequence the ascospores are freed 
and lie intermixed with gelatinous material within the perithecial 
cavity. The gelatinous material absorbs water readily and swells, 
and the spore mass is squeezed out through the ostiole, like tooth- 
paste from a tube. 

The second type, first correctly described by Zopf [de Bary 
(1887)], occurs in Sordaria and Podospora and certain other 
coprophilous species, which develop on the dung of herbivorous 
animals [Griffiths (1901)]. The perithecia are pear-shaped, and 
the ostiolar canal is lined with hyphae directed toward the open- 
ing. The perithecial walls are thin enough for spore discharge 
to be satisfactorily observed. Ingold (1939) mounted entire 
perithecia of Podospora curvula in water in a hanging drop 
and noted that they contain asci in different stages of maturity. 
The asci and interspersed paraphyses are attached to a stroma 
occupying the bottom of the perithecium and remain attached 
to this stroma during discharge. On looking through the peri- 
thecial wall, the observer may note that mature asci elongate by 
growth and by the pressure exerted by the surrounding cells. 
The greatly distended elastic ascus extends into the neck canal, 
and the ascus tip slips through, being "lubricated" by the hyphae 
within the canal, until it protrudes slightly beyond the ostiole. 
At this stage the tip of the ascus opens by a circumscissile rup- 
ture, and the cap formed, together with the mass of 8 ascospores 
and much of the ascus-sap, is shot upward. Immediately after 
discharge the empty ascus, being attached to the basal stroma, 
snaps back inside the perithecium, and another ascus elongates, 
opens, discharges, and is withdrawn seriatim, until the perithecial 
content is exhausted. Since each ascospore of P. curvula possesses 
two terminal gelatinous appendages that become entwined, the 
spore mass is a rather large projectile and can be hurled for a dis- 
tance of 20 cm or more. The neck of the perithecium being 
phototropic, the ascus content is discharged directly toward the 
source of li^ht. 

The third type has asci of "jack-in-the-box" construction, as 
aptly designated by Ingold (1933). This type has many variants, 
but in each kind the elongated ascus extends to the exterior of the 



192 SPORE DISSEMINATION 

perithecium. It was first correctly described by Pringsheim 
(1858) from observations on Fleospora scirpicola (Sjphaeria 
scirpi). Since then this type of discharge has been observed by 
numerous mycologists in various genera, and the accounts of 
Hodgetts (1917), Weimer (1920), Atanasoff (1919), Ingold 
(1933, 1939), and Butler (1939) may well be consulted. In her 
account Butler lists the following fungi as having jack-in-the-box 
dehiscence: Ascospora beijerinckii, A. ruborum, Cucurbit aria 
laburni, Lecanidion atratum, Leptosphaeria acuta, Metasphaeria 
asparagi, Mycosphaerella rubina, Fleospora herbarium, Pl&wrightia 
ribesia, Physalospora malorum, Sphaeria inquinana, S. ellipsocarpa, 
S. lanada, S. lemaneae, Sporormia bipartis, and Venturia iuaequalis. 
Such dehiscence has been noted in many other pyrenomycetous 
£enera and also in several discomycetous ones. 

The essential structure that makes this type of discharge pos- 
sible is the double ascus wall, consisting of an outer, thick, fairly 
riqid, inextensible membrane, sometimes called the ectoascus, and 
an inner, thin, elastic membrane, the endoascus. It may be im- 
possible to distinguish the membranes as entities until the moment 
discharge is begun. At maturity the ascus imbibes water as the 
result of transformation of stored olycooen into osmotically active 
compounds. Endosmosis occurs, but the outer ascus membrane 
does not permit any considerable stretching to increase the di- 
ameter. Enlargement proceeds to the point where the ectoascus 
is ruptured, whereupon the endoascus suddenly elongates to one 
to three times its original intact length. 

There are several types of rupture of the ectoascus. In Lecani- 
dion atratum the tip of the ascus is lifted off, forming a thimble- 
like cap at the tip of the endoascus. The remainder of the ecto- 
ascus slips down toward the base of the ascus, or its edge is 
folded or rolled as stockings are by some wearers. 

In Mycosphaerella a thin place may appear in the ectoascus 
wall, in some species near the tip, in others well down along the 
side. Rupture takes place at this thin area when sufficient in- 
ternal pressure has been developed, and the ectoascus tip persists 
as a flap at the side of the extended endoascus. At any rate, the 
sudden release of the endoascus permits its apex to spring through 
the ostiole. If, as in Sporormia, the ascospores are to be dis- 
charged simultaneously, the further increased pressure ruptures 
the ascus tip, and the spores are squirted en masse. If, as is more 



SPORE DISCHARGE AMONG ASCOMYCETES 193 

common, the spores are to be discharged successively, they be- 
come compressed into a single row with long diameters lying in 
the direction of the longitudinal axis of the ascus. Then a con- 
tractile pore forms in the apex, and each spore is ejected endwise. 
Discharge of the 8 spores requires a period of a few seconds to a 
minute or two. While the first half of the ascospore of Mycos- 
phaerella is passing through the contractile pore, its velocity is 
diminished, and it has been observed to stop momentarily at the 
constriction. Its rate of ejection increases as the second half 
passes through the pore, and the spore is snapped into space, some- 
what after the fashion of a watermelon seed when compressed 
between the fingers. The next spore in line instantly plugs the 
pore, and the process is repeated until all 8 are ejected. The 
empty ascus then contracts, and its place is taken by another 
mature ascus or complement of mature asci. 

A fourth type of discharge is exhibited, especially by long- 
necked or rostrate Pyrenomycetes. In this type, as illustrated by 
Gnomoma riibi, Ophiobolus careciti, Endothia parasitica, and 
Ceratostomella ampallacea [Ingold (1939)], the asci at maturity 
become detached and for a time remain free and intact within 
the perithecial cavity. As more asci are formed from the stro- 
matic tissue within the basal portion of the perithecium, they be- 
come freed and displace those first detached. As a consequence 
a stream of asci is squeezed into the long neck canal, the asci pass- 
ing up in single file. In Ceratostomella ampiillacea the asci swell 
quickly as soon as they protrude from the ostiole, the lower end 
being firmly held by the rigid jaws of the ostiole. The ascospores 
are dispelled by the bursting of the ascus, and the empty ascus is 
pushed out by the next ascus in the series, and so on. In some 
rostrate species, such as Linospora gleditsiae, the asci collect in a 
mucoid droplet at the orifice of the ostiole and must be dissemi- 
nated by water. 

Still another structural mechanism has been described in other 
Pyrenomycetes. In Glomerella, for example, the ascus is apically 
thickened, and exit is provided through a papillar perforation. 
The ectoascus remains intact. As the intra-ascal pressure increases, 
the thickened pore resists stretching, but the ascospores are 
squeezed through the perforation. As each emerges at the tip of 
the pore, it is snapped into space. 



194 SPORE DISSEMINATION 

SPORE DISCHARGE AMONG BASIDIOMYCETES 

Most of our knowledge of spore discharge among the Basidio- 
mycetes comes from the painstaking researches of Buller (1924, 
1933). Throughout this entire group with its numerous species, 
except for the Gastromycetes, which is constituted of relatively 
few species, essentially the same mechanism of discharge prevails. 
This generalized mechanism has been termed the "drop-excretion 
mechanism." As a matter of fact, no satisfactory explanation of 
how this mechanism causes the spores to be forcibly expelled is 
as yet forthcoming, but the problem can be properly appreciated 
if the structure of the basidium is first learned and is kept clearly 
in mind. The hymenium, whether plane or having pores, gills, 
teeth, or other modification to increase the spore-bearing surface, 
is composed of a palisade of basidia. In some species sterile cells 
(paraphyses or cystidia) are interspersed among the basidia. Each 
basidium is a turgid clavate to saccate cell. Apically on this cell 
are formed typically four conical projections, the sterigmata. 
The tip of each sterigma soon becomes slightly bulbous, and the 
inflated portion increases, simulating the appearance of a soap 
bubble being blown. These portions are the basidiospores, which 
vary in shape and surface markings among the different species. 
Mature basidiospores are always inequilateral, with the more plane 
surfaces of the quartet of spores directed toward each other. The 
hilum of each points inward and is thus asymmetrically placed. 
Presumably a wall eventually forms to separate the basidiospore 
from the tip of the sterigma. 

As far as the structural features just recounted are concerned, 
all investigators are in accord. In connection with discharge it- 
self and the forces involved, however, there remain unexplored 
possibilities. For a long time it was generally believed that a 
water-squirting mechanism somewhat comparable to that in Pilo- 
bolus causes discharge. This would be expected to operate most 
effectively if all 4 spores of a basidium were discharged simul- 
taneously. As a matter of fact, the basidiospores are discharged 
successively one at a time. Of course this type of mechanism 
might still be capable of operating to discharge the spores succes- 
sively if the tip of the sterigma were to be sealed before appreci- 
able loss of turgor within the basidium. That there is actually no 



SPORE DISCHARGE AMONG BAS1DIOMYCETES 



195 



loss of turgor of the basidium which can be recognized by change 
in shape and size is shown by Buller's observations on a rather 
large number of species. He found in all cases that the spores 





n 



i ■ \ 




u 



II 




# 



» r 









„M^, 



• 



G 



Fig. 33. Diagrams of apparatus and materials used by Buller to secure 
evidence of forcible discharge of basidiospores by Hymenomycetes. A. 
Threads in ocular of microscope. B. Section of pileus to be mounted in 
compressor cell; C, shown in sectional view. D. Compressor cell in 
vertical position as seen through a horizonally placed microscope. There 
is a bit of pileus near the top of the cell, moist paper at either side, and 
water at the bottom. E. Basidium as seen with such a horizonal microscope, 
one spore discharged. F. Diagram showing paths of discharged basidiospores 
(indicated by arrows) and the necessity of vertical arrangement of gills if 
spores are to fall unimpeded. G. Stages in basidiospore discharge as seen 
when viewed from hymenial surface. (After Buller.) 

are violently discharged from the basidium in succession. More- 
over, just before each spore is expelled, a drop of liquid exudes 
at the hilum. During a period of a few seconds this drop increases 



196 



SPORE DISSEMINATION 



in volume, and when it reaches a definite size, the spore is shot 
away, carrying the drop with it. Drop excretion mav be^in at 
the hilum of one or more of the other members of the quartet 
before the first spore is discharged, and only a few seconds or 
minutes elapse between successive discharge of each member. 
The sterigmata are turgid after discharge, and apparently the tips 




li TTrmniMiii ijj-. 






l—"V/s :••"». 



.-•,»•'*'?. ■'■•**. , "'>v. ; : -'.'£. . : . • 
.•*«.••■.•■.•■.•:»•>. * .;• - . 

■■■?•■ ■.•■:.■.:■>■. \<*i ■ ' ■ 

• "• ■•' --l.^ ■'•• '•■• • • 

• .-j&pmsc .>•••■■ -r 

»«* •--.:■■?;?•. ..." ■ 






■ • >.■ ■ 

■'::•■' 

. u--v. 






Fig. 34. Two tvpes of evidence of spore discharge among Hvmenomvcetes. 
A. Diagram of a spore print of an agaric, made bv placing the ventral 
surface of the pileus horizontally on white paper in a moist chamber. B. 
Diagram to indicate how spore discharge mav be observed in a beam of 
light. A fragment of pileus is attached to cork, fastened to cover of vessel. 
Basidiospores are circulated bv convection currents. 

are sealed. Afterward the basidia mav slowly lose turgor, but 
they collapse only when death occurs. 

This description of the structures involved and the sequence 
of events does not offer any explanation to account for the asym- 
metrical position of the spore on its sterigma, as Ingold (1939) 
points out, nor does it explain how the drops are excreted. Con- 
cerning the force employed to discharge each spore, Bullcr (1922) 
believes that it is caused by surface tension energy. From ingeni- 
ous experiments and from calculations he found that the surface 
energy on a drop of exudate on the spore of Psalliota cavipestris 
is 0.000012 erg. To derive this figure the value of surface tension 
is considered as 72 on a drop 2.3 \x in diameter with a surface area 
of 0.000000166 sq cm. Not all this energy is available for dis- 



SPORE DISCHARGE AMONG BAS1DIOMYCETES 



191 



charge, because the drop is a hemispherical mass in contact with 
the spore. If the surface tension between the surface of the spore 
and the drop is considered negligible, the surface energy of the 
hemispherical drop is 0.0000095 erg. Then the difference between 
0.000012 erg and 0.0000095 erg is 0.0000025 erg. This energy is 
calculated to be seven times that necessary for the actual initial 
velocity of the spore when it is 
liberated. Ingold (1939) explains 
how this energy is mobilized to 
break the connection of the spore 
with the sterigrna and to dis- 
charge it as follows: "At the mo- 
ment of spore discharge the drop 
excreted at the hilum flows to the 
side of the spore, and, while this 
is happening, the spore will tend 
to move in the opposite direction. 
This would involve pressure of 
the spore on the end of the 
sterigma. This pressure, sud- 
denly exerted, might lead to the 
springing of the spore into the 
air just as one jumps from the 
ground by pressing suddenly 
downward. . . . Only a small 
fraction of the available surface- 




Fig. 35. Spore discharge, in dia- 
gram, of Psalliota ccnnpestris. 
(After Ingold.) A, spore on 
sterigma, B, just before discharge. 
The exuded droplet of liquid, C, 
is of full size. During discharge 
the drop, C, takes up position, D, 
and is carried away on the side 
of the basidiospore. 



tension energy is required to im- 
part the necessary initial velocity to the spore, and the remainder 
is available for breaking the connection between the spore and 
its sterigma." 

Spore discharge in smuts. The chlamydospores of smuts are 
pulverulent, except in a few species. Air currents constitute the 
primary factor in the dispersal of these spores. Forcible expulsion 
of sporidia has not been noted among the Ustilaginaceae. Among 
the Tilletiaceae, however, Buller (1933) and his associates have 
studied violent spore discharge in Tilletia tritici, T. laevis, T. hor- 
rida, T. hold, T. asperifolia, Entyloma ?nemspermi ? E. lobeliae, 
and E. linariae. When a chlamydospore of these species germi- 
nates, a short mycelium, generally regarded as the basidium, is 
produced. At the tip of this mycelium a cluster of about a dozen 



198 



SPORE DISSEMINATION 



filiform cells, which have generally been regarded as basidiospores, 
is formed. Buller, however, regards them as steri^mata of a 
highly specialized type for two reasons: (1) they are never shot 
away and therefore do not serve to disseminate the fungus, and 




Fie. 36. Spore discharge by Tilletia tritici. A. Germination of chlamvdo- 
spore, formation of special sterigmata that have fused in H-shaped fashion, 
and stages in formation of true basidiospores or sporidia. B. Tip of sterigma 
with mature basidiospore and a droplet of fluid that serves in forcible dis- 
charge. C. Basidiospore that has just been discharged with droplet clinging 
to base of basidiospore. (After Buller.) 

(2) while still attached to the basidium, they oive rise to sickle- 
shaped spores, asymmetrically placed, that are forcibly expelled. 
These spores may cause infection, and Buller consequently regards 
them as the true basidiospores of the Tilletiaceae. 

As has often been observed, the specialized sterigmata may form 
H-shaped conjugations. Either from these pairs or from a single 
unpaired member, a septate hypha may arise, from which the 
sickle-shaped basidiospores are abstricted. In T. tritici these 



SPORE DISCHARGE AMONG BASIDIOMYCETES 199 

basidiospores are propelled to a vertical height of 1 .0 mm and to a 
horizontal distance of 1.4 mm. 

Spore discharge in rusts. Klebahn (1904) is the first investi- 
gator to point out that the basidiospores of rusts are forcibly dis- 
charged. Dietel (1912) later recorded the same phenomenon in 
connection with Puccinia malvacearum, P. glechomatis, P. annu- 
laris, Coleosporhmi campamdae, C. petasitidis, and Cronartium 
asclepiadeam. In some instances he observed that a tiny droplet 
of water appears at the tip of the sterigma immediately before 
discharge. He determined that under normal conditions basidio- 
spores may be shot about 0.3 mm vertically and 0.6 mm horizon- 
tally. For Gy?nno sporangium juniperi-virginianae Coons (1912) 
recorded a horizontal distance of discharge of 0.26 to 0.36 mm. 
Shortly thereafter Buller (1924) made a detailed study of spore 
discharge, especially in Puccinia graminis and Endophyllum 
euphorbiae-sy haticae. In all essential features the phenomena of 
discharge among the Hymenomycetes, which he had previously 
studied, and those in the Uredinales are alike. As differences be- 
tween the two groups, he notes that the rust basidiospores are 
larger and are usually shot farther, the distances being 0.4 to 0.85 
mm in the rusts and 0.05 to 0.2 mm in the fleshy Hymenomycetes. 
In correlation with these differences he noted that the droplet 
of water exuded at the spore hilum in Uredinales is somewhat 
larger and requires from 10 to 40 seconds to form, whereas in 
Hymenomycetes only 5 to 10 seconds is usually required. 

Later Prince (1943) reported that expulsion of basidiospores by 
Gymno sporangium nidus-avis is accomplished by a different 
mechanism from that described by Buller for other rusts. Prince 
concluded that the mechanism is quite like that among Entomo- 
phthoraceae. The basidiospore arises as an enlargement of the 
apex of the sterigma, so that the primary membrane is common 
to the spore and the sterigma. When the spore attains mature 
size, a septum is formed in the sterigma that delimits the spore and 
leaves an apiculus at its base. Next a wall is laid down inside the 
spore and also one below the septum. Pressures built up in the 
apiculus of the basidiospore and in the apex of the sterigma rup- 
ture the primary membrane at the septum, and the instantaneous 
opposed bulging of the end of the apiculus and of the sterigma 
results in forcible discharge of the spore. 



200 SPORE DISSEMINATION 

From the evidence it seems entirely probable that Uredinales 
generally are capable of violently discharging their basidiospores. 
The urediniospores are powdery and are disseminated by wind. 
Evidently aeciospores in many species are projected out of the 
aecia with considerable force. Attention was first directed to the 
matter of forcible ejection of aeciospores by Zalewski (1883) 
from observations on Uromyces pisi, whose aecia are borne on 
Euphorbia. He also showed from experiments that Puccinia 
graminis, P. calystegia, P. coronata, and Aecidium symphyti dis- 
charge their aeciospores, the oldest, outermost aeciospores of the 
chain being discharged first. Dodge (1924) observed the same 
phenomenon in Gymnotelium myricatum and Puccinia podo- 
phylli. From similar studies Buller (1924) recorded its occur- 
rence in XJroviyces poae, Puccinia clematidis, P. fraxinata, P. gros- 
sulariae, P. graminis, P. hieraciata, P. impatientis, P. poarum, P. 
pulverulenta, and P. urticata. The mechanism by which expulsion 
is made possible consists of thickenings of the spore walls, which 
push into the spore wall opposite. These thickenings serve as 
fulcra, against which the elastic spore walls react. As the upper- 
most cells approach maturity, the pressure may be suddenly re- 
leased above, whereupon the aeciospore is shot out. Sometimes 
masses of spores are expelled. The aeciospores of P. graminis 
may be discharged to a height of 7 to 8 mm, of Uromyces pisi, 
15 to 20 mm. Thus far observations have dealt with cupulate 
and caeomoid aecia, no studies having been made of rostelioid 
and peridermioid aecia, in some of which the peridial layer reacts 
to moisture, and the hygroscopic movements of peridial segments 
expel the aeciospores. 

Spore discharge among Hymenomycetes. Of course the 
simplest procedure to demonstrate that Hymenomycetes shed 
their spores is to place the pilei with undersurface downward on a 
piece of white paper to secure a spore print or to focus a beam 
of licrht below the fruit body suspended in a closed glass vessel. 
Practically all our knowledge of violent spore discharge among 
Hymenomycetes has come from the studies by Buller, conducted 
over a period of about 30 years and recorded in his Researches on 
Fungi. In this period he examined numerous genera and species, 
including such well-known and widely distributed species as 
Psalliota cavipestris, Coprinus comatus, C. atramentarius, Poly- 
porus squaviosus, Lentinus lepideus, Psathyrella disseminata, Ar- 



SPORE DISCHARGE AMONG BASIDIOMYCETES 201 

miliaria mellea, Amanitopsis v agin at a, Rnssnla emetic a, Panus stip- 
ticns, and Pleurotiis ostreatus. All exhibit the following features 
during basidiospore discharge: (1) the four spores are discharged 
in succession, not simultaneously; (2) a droplet of exudate appears 
at the hilum of the basidiospore just before discharge and is absent 
on the sterigma after discharge. It is carried along with the spore 
and disappears as the spore strikes, causing it to adhere; (3) the 
sterigmata and basidium do not collapse as the spores disappear. 

Violent basidiospore discharge is an important phenomenon in 
this group because the spores, when liberated into the space be- 
tween gills or spines or into pores, are prevented bv the position of 
the pileus from touching each other or the hymenial surface. 
Thev thus escape from the pilei. Each is shot horizontally for a 
short distance, the motion being rapidly terminated because of 
resistance of the air. In Amanitopsis vaginata horizontal move- 
ment of the spore is completed in 1/400 second [Builer (1909)], 
and the initial velocity approximates 40 cm per second. When 
horizontal movement is at an end, the spores react in response to 
gravity. Builer observed the rate of fall of basidiospores by use 
of a horizontally placed microscope. He mounted sections of 
hymenium in a chamber and placed the chamber on the micro- 
scope stage. The hymenium was thus vertically disposed. Three 
silk threads were then attached to the eyepiece at equal distances 
from each other across the field of view. Records of the velocity 
of spores passing through the field of view could then be made on 
an electrically rotated drum connected with a tapping key that 
could be depressed by the observer. By this means Builer (1909) 
found that the velocity of fall in millimeters per second for 
Colly bia dryophila was 0.37, for Plntens cervinus, 0.67, for Psal- 
liota campestriSy 1.61, for Poly poms squamosns, 1.03, for Boletus 
felleus, 1.22, for Rnssnla emetica, 1.64, for Amanitopsis vaginata, 
2.95, and for Coprinns commatus, 3.96. Small spores fell at a 
slower rate than larger spores. These rates of fall were found 
to be considerably greater than expected from calculation by 
Stokes' law, a discrepancy for which Builer was unable to offer a 
satisfactory explanation. Presumably it is in part related to dimi- 
nution in volume of the mass (spore plus droplet) as fall proceeds. 

Among other interesting facts established by these studies on 
violent spore discharge among Hymenomycetes is that, so long 
as corky and woody pilei have sufficient moisture, they may con- 



202 SPORE DISSEMINATION 

tinue to shed spores. Species of Lenzites, Daldalea, Schizophvl- 
lum, Polvstictus, and Stereum, after having been dried for as long 
as a year or two, may be revived in the presence of moisture, 
\\ hereupon spore discharge is renewed. In the presence of vapors 
of ether pr chloroform spore discharge ceases. Such reactions 
leave no doubt that discharge is a vital phenomenon. 

The pilei of species of Coprinus are bell- or thimble-shaped. 
Their srills undergo autodigrestion, commonly regarded as del- 
iquescence. This process is a very important adaptation to insure 
escape of the spores into the air, which is accomplished because 
the spores on each gill mature and are discharged progressively 
from the outer edge of the gill toward the stipe. Those portions 
of the gills from which the spores have been shed are digested and 
removed soon after discharge, and in consequence space is pro- 
vided for the shedding of spores just above, as the pilei continue 
to open outward like the opening of an umbrella. 

Spore discharge among Gastromycetes. The Gastromycetes 
include a group of species whose best-known members are called 
"purTballs" or "snuffboxes." The spore mass of the larger propor- 
tion of species in this subclass is dry and powdery and therefore 
admirably adapted for dissemination by air currents. The hygro- 
scopic movement of capillitia aids in spore expulsion in certain 
species. Some few are subterranean, and their spores are scat- 
tered by rodents or burrowing animals that find the fruit bodies 
attractive as food. Another group, the stinkhorns, possesses a 
glebal or spore-bearing portion which is attractive to carrion 
flies because of its putrid odor. These stinkhorns appear to de- 
velop overnight, but actually the "eggs," encased in a protective 
membrane or volva, have gradually been developing in the decay- 
ing leaf mold. When the volva is ruptured, the spongy stalk or 
receptacle, capped with the gleba, rather suddenly elongates in a 
jack-in-the-box fashion. De Bary thought that this straightening 
out or elongation of the stalk was caused by inflation from gas 
within the tissues. Burt (1897) determined, however, that the 
stretching is an osmotic phenomenon and that it occurs coincident 
with the disappearance of a reserve of glycogen in and about the 
receptacle, whose cells merely increased rapidly in size. 

These modifications in stinkhorns to insure spore dispersal are 
much less spectacular and remarkable than those in Sphaerobolus. 
Members of this genus occur on rotton wood and on the dung of 



SPORE DISCHARGE AMONG BASIDIOMYCETES 



203 



such herbivors as rabbit, horse, cow, and elephant. In 1729 Micheli 
in his Nova Plantarum Genera first described and illustrated 
Sphaerobolns stellatus in his Plate 86, but he employed for it the 
name "carpobolus." Fischer (1884^ gave an account of the struc- 





Fig. 37. Structure of Sphaerobolus stellatus in diagram. A. Section of 
mature sporocarp, with six (1-6) layers that invest the central peridiole (7). 

B. Dehiscence of sporocarp at apex. The inner membrane has liquefied. 

C. Eversion of the remaining two inner layers, by which the peridiole, D, is 

hurled away. 

tural mechanism by means of which the gleba, about the size of a 
BB shot, is discharged. Later Walker (1927), Walker and Ander- 
sen (1925), and Buller (1933) have painstakingly and graphically 
worked out the details of the mechanism of this veritable fungus 
"trench mortar." 

The peridium or wall consists of six layers: (a) an outer layer 
of loosely interwoven hyphae, (b) a gelatinous layer penetrated 
by hyphae, (c) a compact pseudoparenchymatous layer, (d) a 
narrow layer of tangentially ramifying threads, (e) a layer of 



204 



SPORE DISSEMINATION 



radially arranged pseudoparenchyma, and finally (f) a thin layer 
of small-celled pseudoparenchyma. 

Within this multiple periderm is the glebal mass. At maturity 
the peridium splits and bends outward to expose the gleba. The 
fruit body now consists of two tooth-rimmed cups, one fitting 
inside the other and the two joined at the tips of the teeth. The 
outer cup consists of the three outermost layers. The innermost 




Fig. 38. Feeding tracks made by the snail Polygyra thyroidcus on lilac 

leaves infected with powdery mildew. 

layer liquefies, and this liquid accumulates around the spherical 
glebal mass, which is now free and can be rolled around in the 
cup. The tanoentially arranged layer and the radially elongated 
layer are left to constitute the inner cup. Everything is now ready 
for discharge, provided that there is light, a temperature approxi- 
mating 90° F, and high relative humidity. If these conditions pre- 
vail, the inner cup suddenly everts itself and in so doing hurls the 
gleba away. If the fruit body is tilted to get the most suitable 
trajectory, the gleba may be projected a horizontal distance of 546 
cm. Sphaerobolus holds the long-distance record among fungi 
for spore projection. 

The inner cup may be ejected along with the glebal mass, but 
more often it remains as a p;listenin«; dome. i\fter a time it may 
assume its original position. Wax or plasticine spheres can then 



IMPLICATIONS 205 

be substituted for the natural projectiles, and the "trench mortars" 
can be repeatedly operated with these artificial projectiles. 

The tensions that are responsible for eversion of the inner cup 
arise from transformation of glycogen [Errera (1885), Walker 
and Andersen (1925)] into reducing sugars. Up to the time that 
the peridium splits open, the radially arranged palisade tissues are 
filled with glycogen, but it has all been digested by the time dis- 
charge takes place. In conclusion it may be mentioned that spe- 
cies of Sphaerobolus are to be regarded as among the most fasci- 
nating objects of study among fungi. 

Evidence indicates that the peridioles of Nidulariaceae are dis- 
seminated by rain splash and adhere to near-by objects by means 
of their glutinous coating, as was noted by Diehl (1941). He 
found that the lower leaves of camellia bushes beneath which 
Cyathus pallidus was fruiting on fragments of wood were studded 
with black, button-shaped peridioles. xMore remarkable, perhaps, 
is Diehl's (1941) re-examination of specimens of a fungus on 
camellia leaves collected over 100 years ago and identified as 
Leptostroma camelliae. This fungus proved to be not a pycnidial 
form but the peridioles of Cyathus st ere or ens. 

IMPLICATIONS 

The studies that have been made on dissemination of fungus 
spores are essentially of two types: (1) those that deal with the 
structural mechanisms involved and with the manner in which 
these mechanisms function; and (2) those that deal with the 
vectors or agencies of dissemination. Spore dissemination is of 
consequence to each particular fungus because the perpetuation 
of that species requires that spores be dispersed. Perpetuity is in- 
sured, all other factors being favorable, if spores are brought into 
contact with new sources of food. - 

A study of spore dissemination becomes meaningful mainly in 
relation to the occurrence and relative abundance of diseases of 
plants and animals and in relation to the geographical distribution 
of the given fungus. The student may even wonder why plant 
and animal diseases are not more prevalent and why more fungi 
are not ubiquitous in distribution, when once he appreciates that 
many species are incredibly profligate in the production of spores. 



206 SPORE DISSEMINATION 



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Zalewski, A., "Uber Sporenabschnurung und Sporenabfallen bei den Pil- 

zen," Flora, 66: 268-270, 1883. 
Ziegenspeck, H., "Schleudermechanismen von Ascomyceten," Botan. Arch., 

75:341-381, 1926. 



Chapter 9 
GERMINATION OF SPORES 

The process of germination of spores is generally regarded as 
belonging among growth phenomena and hence being subject to 
modification by all those factors that influence growth. Spore 
germination has much in common with seed germination, as might 
be anticipated, and much of value has been learned by mycologists 
from the techniques and interpretations of those who have studied 
the termination of seeds. Manifestly the factors that affect the 
orermination of spores, just as that of seeds, are of two types: 
hereditary or internal, and environmental or external. Hereditary 
factors include the maturity", longevity, dormancy, and vitality 
of spores. The environmental factors include the influence of 
moisture, temperature, pH, kind and concentration of nutrients, 
light, and the presence of oxygen and carbon dioxide. 

Both saprophytic and parasitic fungi have been used in spore- 
germination studies, more especially the parasitic, because weather 
conditions are known to influence the incidence and relative 
prevalence of plant-disease outbreaks. In attempts to evaluate the 
relative importance of environmental factors to plant diseases, 
the pathogens have been grown in culture, and, as an incidental 
result, our knowledge of spore germination has been increased. 

GERMINATION TYPES 

Different kinds of spores germinate differently. Sometimes 
the type of germination is characteristic of a large number of 
closely related species. In other cases environmental factors exert 
a controlling influence on the type of germination within the 
same species. Among the aquatic Phycomycctes each propaga- 
tive element is at first a mother cell whose content breaks up 
into intracellular units of protoplasm, which, after escape from 

210 



GERMINATION TYPES 211 

the mother cell and after one or more motile stages, become 
transformed into the assimilatory phase or thallus. In the simplest 
Phycomycetes this transformation is accomplished by the imbi- 
bition of water, after which the protoplast increases in volume, 
although remaining within the stretched parent-cell membrane; 
more nuclei and more protoplasm are then formed. The spore 
thus becomes the unicellular, coenocytic, spherical thallus. The 
transition from germination to subsequent growth is so imper- 
ceptible that there is little or no evidence of delimitation of the 
two. Among the higher Phycomycetes, for example, species 
of Albugo, Phytophthora, Plasmopara, and Sclerospora, the con- 
tent of the mother cell (sporangium) may break up into intra- 
sporangial elements (spores), or the sporangium may germinate 
by the production of a germ tube, depending upon temperature, 
as one of the controlling factors. 

All spores absorb water and swell as an initial step in germina- 
tion. In most species a germ tube, the primordium of the myce- 
lium, is then formed. In some, however, reserve food in the form 
of droplets of oil can be noted to disappear as the protoplasm 
moves into the developing germ tube. If the spore contents in 
their entirety migrate into the hypha, an empty spore cavity 
devoid of living content is formed. 

In the Erysiphaceae, Peronosporaceae, and Uredinales the germ 
tube ceases to grow as soon as the reserve food is exhausted un- 
less a nutritive relationship with an appropriate host plant has 
been established. If nutrient is available, either from the host 
tissues or from the culture medium, the germ tube continues to 
grow, becomes branched, and otherwise assumes the character- 
istics of the parent thallus. 

The germ tube of the chlamydospores of Ustilaginales and of 
the teliospores of the Uredinales is, however, a promycelium or 
basidium. Its growth is determinates The promycelium produces 
sporidia which may germinate by tube formation. Among the 
Ustilaginaceae the sporidia may germinate by budding. Among 
the Tilletiaceae the thread-like elements (so-called sporidia) are 
regarded by Buller as sterigmata of a specialized type, wmich pro- 
duce the true sporidia. In species of Taphrina and in Gloeo- 
sporhtm aridum, Microstroma juglandis, Protocoronospora (Ka- 
batiella) nigricans , Catenophora pruni, Dematium pallulans, Poly- 



212 GERMINATION OF SPORES 

spora lini, and many other fungi, the spores may germinate by 
buddine and may continue to grow by budding. In a goodly 
number of species the spores first form a tube, and subsequent 
growth is wholly or in large part by budding. 

Some spores require both water and a supply of nutrient sub- 
stances before they can be made to germinate. A lining of proto- 
plasm remains in their spore cavity, and the spore becomes an 
integral part of the mycelium. 

Spores seldom form more than a single germ tube or at most 
a few. Exceptions are the multicellular spores or large spores. 
Each cell of a multicellular spore normally behaves as an entity. 
Lar«e spores, such as those of Pertusaria and Megalospora, may 
develop simultaneously fifty or more germ tubes. The tubes in 
these genera are emitted through pores in the thick wall, but they 
possess no other peculiarities. 

METHODS OF TESTING SPORE GERMINATION 

Hoffman (1860) initiated the hanging-drop technique, which 
now employs Van Tieghem cells and which is now widely used 
in spore-germination tests. It is employed successfully not only 
with liquid but also with semisolid media. This method has cer- 
tain obvious advantages over the use of drops of water containing 
spores and placed on microscopic slides and over the implanting 
of spores on media in Petri dishes. None of these features seems 
to be so important as an understanding of how to secure repro- 
ducible results of germination trials. It is quite apparent that 
there is little accord among the results of investigations on spore 
germination. As McCallan and Wilcoxon (1932) have shown, 
the variations in spore germination that have been reported may 
be attributed to two causes: faulty technique and variation in 
sampling. Among the common errors which McCallan and Wil- 
coxon enumerate are: (j) failure to state the number of spores 
counted, either germinated or not germinated, making it impos- 
sible to determine from statistical analysis the decree of siimiri- 
cance to attach to the results; (b) counting the control germina- 
tion as 100 c ; and adjusting the treatments accordingly, thus pre- 
venting adequate comparison, because small differences between 
controls will result in large differences between treatments; (c) 



METHODS OF TESTING SPORE GERMINATION 213 

expressing the results of progressively changing treatments plotted 
against germination of spores as a jagged curve, whereas this curve 
should be smooth if a sufficient number of spores has been counted. 

In regard to faulty technique it is essential that such environ- 
mental factors as temperature, time, nature of the medium used 
in germination, and cleanliness of glassware be controlled. More- 
over, uniformity of source and age of spores and density of spore 
suspension should be given attention. Repetitions of tests on 
different days may yield variations whose causes are not well 
understood. 

In connection with differences in results between duplicated 
germination tests, it is possible to determine whether these differ- 
ences are real and also to compute the degree of significance of 
differences. Differences attributable to variations in sampling have 
been found to follow a mathematical law. By use of a formula, 
that is, by making the Chi-square test (X 2 ), and by reference to 
the tables of Fisher (1930, pp. 75-98), which give the probability 
of occurrence of such values of X 2 , the variations caused by errors 
in sampling can be evaluated. The procedures involved in these 
computations are not complicated, although detailed explanation 
of them is wholly beside the purpose of this chapter. 

Some fungi do not seem to require that energy-yielding ma- 
terials be present for germination, as noted by Lin (1940), using 
conidia of Sclerotinia fructicola. Other species, however, are 
found dependent upon the presence of sugars and minerals. Lin 
(1945) demonstrated that the conidia of Glomerella cingiilata re- 
quire carbon, magnesium, nitrogen, and phosphorus. He supplied 
carbon as dextrose in 0.01% solution and minerals in 1.0 millimols 
with the results shown in Table 16. 

TABLE 16 

Nutritional Requirements for Germination of Glomerella cingulata 



Substance Supplied 

Redistilled water 

Dextrose 

KXO3 + KH2PO4 + MgS0 4 

Dextrose + KNO3 + KH 2 P0 4 + Na 2 S0 4 

Dextrose + KXO3 4- KC1 4- MgS0 4 

Dextrose + KC1 + KH 2 P0 4 4- MgS0 4 

Dextrose + KNO3 4" KH 2 P0 4 4" MgS0 4 





Percentage of 


Element Lacking 


Germination 


Carbbn and minerals 


0.0 


Minerals 


0.0 


Carbon 


0.7 


Magnesium 


0.9 


Phosphorus 


1.5 


Nitrogen 


3.9 


None 


92.8 



214 GERMINATION OF SPORES 

HEREDITARY FACTORS AND GERMINATION 

Many observations on the maturity, longevity, dormancy, and 
vitality of spores as factors in germination have been recorded, 
but very little of a fundamental nature is known regarding them. 
In general, conidia are capable of germination as soon as they are 
abstricted from the parent cell, just as many seeds may be germi- 
nated as soon as they are mature. The zygospores and oospores 
of Phycomvcetes, the chlamvdospores of many smuts, and the 
teliospores of certain species of rusts, however, are known to re- 
quire a period of dormancy, which, as in seeds, is characterized by 
thick, hard, protective walls that are presumably quite impervious 
to water and oxygen. 

It may well be that some spores must undergo a period of after- 
ripening also, as has been reported in connection with Ustilago 
longissima, U. striaeformis, Urocystis anemones, and U. cepnlae. 
Davis (1924) stored spores of U. striaeformis in the laboratory in 
a damp atmosphere at 20° C for about 240 days before he could 
secure germination. About 265 days were required if the material 
was stored out of doors. Davis was able to hasten after-ripening 
by exposing fresh smut spores to fumes of chloroform for 1 min- 
ute, then submerging them for 5 minutes in a 10° £ solution of 
citric acid, and washing them before placing them in storage. 

The oospores of certain Peronosporaceae, for example, Plasmo- 
para viticola and Sclerospora graminicola, and the teliospores of 
certain rusts that normally hibernate and then germinate in spring 
may be induced to germinate during the preceding autumn. By 
floating teliospores on water or by alternate wetting and drying, 
Maneval ( 1922 ) was able to secure germination during November 
and December. As the season advanced, there was a marked 
increase in the percentage germination and a decrease in the time 
necessary for germination to begin. He used Puccinia asparagi, P. 
helianthi, P. menthae, P. peridermiospora, P. riielliae, P. sorghi, P. 
sydowiana, P. windsoriae, and Phragmidium potentillae. Still 
other species appear completely to lack a more or less fixed period 
of dormancy. Spaulding and Rathbun-Gravatt (1925) noted that 
under outdoor conditions one collection of teliospores of Cvonav- 
tium ri hi cola from Ribes rotundifolium retained longevity for 19 
days, and one from jR. nigrum for 87 days, and that urediniospores 






HEREDITARY FACTORS AND GERMINATION 215 

accompanying the teliospores remained viable for a maximum 
period of 59 days. 

Horner (1921) attempted to germinate the aeciospores of Puc- 
cinia coronata avenue on leaves of Rhamnus kept in the herbarium 
and found them non-viable 167 days after collection, whereas 
urediniospores on Avena sativa, under the same conditions of stor- 
age, were viable 87 days after collection. He also placed rust-in- 
fected oat leaves in Petri dishes and stored them as follows: Five 
collections were stored outdoors under a thick covering of leaves 
and snow, at a temperature range of 27° to 42° F. Two of these 
collections showed viable urediniospores after 44 days. Of four 
collections placed unprotected outdoors, none showed viable 
spores after 22 days. Both of the collections wrapped in paper 
and stored in the dark at temperatures ranging from 29° to 86° F 
had viable urediniospores after 79 days. Neither of two collec- 
tions exposed to sunlight at 29° to 86° F had viable spores after 
23 days. The urediniospores of this species kept outdoors in 
Arkansas under the natural variations of temperatures and humid- 
ity succumbed in 15 days [Rosen and Weetman (1940)]. Under 
controlled conditions Rosen and Weetman found that spores were 
short-lived at relative humidities below 25% or above 50%, irre- 
spective of temperature. At higher temperatures and humidities 
viability was lost in 15 days, and at lower temperatures and hu- 
midities the spores survived for over 300 days. These results with 
crown rust of oats and other similar ones with Puccinia graminis 
triticij both heteroecious species, have an important bearing on the 
problem of the source of inoculum in spring for infections on 

these cereals. 

Hart (1926) found a similar relationship between temperature 
and humidity in the retention of viability of urediniospores of 
Melampsora lint. They retained ability to germinate for almost 
3 months at favorable temperature and humidity. At relative 
humidities of 40% and 60% they were viable longer than at 20% 
or 80%. When stored at high temperatures, they lost viability 
more rapidly than when kept at low temperatures. 

Raeder and Bever (1931) recorded that urediniospores of Puc- 
cinia glumarum, P. graminis phlei-pvatensis, and P. graminis tritici 
remained germinable 88, 120, and 128 days, respectively, when 
kept at a relative humidity of 49% and at a temperature range 
between 9° and 13° C. At the same relative humidity and at 



216 GERMINATION OF SPORES 

temperatures between 3° and 11° C, P. triticina remained viable 
124 days. 

Smut fungi are known to retain their viability for long periods. 
.McAlpine found Tolyposporium bursum on kangaroo grass viable 
after 4 years 1 storage in the laboratory. Long ago Brefeld noted 
that Tilletia tritici, when kept dry in the herbarium for 8% 
years, was still germinable. Urocystis cepulae is reported to re- 
main viable in the soil for at least 5 years. Many root-invading 
pathogens are well known to persist in the soil not only from one 
year to the next but also for a term of years. 

It has been indicated that, as teliospores of certain rusts become 
older, less time may be necessary for their germination, especially 
in species in which the teliospores constitute the overwintering 
stage. The converse is true in many conidial forms. Brown 
(1922) observed that 6-week-old conidia of Botrytis cinerea, for 
instance, require twice as long to germinate as do 10-dav-old ones. 
On the other hand, a larger percentage of conidia of Phyllosticta 
solitaria are capable of germination 10 to 14 days after they are 
of mature size than can germinate immediately after they have 
attained this size [Burgert (1934)]. It thus appears that an in- 
terval may exist between morphological and physiological ma- 
turity of spores. 

The retention of viability by spores is in some instances mated 
to their separation from the parent cell and from the host tissues 
and to their isolation from each other. The ascospores of bark- 
inhabiting and leaf-inhabiting species are known to retain their 
ability to germinate for a longer time if they remain within the 
host tissues than if they are removed. Similarly, the conidia of 
Gloeosporium, Colletotrichum, Lecanosticta, and other genera in 
which a mucilaginous matrix holds the conidia together in mucoid 
masses succumb much more quickly after they have been dis- 
persed by contact with water. Desiccation is undoubtedly the 
primary cause of loss of viability in such cases. 

The studies by Goddard ( 1935) and Goddard and Smith (1938) 
constitute an interesting approach to the problem of dormancy 
in spores. Goddard (1935) induced the dormant ascospores of 
Xcuvospora tetrasperma to germinate by heating them for a few 
minutes at temperatures of 50° C or higher. Germination occur- 
red within 2 or 3 hours if such heat-treated spores were placed 
in water at room temperature. If the spores were stored under 



HEREDITARY FACTORS AND GERMINATION 



211 



anaerobic conditions for a few hours after treatment and then 
placed under conditions favorable for germination, however, they 
failed to grow. Activation and deactivation were therefore re- 
versible reactions. In later work Goddard and Smith (1938) 
sought to explain what portion of the respiratory mechanism is 
activated by heat and what constitutes the respiratory block in 
dormant spores. By subjecting spores to various partial pressures 
of oxygen and carbon dioxide, they determined that the respira- 
tory rates are not limited bv permeability of the spore membranes 
to passage of these gases. Under anaerobic conditions carbon 
dioxide was not evolved by dormant spores, an observation which 
led Goddard and Smith to suggest that active carboxylase is not 
present in such spores. On heating, however, this enzyme is re- 
versibly activated. They interpreted their results to show that 
two qualitatively different respiratory systems are present in the 
ascospores of N. tetrasperma, the dormant system which func- 



TABLE 17 




Viability of Spores of M 1 


fXOMYCETES 




Germinated at Indicated 




Interval after Collection 




(approximate number 


Species 


of years) 


Stemonitis favogenita 


5 


Fuligo septica 


6 


Reticularia lycoperdon 


10 


Lamproderma violaceum 


13 


Trichia favoginea 


16 


Enteridium olivaceum 


17 


Badhamia utricularia 


20 


Stemonitis ferruginea 


21 


Dictydiaethalium plumbeum 


22 


Badhamia panicea 


23 


Trichia botrytis 


26 


Lepidoderma tigrinum 


26 


Physarum straminipes 


26 


Trichia scabra 


27 


Trichia later it ia 


28 


Physarum cinereum 


29 


Didymium squamulosum 


30 


Fuligo septica 


30 


Diachea leucopoda 


30 


Hemitrichia clavata 


32 


Stemonitis ferruginea 


32 



218 GERMINATION OF SPORES 

tions in the absence of the enzyme carboxylase, and a second sys- 
tem which is active in heated spores. Inactivity- of carboxylase 
thus constitutes the respiratory block. 

The age of spores of Myxomycetes has been shown by Smith 
(1929) to be of little significance in germination. Using herbarium 
specimens, he secured germination in spores from 5 to 32 years 
after collection, as is shown in Table 17. 

WATER RELATIONS AFFECTING GERMINATION 

Since water is profoundly important in all vital phenomena, it 
may be anticipated that its presence is a primary requirement in 
initiating spore germination. Spores, like seeds, do not all become 
wet with equal facility, an observation that has been made by 
everyone who has attempted to suspend spores in water for use as 
inoculum. Ziegenspeck (1934) has clarified the physico-chemical 
principles involved in the problem of wetting spores. Wetting 
must be regarded as the displacement of a gas film at the surface 
of a solid (spore) by a liquid (water). It implies an affinity of the 
solid for the liquid and is governed by solid-liquid, solid-gas, and 
liquid-gas tensions. The resultant forces are measurable in terms 
of the angle of contact made by the solid with the liquid, as 
Ziegenspeck shows. 

An examination of the literature on moisture requirements for 
germinating spores shows conflicting results concerning whether 
a film of water is necessary, since at high relative humidity a 
slight decrease in temperature causes condensation. Experimenta- 
tion becomes difficult if the effects of an aqueous film and of hu- 
midity are to be distinguished. Far too little careful work has 
been done on this problem. 

Doran (1922, pp. 334-335) recorded that Sclerotinia fructigena, 
Peronospora pygmaea, Phyllosticta antirrhini, Cylindrocladium 
scoparium, and urediniospores of Puccinia coronata germinate 
only when in direct contact with water. On the other hand, his 
observations show that aeciospores of Gy?tmosporangium clavipes 
and conidia of Alternaria solani and Venturia inaequalis may germ- 
inate in moist air. Stock (1931) failed to secure germination of 
urediniospores of Puccinia graminis and P. coronata when the 
spores were dusted on glass slides at relative humidities of 99% 
or below. 






WATER RELATIONS AFFECTING GERMINATION 219 

Hemmi and Abe (1933) controlled humidity by exposure over 
varying concentrations of sulphuric acid with the results shown 
in Table 1 8 for urediniospores of P. glwnarum. 

TABLE 18 

Germination of Puccinia glumarum as Modified by Various Relative 

Humidities 





H 2 S0 4 




Number 


Percentage 


Relative 


{specific 


Condition 


of 


of 


Humidity 


gravity) 


of Spores 


Spores 


Germination 


100 


1.0 


In drops 


1247 


44.5 


100 


1.0 


Dry 


684 


12.4 


99 


1.020 


Dry 


646 


1.5 


95 


1.090 


Dry 


739 





90 


1.158 


Dry 


904 






Although fungi generally respond to humidity in a manner 
similar to that shown by P. glumarum in Table 18, certain of them 
germinate independently of the moisture content of the surround- 
ing air. The conidia of Erysiphe polygojii, for instance, were 
found by Brodie and Neufeld (1942) to germinate through a 
range of relative humidity from approximately zero to 100%. 
These observations find support in the fact that powdery mildews 
are known to grow luxuriantly in areas where low relative hu- 
midities prevail. 

By means of apparatus in which he was able to control relative 
humidities accurately, Clayton (1942) found that the mean per- 
centage germination of urediniospores of Puccinia coronata, P. 
graminis tritici, and P. graminis avenae was lower at a relative 
humidity of 100% than in water, was considerably less at 99% 
relative humidity, and was practically nonexistent at 98%. The 
conidia and ascospores of Venturia inaequalis germinated on dry 
glass if the relative humidity was 99 to 100%. When chlamydo- 
spores of Ustilago hordei and 17. nuda were similarly placed on 
dry glass, they germinated at relative humidities of 95 to 100% 
but not at 93% or below. Furthermore he was able to germinate 
the conidia of Erysiphe polygoni on dry glass at relative humidi- 
ties of zero to 100%, thus verifying the results of Brodie and 
Neufeld (1942). 

Rippel (1933) presented evidence in connection with his studies 
on the germination of conidia of Cladosporium fulvum that the 



220 GERMINATION OF SPORES 

humidity gradient between the air and the spore membrane is a 
more decisive factor than relative humidity in influencing germi- 
nation. In C. fulvum moisture content of the spores is low. He 
concluded that the higher is the gradient, the better are the 
chances of germination. 

In many of the studies concerned with germination of rusts, 
infected host tissues or the spores themselves are floated on water. 
Blackman (1903) noted that the submerged germ tubes (promy- 
celium)"of Uromyces fabae, Fncchiia graminis, and Phragnridium 
ruin grew to considerable length with the protoplasm collected 
near the apex and that basidiospores were not formed unless the 
tube reached the air, whereas in moist air the tubes were short and 
4-celled, and each cell possessed a sterigma upon which a basidio- 
spore was borne. This morphological modification in type of 
termination is now known to be related to the fact that rusts 
forcibly expel their basidiospores, which are adapted for dispersal 
by air. 

Evidently alternate wetting and drying play an important part 
in the spore germination of some species. Jahn ( 1905) stated this 
to be true of certain slime molds. Alternate wetting and drying, 
he believed, activated the glvcogen-cleaving enzymes, thus caus- 
ing glycogen in the spore to be converted into maltose with re- 
sultant increase of osmotic pressure. This explanation may well 
apply to other kinds of fungi, but it is conceivable that modifica- 
tion of the spore wall itself may result from alternate wetting and 
drying and that this change is an important factor in germination. 

Little is known concerning the application of findings from 
laboratory studies on the relation of moisture to spore germination. 
A body of data is much needed, especially on the relation of mois- 
ture to germination and infection by plant pathogens. Observa- 
tional evidence, which is insufficient and which may indeed be 
misleading, has led to the conclusion that outbreaks of some plant 
diseases are caused by dry weather, others by wet weather. 
Among studies of this kind is that of Jones ( 1923 ), who attempted 
to correlate the moisture-holding capacity of the soil with germi- 
nation by Ustilago avenae. She placed chlamvdospores on agar 
between filter papers and then placed them in soils containing 30, 
60, or 80° \ of their water-holding capacitv. At favorable temper- 
atures germination was highest at 30 , slightly less at 60%, and 
markedly less at 80%. At 80 , which is also unfavorable for in- 



EFFECTS OF TEMPERATURE ON GERMINATION 221 

fection, lack of sufficient oxygen, as Jones points out, is undoubt- 
edly a controlling factor. 

Heavy water, deuterium oxide, as it affects germination of 
conidia of Erysiphe gravnms tritici, was studied by Pratt (1936). 
He varied the concentrations of D 2 from 0.02 to 100%, with 
phosphates as buffers. Conidia germinated in all concentrations, 
but the rate of elongation of the germ tube and its final length 
were found to be inversely proportional to the concentration of 
DoO. Deuterium oxide seems to limit the amount of solutes and 
colloids within the conidia that is utilizable in growth. 

EFFECTS OF TEMPERATURE ON GERMINATION 

Temperature is known to be one of the factors that modify the 
severity of plant diseases. It may also be the limiting factor in the 
prevalence of diseases of crop plants in certain areas. As examples 
it may be recalled that apple scab and late blight of potatoes are of 
rare occurrence and are never of consequence in the Coastal 
Plain area of the southeastern United States. Anthracnose-free 
bean seed can be produced in portions of this area by planting at 
such seasons that high temperatures will prevail at the critical 
period during maturing of the crop. Blue-staining fungi are an 
important cause of the degrading of lumber in the warmer parts 
of the United States. 

For every fungus there is a minimum, an optimum, and a maxi- 
mum temperature, the cardinal temperatures, for germination and 
for subsequent growth of the fungus. The metabolic activities 
or rate of reaction of each species increases with an increase of 
temperature up to a certain limit. These cardinal temperatures 
must be understood to mean both the extreme temperature limits 
of metabolic activity, all other factors being kept constant, and 
the temperature at which metabolism proceeds at the best rate. 

The effect of temperature on germination of urediniospores of 
Puccinia coronata and on rate of qrowth of the s^erm tube is shown 
by the work of Melhus and Durrell (1919), the rate being greatest 
at the optimum temperature. At either extreme, there is no 
growth. Rate of growth may, therefore, be regarded as a direct 
function of (t — t°), if t represents any particular temperature, 
and t°, the minimum temperature. In some instances, as could 
be expected, the temperature which is optimum for germination 



222 



GERMINATION OF SPORES 



of spores may not be optimum for subsequent development. 
These adaptations may be hereditary and may account for the 
geographical distribution of the organisms concerned and for 
their seasonal incidence. 

One type of influence of temperature upon the method of spore 
termination was shown by Melhus (1915). He noted that the 
sporangia of Phytophthora infestans germinated by either forma- 
tion of a tube or formation of swarm spores. A temperature of 



30 



0) 

to 
3 

| 20 

a 

c 
o 

I 10 



a 



1 






















\ 
\ 








' 


4 






\ 








/ 

/ . 








\. \ 

- ^s\ — 






IS 








V 






'I 

// 

/ / 










1 


H\ 














\\ 



600 « 
c 
o 
E 
5 



400 g 

3 

s 

200 & 

O 

X. 

& 

c 

_3 







30 



35 



5 8 13 17 20 25 

Temperature (degrees Centigrade) 

Fig. 39. Effect of temperature upon percentage of germination and upon 
length of cjcrni tubes in urediniospores of Viiccinia coronata. (After Melhus 
and Durrell.) A. Percentage of germination. B. Length of germ tubes in 

microns. A close correlation is shown. 

23° C was optimum for tube formation and of 13° C for produc- 
tion of swarm spores. These critical temperatures, as given by 
Crozier (1933), were 24° C and 12° C, respectively. Other 
Peronosporales, notably Peronophmnopara cnbensis, are known 
to behave similarly. 

Cardinal temperatures for germination. From his own ob- 
servations and those of other workers, Doran (1922) assembled in 
tabular form data on the influence of temperature on spore germi- 
nation among a variety of pathogenic fungi. These data consti- 
tute the bases from which Table 19, supplemented by more recent 
observations, has been prepared. Xo generalizations appear war- 
ranted from these data, except perhaps that the temperatures 
which normally prevail when the spores of these species are be- 
in<T dispersed are favorable for germination. The data have a 
direct bearing, however, on problems that concern the relation 



EFFECTS OF TEMPERATURE ON GERMINATION 



223 



TABLE 19 

Cardinal Temperature and Spore Germination 



Cardinal Temperatures 



Organism 

Plasmodiophora brassicae 
Plasmopara viticola (sporangia) 
Cysiopus candidus (sporangia) 
Phytophthora injestans (sporangia) 
(indirect method) 
(direct method) 
Peronospora parasitica (sporangia) 
Sclerospora graminicola 

(sporangia) [Tsaugi (1933)] 
(oospores) [Tsaugi (1933)] 
Rhizopus nigricans [Ames (1915)] 
Glo?nerella rujormaculans 
Gymnosporangium clavipes (aeciospores) 
Gymnosporangium juniperi-virginianae 
(teliospores) 
(teliospores) 
Cronartium ribicola 
(aeciospores) 
(urediniospores) 
Melampsora lini (urediniospores) [Hart (1926)] 
Puccinia antirrhini (urediniospores) 
Puccinia coronifera (urediniospores) [Stock (1931)] 
Puccinia coronata 
(urediniospores) 
(urediniospores) 
(urediniospores) 
(urediniospores) [Stock (1931)] 
Puccinia dispersa (urediniospores) 
Puccinia graminis (basidiospores) 
(urediniospores) 
(urediniospores) [Stock (1931)] 
(teliospores) 
Puccinia malvacearum (teliospores) 
Puccinia phlei-pratensis (urediniospores) 
Puccinia rubigo-vera (urediniospores) 

(urediniospores) [Johnson (1912)] 
Puccinia sorghi (urediniospores) 
Puccinia triticina (urediniospores) [Stock (1931)] 
Uromyces caryophyllinus (urediniospores) 
Uromyces trifolii (urediniospores) 
Ustilago avenae [Jones (1924)] 
Ustilago striaeformis [Davis (1924)] 
Urocystis cepulae [Walker and Wellman (1926)] 
Urocystis tritici [Noble (1923)] 
Urocystis occulta [Ling (1940)] 
Alternaria solani [Doran (1919), p. 392] 
Colletotrichum lagenarium 

Cylindrocladium scoparium [Doran (1919), p. 392] 
Phyllosticta solitaria [Burgert (1934)] 
Monilia fructigena [Ames (1917)] 
Cephalothecium roseum [Ames (1917)] 
Penicillium digitatum [Ames (1917)] 



r 

Mini- 


Opti- 


1 

Maxi- 


mum 


mum 


mum 


(°C) 


(°Q 

27-30 
25-35 


(°C) 





10 


25 


2-3 


12-13 


24-25 


12-13 


24 


30 




8-12 


29 


5 


17.5 


33.5 


11.5 


20-23.5 


35 


5 


36 




5 


30 




8 


14 


25 


11 


15 


29 


7 


23-24 


29 


5 


12 


19 


8 


14 


25 





6-23 


26 


5 


10 


20 


5 


14-25.5 


32.5 




18 


30 


7 




30 


1 


17-22 


35 


7-8 


12-17 


30 ' 


10-12 


18-20 
15-20 


25-27 


2 




31 


2-3 


5-20 


29-30 


9 


22 


23 


3 


14 


30 




18 


30 


2 




31 


2 


12-17 


30 


4 


14 


25 


2-3 


5-20 


29-30 


4 


14 


29 




16 


34 


4-5 


15-28 


29-30 


7 


22 


35 


9 


15-22 


29 


5 


24 


32 


5 


15 


30 


1-3 


26-28 


37-45 


7 


22-27 




8 


12-30 


36 


5 


23 


39 





25 




5 


30 







25 





224 GERMIXATIOX OF SPORES 

of temperature to infection, escape from infection, resistance, 
and cardinal temperatures of the host. Such matters are beside 
the present purpose but are comprehensively dealt with bv 
Lauritzen (1919) in his studies of Ascochyta fagopyrum on buck- 
wheat, Colletotrichum Undentuthianum on bean, and Pucc'mia 
grandnis on wheat. It may be mentioned, how ever, that tem- 
peratures permitting spore germination generally also permit 
infection. In soil-borne pathogens and smuts that infect seed- 
lings, temperature interacts with soil moisture and soil reaction, 
and each factor is interdependent. 

The temperature relations of those fungi that produce decay, 
especially of fruits and vegetables, have been extensively studied 
because of their bearing on problems of storage and refrigeration. 
YVeimer and Harter (1923) determined the cardinal temperatures 
of several species of Rhizopus, all of which cause decay of sweet 
potatoes in storage, to be as shown in Table 20. The first four 

TABLE 20 

Cardinal Temperatures of Species of Rhizopus Associated with Soft Rot 

of Sweet Potatoes 

Temperature {degrees C) 



Species 


r 

Minimum 


Optimum 


Maximum 


R. artocarpi 


1.5 


26-29 


33.5 


R. nigricans 


1.5 


Zh-28 


33.0 


R. reflex us 


1.5 


30-32 


36.6 


R. microsporus 


1.5 


26-28 


33.0 


R. tritici 


1.5 


36-38 


44.0 


R. delemar 


8.7 


36-38 


44.0 


R. nodosus 


1.5 


36-38 


44.0 


R. oryzae 


9.0 


36-38 


44.0 


R. arrhizus 


1.5 


36-38 


43.6 


R. chinensis 


10.0 


43-45 


51.0 



species in the list may be set apart as a low-temperature group, 
R. chinensis is a high-temperature species, and the others are 
intermediate. 

In general, the studies on minimal temperatures that prevent 
germination and growth of species causing decay of perishable 
foods show that storage temperatures near 0° C must be main- 
tained if losses are to be prevented. Hoffman ( 1860) found that 
conidia of Penicillium glaucuvi, Botrytis vulgaris, and Trie hot he- 



EFFECTS OF TEMPERATURE ON GERMINATION 22$ 



chim roseum, all essentially omnivorous species, germinate very- 
near the freezing point. In similar studies Ames (1915) employed 
Glovierella ntfomaculans and Cephalotheciwn roseum from apple, 
Thielaviopsis paradoxa from pineapple, Fenicillhnn digitatam from 



be 

S 
c 
a> 
v 

- 

C 

o 

-t-> 
cs 

e 

■ <*-« 

6 

- 

O 



100 

90 

80 

70 

60 

50 

40 

30 

20 

10 
























































































































s s 
— _< * 
















\ 




4 


/ 
/ 

f 












y — 


t 

/ 
/ 

— -i 


* 


s/ 


- -^ V 


4* 
* 












4 

/ 
/ 
y 


f 


4 

t 

/ 
/ 
/ 




















/ 




f 

1 

/ 

t 

f 























/ 
i 





















8 10 12 14 
Time (hours) 



16 



18 20 



22 



24 



Fig. 40. Effect of storage indoors for different periods on germination of 
teliospores of Cronartium ribicola. The curves are based on 3-hour moving 
averages. Solid line, from teliospores taken from Ribes nigrum and stored 
for 5 days; dash line, from teliospores taken from R. americanum and 
stored for 15 days; dot-dash line, from teliospores taken from R. nigrum 
and stored for 25 days. (After Spaulding and Rathbun-Gravatt.) 

orange, Rhizopus nigricans from sweet potato, and Monilia fruc- 
ticola from plums and found that near-freezing temperatures 
must be maintained in storage if germination of these species is 
to be prevented and development of decay by them entirely 
avoided. 

The most extensive data on cardinal temperatures among Alyxo- 
mycetes are those of Smart (1937). He germinated the spores 
of 70 species, finding that the range 22° to 30° C is optimum for 
all. At 10° C or lower and above 30° C percentage germination 



226 GERMINATION OF SPORES 

and rate of germination are greatly reduced. The temperature 
range for germination extends from 2° to 36° C. 

Thermal death point. In bacteriology the term thermal 
death point or, more appropriately, thermal death time has b'een 
employed to express that minimal temperature fatal to all bac- 
teria after exposure for 10 minutes. The method used is to sub- 
ject a suspension of bacteria to a series of selected temperatures 
and at definite intervals to plant out portions to determine the 
number of survivors. If the operation is repeated sufficiently 
often, it will be found that at a particular temperature all organ- 
isms are dead after an exposure of 10 minutes. All other factors 
must be identical in thermal-death-time measurements, because 
age of organisms, concentration of organisms, and pH are modi- 
fying factors. Essentially the same method, using suspensions of 
spores, may be employed for fungi. Smith (1923) made such a 
study with conidia of Botrytis cinerea exposed at a range of tem- 
peratures between 31° and 50.3° C. When he plotted the propor- 
tion surviving at different times for each temperature, he got a 
series of approximately symmetrical sigmoid curves all exactly 
alike except for the rate of speed of killing at different tempera- 
tures. If the observations at each temperature employed by 
Smith are plotted, they will be seen to fall closely on a typical fre- 
quency-distribution curve. 

Spores retain their viability at higher temperatures when sub- 
jected to dry heat than to moist heat. These differences in toler- 
ance become greater if the temperature is very slowly elevated 
during dry heating. In explanation it may be pointed out that 
heat coagulates proteins more readily when the moisture content 
is high than when a small percentage of water is present. The 
observations of Tsaugi (1933) on retention of germinabilitv by 
oospores of Sclerospora graviinicola are concerned with this point. 
Those subjected to dry heat at 50° C, 55° C, and 60° C remained 
viable, whereas moist heat at these temperatures was lethal. 

Ames ( 1915) determined that the thermal death point of Thiela- 
viopsis paradoxa is 52.5° to 53.5° C, of Rhizopus nigricans, 60° C, 
of Monilia fructicola, 52.0° to 52.5° C, of Glomerella rufomacu- 
lans, 53.0° to 5 3.5° C, of Cephalothecium roseum, 47° to 48° C, 
and of Penicillin//; digitatum, 58.0° to 58.5° C. 



EFFECTS OF TEMPERATURE ON GERMINATION 221 

The tolerance of fungus spores to low temperatures should be 
subjected to study by methods patterned after those dealing with 
thermal death points. Such studies appear not to have been ac- 
complished, except for relatively few species. This topic is sum- 
marized by Luvet and Gehenio (1941) and is briefly discussed 
in Chapter 5. Investigations of the effects of cold on fungi, espe- 
cially rusts, have been largely concerned with overwintering, as 
related to the source of inoculum for the development of disease 
outbreaks. Christman (1905) and Horner (1921) are among 




Fig. 41. Effect of temperature upon survival of Botrytis cinerea. Percent- 
age surviving plotted against time in minutes, except for the 37° C curve, 
the intervals of which are 30 minutes. (After J. H. Smith.) 

those who have investigated survival of urediniospores of cereal 
rusts. Ewert (1910) noted that a small proportion of conidia of 
Psendopeziza ribls survived the winter when exposed to outdoor 
temperatures as low as — 22° C. Several exposures of Fusicladhim 
dendriticum and F. pirinum to freezing greatly reduced their 
percentage of germination. The conidia of Mycosphacrella sen- 
tinel, however, artificially subjected to alternate freezing at tem- 
peratures as low as -16° C and thawing, retained germinability 
as well as did untreated ones. 

Many Ascomycetes known to possess a conidial stage can over- 
winter in this stage. The ascospores of others are mature in fall 



228 GERMINATION OF SPORES 

and remain dormant throughout winter. The ascogenous stage of 
a third group develops slowly during winter and matures in 
spring. In any event the overwintering of conidia of a species 
not known to possess an ascogenous stage is not a criterion upon 
which to predicate the possession of such a stage. 

Temperature and percentage germination. Obviously the 
proportion of the total number of spores of a given species which 
(terminate is correlated with temperature and with time as external 
factors of primary importance. Temperature as a correlated 
factor in percentage germination is illustrated by the observations 
of Kaufmann (1934), presented in Table 21. Germination was 

TABLE 21 

Proportion of Spores of Certain Basidiomycetes That Germinate at 

Different Temperatures 

Percentage of Germination at 



Organism 


15° C 


20° C 


25° C 


30° C 


35° C 


40° C 


45° C 


Coprinus micaceus 


1.72 


3.30 


15.09 


15.05 


77.73 


39.54 


27.48 


Coprinus comatus 


3.37 


6.72 


12.69 


17.18 


16.29 


14.38 


7.58 


Lepiota cepae stipes 








3.39 


8.97 


2.71 








Cyathus olla 





2.59 


4.26 


10.52 


4.53 








Cyathus striatus 





1.99 


3.93 


7.65 


3.19 









relatively poor with each of these species, so that the relationship 
is not so striking as may have been desired. Doran's (1922) re- 
sults with a series of trials employing conidia of Venturia inaequa- 
lis are more representative. The averages of his tests are 0% at 
2° C, 3% at 5° C, 21.5% at 8° C, 56.2°fat 10° C, 76.5% at 12° C, 
100% at 15° C, 77.2% at 18° C, 56.5\ at 20° C, 41.5% at 24° C, 
22.2% at 28° C, 11% at 30° C, and 0% at 32° C 

Time required for germination at different temperatures. 
As has previously been stated, the spores of some fungi are capable 
of germination as soon as they are produced, whereas others un- 
dergo a period of dormancy. In any event temperature is a factor 
correlated with the time required for spore germination. In some 
species germination can be secured within an hour; at the opposite 
extreme, others may require exposure for several weeks to condi- 
tions favoring germination. The time-temperature relationships 
in spore germination are illustrated by Ames's (1915) results of 
studies on fruit-rottino- funm and are shown in Table 22. 



INFLUENCE OF REACTION ON GERMINATION 229 

TABLE 22 

Time (Hours) Required for Germination of Spores at Different 

Temperatures 



Fungus 


1° C 


5°-6° C 1 


0°-12° ( 


: 15° c 


20° C 


25° C 


30° 


Thielaviopsis paradoxa 
Rhizopus nigricans 
Monilia jructigena (jructicola) 
Penicillium digitatum 


i 
i 

245 
120 


168 
168 
100 

48 * 


23 
43 

7 
15 


13 
36 

7 
8 


8 
16 

7 
8 


8 
13 

7 
8 


8 
16 

7 
8 


Glomerella rujormaculans 


i 


174 


15 


8 


7 


7 


7 


Cephalothecium roseum 


i 


i 


24 


15 


7 


7 


7 



1 Failed to germinate. 

INFLUENCE OF REACTION ON GERMINATION 

The pH which limits germination has been determined for many 
species of fungi. A divergence of opinion exists concerning the 
proper appraisal of the value of such knowledge. No such dis- 
agreement exists, however, regarding; the usefulness of data in- 
volving the influence of reaction on mycelial development of 
fungi in cultures or in soil and other natural habitats. 

Most fungi germinate and develop best in acid media. The kind 
of nutrient, however, exerts a profound influence upon the re- 
sponse to the reaction of the medium. Webb (1921) made. a 
comparative study of the effects of hydrogen- and hydroxyl-ion 
concentration upon the germination of B'otrytis cinerea, Asper- 
gillus niger, Penicillium italicum, P. cyclopium, Lenzites saepiaria, 
Puccinia graminis, Fusarium sp., and Colletotrichum gossypii in 
four liquid media, namely, solutions of mannite and of peptone, 
Czapek's nutrient, and sugar-beet decoction. All except Fusarium 
sp. and C. gossypii responded favorably to successively increasing 
concentrations of hydrogen ions in all media within the range pH 

7.0 to 3.0-4.0. Colletotrichum gossypii responded best within the 
alkaline range. Specificity of response in each nutrient is illus- 
trated by the fact that Botrytis cinerea germinated in mannite 
from pH 1.6 to 6.9, but did best at pH 3.0; in Czapek's nutrient 
from pH 2.5 to 9.6, but best at pH 3.0 to 3.6; in peptone from pH 

2.1 to 8.7, but best at pH 4.0 to 5.3; and in beet decoction from 
pH 2.0 to 9.8, but best at pH 4.0 to 7.0. 

Similar studies on other organisms show that pH 6.86 is opti- 
mum for germination of Urocystis occulta, with no germination 



250 GERMINATION OF SPORES 

at pH 3.80 and sparse germination at pH 8.95 [Ling (1940)]. 
Tsau^i (1933) secured best germination of oospores of Sclero- 
spora graminicola at pH 2.9 to 3.1, with very little germination at 
pH 9.3. Kaufmann (1934) found pH 7.5 optimum for Coprimis 
mi c ace us, C. comatus, and Cy at hits olla, pH 7.0 optimum for 
Lepiota cepaestipes, and pH 6.5 optimum for Armillaria mellea. 
Smart (1937) made a study involving the effect of reaction on the 
germination of 70 species of slime molds. All species germinated 
within the range pH 4.0 to 8.0. Full go septic a germinated within 
the range pH 2.0 to 10, and Fhysarum serpula, pH 2.0 to 8.5. The 
optimum pH for germination in all species was 4.5 to 7.0. 

INFLUENCE OF OXYGEN ON GERMINATION 

De Bary (1887) noted that spores in a drop of water between 
the cover glass and slide germinate better near the periphery of 
the cover glass than near the center. He attributed this effect 
to the relative amounts of air available. This observation has been 
verified by everyone who has attempted to repeat the experiment. 
Dus^ar (1901) gave special consideration to reduced 2 tension 
as a factor in retarding germination. Blackman (1903) pointed 
out the occurrence of morphological differences in germination 
of teliospores in water and in air. Melhus and Durrell (1919) 
recorded that few urediniospores of Puccima coronata germinate 
if submerged in comparison with the number germinating if 
they float at the surface. Abundant evidence shows that a 
smaller percentage of germination is secured in drops of water 
containing many spores than in those containing few. Oxygen 
relations must therefore be considered in studies of spore germi- 
nation, and they may be expected to be correlated with the ability 
to become wet and with the specific gravity of the spores. 

The absence of oxygen may not inhibit germination, as is 
shown by UppaFs (1926) studies on certain Peronosporales. 
When he removed the oxygen ( by a vacuum pump or by alkaline 
solutions of pvrogallic acid) from the environment in which 
sporangia of Phytophthora colocasiae, P. infestans, P. parasitica, 
and P. pahmvora were placed for germination, these species germi- 
nated by formation of swarm spores. On the other hand, Albugo 
Candida, Flasmopara viticola, and Sclerospora graminicola, which 
germinate in the same manner, require the presence of oxygen 



INFLUENCE OF LIGHT ON GERMINATION 231 

for germination, as do also Peronospora parasitica and P. trifolio- 
rz/777, which germinate by formation of germ tubes. 

INFLUENCE OF CARBON DIOXIDE ON GERMINATION 

The observations of Brown (1922) on volatile materials pro- 
duced by apples and potatoes in storage led him to conclude that 
volatile substances may have considerable influence in control 
of organisms which produce decay. Ethyl acetate, a common 
fruity ester evolved by apples, exerted either a stimulatory or an 
inhibitory effect on the germination of Botrytis cinerea, depend- 
ing upon the concentration. Volatile substances from leaves of 
apple, Ruta, Eucalyptus and other aromatic plants increased germi- 
nation of this fungus, whereas vapors from potato tubers and 
onions were inhibitory. Platz, Durrell, and Howe (1934) con- 
cluded that stimulation of germination of Ustilago zeae in the 
presence of plant tissues is the result of increased carbon dioxide 
tension, the carbon dioxide being generated by the plant tissues. 
The presence of corn leaves in their germination chambers in- 
creased the carbon dioxide content to 15%, the optimum for ger- 
mination of the corn smut. Platz, Durrell, and Howe reported 
that carbon dioxide acts by changing the reaction, and that 15% 
carbon dioxide in the air produces hydrogen-ion concentrations 
ranging from pH 4.9 to 5.6, which is optimum for U. zeae. 

INFLUENCE OF LIGHT ON GERMINATION 

Too little is known regarding the effect of radiations on the 
germination of fungi, and published reports frequently contain 
conflicting conclusions. De Bary [Doran (1922), p. 333] and 
Farlow [Doran (1922), p. 333] state that light inhibits germina- 
tion of spores of Oomycetes. Melhus (1915), on the other hand, 
found that light does not inhibit germination of sporangia of 
Phytophthora infestans. Doran (1922) noted that Alternaria 
solani and conidia of Sclerotinia fructigena germinate equally well 
in direct light, diffuse light, or darkness. Dillon-Weston (1932) 
germinated urediniospores of Puccinia graminis avenae, P. graminis 
tritici, and P. coronata under standardized Wratten green and blue 
filters, which permit the passage of wavelengths of 450 to 555 m^, 



232 GERMINATION OF SPORES 

but ^Termination was inhibited under the red, orange, yellow, and 
purple filters. 

INFLUENCE OF NUTRITION ON GERMINATION 

As has been emphasized, the intake of water by the spore is the 
sine qua 11 on for the initiation of germination. Apparently, how- 
ever, not all species can be made to germinate in pure water. 
Since water is a universal solvent, spores do not come in contact 
with pure water under natural conditions. Whether they lodge 
on living plants or animals, on decaying tissues, on the soil, or in 
water, thev come in contact with soluble organic materials. Ad- 
vantage may be taken of this fact in germination trials, especially 
with species that thus far have proved impossible to grow in arti- 
ficial culture, for example, Peronosporaceae, Erysiphaceae, and 
Hvpodermataceae. The germination of some species in those 
families appears to be hastened by the presence of the green tissues 
of their appropriate hosts. Similar experiences have also been 
recorded with Rhytisma acerinum, Gnomonia ulmea, Cymadothea 
trifolii, Diplocarpon rosae, and Lhwspora gleditsiae. Germination 
of the spores of Merulius lacrymans is hastened by the presence 
of urine. In general, with species whose germination is attended 
with difficulty in potable water, an attempt should be made to 
approximate natural conditions of germination. 

RESUME 

It is plainly apparent from the foregoing discussion that both 
hereditarv and environmental factors influence the germination 
of fungus spores. It is not evident, however, that anything of 
fundamental importance is likely to be established by additional 
studies of this sort involving either these same species or other 
species. Perhaps attention might better be centered on determin- 
ing the causes of dormancy in spores and the means whereby dor- 
mancy may be broken. Such inquiries are likely to be most fruit- 
ful if they are patterned after studies on the germination of seed. 

Studies involving the presence of growth factors to hasten or 
to increase germination might conceivably yield results of value, 
especially with species that require the given growth factor for 



RESUME 233 



mycelial development. Conceivably such information might have 
a bearing on problems of obligate parasitism. 



LITERATURE CITED 



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rots," Phytopathology, 5: 11-19, 1915. 
Bary, Anton de, Comparative morphology and biology of the fungi, myce- 

tozoa, and bacteria. Oxford Press, 1887. 
Blackman, V. H., "Conditions of teleutospore germination and of sporidia 

formation in the Uredinales," New Phytol., 2: 10-14, 1903. 
Brodie, H. J., and C. C. Neufeld, "The development and structure of the 

conidia of Erysiphe polygoni DC and their germination at low humid- 
ity," Can. J. Research, 20:41-61, 1942. 
Brown, William, "On the germination and growth of fungi at various 

concentrations of oxygen and of carbon dioxide," Ann. Botany, 36: 257— 

283, 1922. 
"Studies in the phvsiologv of parasitism. IX. The effect on the germi- 
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Ann. Botany, 36:285-300, 1922a. 
Burgert, Irma A., "Some factors influencing germination of spores of 

Phyllosticta solitaria," Phytopathology, 24: 384-396, 1934. 
Christman, A. H., "Observations on the overwintering of grain rusts," 

Trans. Wis. Acad. Sci., IS: 98-107, 1905. 
Clayton, C. N., "The germination of fungous spores in relation to con- 
trolled humidity," Phytopathology, 32:921-943, 1942. 
Crozier, Willard, "Studies in the biology of Phytophthora infestans (Mont.) 

de Bary," Cornell Agr. Expt. Sta. Mem., 755:40 pp. 1933. 
Davis, W. H., "Spore germination of Ustilago striaeformis" Phytopathology, 

14:251-267, 1924. 
Dillon-Weston, W. A. R., "The reaction of disease organisms to certain 

wavelengths in the visible and invisible spectrum," Phytopath. Z., 4: 229- 

246, 1932. 
Doran, W. L., "The minimum, optimum, and maximum temperatures of 

spore germination in some Uredinales," Phytopathology, 9: 391-402, 1919. 
"Effect of external and internal factors on the germination of fungous 

spores," Bull. Torrey Botan. Club, 49: 313-336, 1922. 
Duggar, B. M., "Physiological studies with reference to the germination of 

certain fungous spores," Botan. Gaz., 31: 38-66, 1901. 
Ewert, R., "Die Uberwinterung von Sommerkonidien pathogener Ascomy- 

ceten und die Widerstandfahigkeit derselben gegen Kalte," Z. Pflanzenk., 

22: 129-141, 1910. 
Fisher, R. A., Statistical methods for research workers 3rd ed. 283 pp. 

Oliver and Boyd, Edinburgh. 1930. 



234 GERMINATION OF SPORES 

Goddard, D. R., "The reversible heat activation inducing germination and 

increased respiration in the ascospores of Neurospora tetrasperma," 

J. Gen. Physiol., 19: 45-60, 1935. 
Goddard, D. R., and P. E. Smith, "Respiratory block in the dormant spores 

of Nettrospora tetrasperma," Plant Physiol., 75:241-264, 1938. 
Hart, Helen, "Factors affecting the development of flax rust, MeLvnpsora 

lini (Pers.) Lev," Phytopathology, 16:185-205, 1926. 
Hemmi, H., and T. Abe, "On the relation of air humidity to germination 

of urediniospores of some species of Puccinia parasitic on cereals," 

Forsch. Gebiete Pflanzenk., 2: 1-10, 1933. 
Hoffman, H., "Untersuchungen iibcr Kcimung der Pilzsporen," Jahrb. 

wiss. Botan., 2: 267-297, 1860. 
Horner, G. R., "Germination of aeciospores, urediniospores, and teliospores 

of Puccinia coronata" Botan. Gaz., 12: 173-177, 1921. 
Jahn, E., "Mvxomycetenstudien 4. Die Keimung der Sporen," Ber. dent. 

botan. Ges., 23: 489-497, 1905. 
Johnson, E. C, "Cardinal temperatures for the germination of uredinio- 
spores of cereal rusts (abst.)," Phytopathology, 2:47^8, 1912. 
Jones, Edith S., "Influence of temperature, moisture, and oxygen on spore 

germination of Ustilago avenae" J. Agr. Research, 24: 577-590, 1923. 
Kauf.m ann, F. H. O., "Studies on the germination of the spores of certain 

Basidiomycetes," Botan. Gaz., 96: 282-297, 1934. 
Lauritzen, J. I., "Relation of temperatures and humidity to infection by 

certain fungi," Phytopathology, 9: 7-35, 1919. 
Lin, C. K., "Germination of the conidia of Sclerotinia fructicola, with special 

reference to the toxicity of copper," Cornell Agr. Expt. Sta. Mem., 

233: 1-33, 1940. 
"Nutrient requirements in the germination of the conidia of Glomerella 

cingnlata," Am. J. Botany, 32: 296-298, 1945. 
Ling, Lee, "Factors affecting spore germination and growth of Urocystis 

occulta in culture," Phytopathology, 30: 579-591, 1940. 
Luyet, B. F., and P. M. Gehenio, Life and death at low temperatures. 341 

pp. Biodvnamica, Normandv, Mo. 1941. 
M \neval, W. E., "Germination of teliospores of rusts at Columbia, Mis- 
souri," Phytopathology, 72:471-488, 1922. 
McCaj i an, S. E. A., and Frank Wilcoxon, "The precision of spore-germi- 
nation tests," Contrib. Boyce Thompson Inst., 4: 233-243, 1932. 
Melhus, I. E., "Experiments on spore germination and infection in certain 

species of Oomvcctes," Wis. Agr. Expt. Sta. Research Bull., 15: 25-91, 

1911. 
"Germination and infection with the fungus of the late blight of potato," 

Wis. Agr. Expt. Sta. Research Bull., 57:64 pp. 1915. 
Mi i in s. 1. 1 .. and I.. \V. Dl krell, "Studies on the crown rust of oats," Iowa 

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Noble, R. J., "Studies on Urocystis tritici Koern., the organism causing flag 

smut of wheat/' Phytopathology, 13: 127-139, 1923. 
Platz, G. A., L. W. Durrell, and Mary E. Howe, "Effect of carbon dioxide 



LITERATURE CITED 235 

upon the germination of chlamydospores of Ustilago zeae (Beckm.) 

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Pratt, R., "Growth of germ tubes of Erysiphe spores in deuterium oxide," 

Am. J. Botany, 25:422-431, 1936. 
"The influence of the proportions of KH 2 P0 4 ,i\IgS04, and NaNCX in 

the nutrient solution on the production of penicillin in surface cul- 
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Chapter 10 

HOST PENETRATION 

The production of disease by pathogenic fungi involves the fol- 
lowing sequential processes: inoculation, incubation, and infec- 
tion, "in the first process is included distribution of the pathogen 
bv any agencies whatsoever that bring into contact inoculum and 
suscept. The inoculum may take an active part in this process, as 
occurs among swarm spores of certain Phycomycetes and among 
ascospores and basidiospores that are forcibly expelled. On the 
other hand, the inoculum may be entirely passive and therefore 
be dependent upon water, currents of air, and insects or other 
bioloo-ical agencies. In incubation are included penetration of 
the tissues of suscepts by germination and growth of the inoculum 
to set up the parasitic relationship. Penetration may be accom- 
plished by entrance through natural openings, such as stomata, 
lenticels, and hvdathodes, by direct passage through cuticle and 
epidermal walls, or bv entrance through wounds. Length of the 
incubation stage is definite for each specific pathogen and termi- 
nates when symptoms appear. All of those physiologic and 
morphologic responses (symptoms) that express the interaction 
of pathogen and suscept are infection phenomena, and they con- 
stitute a "continuous process. It may be impossible to determine 
when the incubation stage ends and the infection stage begins, the 
disturbances being imperceptible when first initiated. 

In the present instance concern centers upon the beginning of 
the incubation stage, especially upon the phenomena of penetra- 
tion and the subsequent relationship of the fungus to the invaded 
tissues. Numerous studies have been made of this complex prob- 
lem, beginning with those by de Bary (1886) and carried forward 
by Ward (1888) and later by Blackman and by Brown and his 
associates. The status of this problem has been summarized by 

Blackman (1924) and Brown (1936). 

236 



DIRECT PENETRATION 231 

DIRECT PENETRATION 

Pioneer work on the initiation of attack by direct penetration 
was done by de Bary (1886) in a study dealing with invasion by 
Sclerotinia libertiana. He maintained that, when he applied asco- 
spores in drops of nutrient solution to suitable intact plant tissues, 
they were able to penetrate directly; whereas, if ascospores were 
placed in drops of water, they formed organs of attachment by 
means of which they intimately applied themselves to the surface 
of the host. These organs of attachment secreted a principle 
which killed the underlying cells, and in consequence nutrients 
diffused from the dead cells. As a parasite, therefore, S". libertiana 
was able to penetrate directly, and as a saprophyte it must first 
kill the underlying host cells. When he prepared an extract from 
infected tissues, he was able to demonstrate that this extract could 
cause the cells to fall apart, that is, to rot, and could kill the proto- 
plasts. Boiling destroyed the activity of the extract; from this 
fact he concluded that rotting was caused by an enzyme, but he 
was unable to determine the nature of the lethal substance. Al- 
though he expressed the opinion that oxalic acid produced by 
the fungus killed the cells, he did not know whether this acid was 
solely responsible for the death of the tissues. Several reports 
have subsequently appeared, the authors of which accepted the 
de Bary hypothesis that parasitic fungi and bacteria secrete a fer- 
ment that enables them to penetrate cell walls. Ward (1888) 
expressed this opinion regarding Botrytis cinerea, the cause of a 
disease of lilies. 

In regard to the cause of killing in advance of penetration, 
Smith (1902) found in connection with Botrytis cinerea, parasitic 
on lettuce, that it produced a thermostable toxic substance and 
expressed the opinion that this substance was oxalic acid. Peltier 
(1912), on the other hand, in a study involving presumably the 
same fungus on pepper and lettuce, concluded that the toxic 
thermostable substance was not oxalic acid but some other organic 
acid or acids. Higgins (1927) demonstrated production by 
Sclerotinm rolfsii of oxalic acid in certain nutrient solutions. 
Considerable quantities of oxalates were also found in the dead 
cells underlying the holdfasts of soybeans and peppers, but none 
occurred in healthy cells of the same hosts. Moreover, the tox- 



238 HOST PENETRATION 

icitv of filtrates from cultures in which 5. rolfsii had been grown 
became greater with increase in oxalic acid content. For these 
reasons Higgins stated that the evidence appears conclusive in 
showing that oxalic acid, secreted by fungus hvphae, causes death 
of cells in advance of actual penetration. 

Other pertinent evidence was presented by Brown (1915) from 
his experiments with extracts from germ tubes of Botrytis cinerea. 
These extracts are highly active in decomposing parenchymatous 
tissues of manv kinds of vegetables and fruits. Heating to 60° to 
70° C inactivated this extract, and he was unable to separate 
enzyme from toxic principle. Drops of extract, when placed on 
delicate rose petals, were quite innocuous, provided that the cu- 
ticle was intact. Brown found no oxalic acid in the extract and 
was forced to conclude as follows: (a) that the only active con- 
stituent of the extract was pectinase, and (b) that he had failed 
to extract a toxic principle, leaving unfounded the killing in ad- 
vance of penetration described by de Bary. 

In early stages of invasion bv Diplocarpon rosae, browning of 
the host cells has been observed [Aronescu (1934)1, but the im- 
mediate cause has not been determined. 

When Blackman and Welsford (1916) and Boyle (1921) made 
cytoloeical examination of tissues attacked bv Botrytis cinerea 
and by Sclerotinia libertiana, thev noted that the staining reactions 
of the host cell beneath the germ tube were early modified and 
that a very slender "infection hvpha" always penetrated the cells 
in advance of killing-. Once this had been established, Brown 
(1922) determined bv conductivity tests that rapid exosmosis of 
solutes from the tissues does not occur unless the infection hvphae 
have penetrated, and thus he was able to establish with some de- 
gree of finality how facultative parasites are able to attack host 
cells. 

The role of appressoria. Certain pathogenic fungi, notably of 
the genera Colletotrichum, Gloeosporium, and Marssonia, form pe- 
culiar structures called appressoria, which function in penetration 
of the suscept. Frank (1883) first recognized the true nature of 
appressoria in connection with observations on Fusicladium trevi- 
ulae, Poly stigma nibrnm, and Colletotrichum lindeinuthiamnn. 
He interpreted them to be adhesion disks which applied them- 
selves closely to the surface to be penetrated and there served to 
anchor the pathogen while the membranes immediately beneath 



DIRECT PENETRATION 239 

were being pierced by the infection hypha. Biisgen (1893) veri- 
fied Frank's observations and concluded that appressorium forma- 
tion results in response to contact of germ tubes or hvphac with 
a solid body. In hanging drops or in drops of liquid on glass 
slides, appressoria generally form as soon as the tube emerges. 

Hasselbring (1906) noted the existence of appressoria 12 to 18 
hours after inoculation in Gloeosporhim fructigenum, Allen 
(1923) on the day after inoculation with wheat rust, and Aro- 
nescu (1934) as early as the ninth hour after inoculation with 
Diplocarpon rosae. Appressoria become separated from the tube 
by a septum, their wall thickens, and eventually they become cir- 
cular in outline, being flattened, however, on the side in contact 
with the solid body. These characteristics led various American 
workers who early studied the organisms causing cotton anthrac- 
nose, apple bitter-rot, ripe-rot of grapes, and pepper anthracnose 
to regard appressoria as secondary spores. Attention was directed 
to this error and to their true function among anthracnose-pro- 
ducing species by Hasselbring (1906). He also observed that 
lack of food is a factor in their formation, since they may not 
develop in the presence of a supply of nutrients. Similar studies 
involving appressoria of Collet otrichum lindemathiamim and C. 
gloeosporioides were made by Dey (1919, 1933). His evidence 
indicated that appressoria can withstand drying and that they give 
rise to the penetration tube only when nutrients are available. 
It is indicated that substances diffuse out through the cuticle to 
stimulate germination. 

In 1886 de Bary described organs of attachment that facilitated 
penetration among species of Sclerotinia. Details of the penetra- 
tion by 5. libertiana were presented by Boyle (1921). When this 
fungus was placed on bean leaves, appressoria formed near the 
hypha tips. Thev became fixed to the leaf surface by means of a 
mucilaginous sheath. From beneath the appressoria a very slender 
"infection hypha" then developed, which indented the cuticle at 
the point of contact. There was no evidence of dissolution of 
the cuticle, but eventually the infection hypha penetrated this 
membrane by mechanical pressure. 

Diplocarpon rosae pierces rose cuticle wholly by mechanical 
pressure, but the enlargement of the infection peg into an infec- 
tion hyphae is interpreted by Aronescu (1934) to indicate that 
further penetration occurs in a different manner. 



240 HOST PENETRATION 

Penetration by infection hvphae of the same type is known 
among other pathogenic fungi. Blackman and Welsford (1916) 
noted that Botrytis cinerea pierces the cuticle of Vic'ia faba by 
mechanical pressure exerted on a narrow outgrowth from the 
germ tube. Waterhouse (1921) recorded the occurrence of a 
mucilaginous envelope on the germ tubes of sporidia of Puccinia 
graminis germinating on leaves of Berberis vulgaris. The muci- 
laginous matrix fixed the sporidium and germ tube to the leaf. 
Penetration of the cuticle was accomplished by mechanical pres- 
sure exerted upon a beak-like outgrowth from beneath the spo- 
ridium or upon a very tenuous style-like hypha beneath the germ 
tube. After penetration the tip of the infection tube swells into 
a vesicle, and from it the mycelium forms. 

Penetration by boring through the host-cell membranes has 
been observed and described in several Phycomycetous species. 
Curtis (1921) noted entrance of zoospores that had come to rest 
and of young zygotes of Synchytrhnn endobiotiaim into potato 
tissues. A small protuberance develops on the side in contact 
with the host, which eventually perforates the wall. The tip of 
the tube then enlarges into a vesicle within the lumen of the host 
cell, into which the entire protoplast flows, leaving the empty 
wall of the resting cell or of the zygote outside the host. Similarly 
Tisdale (1919) noted migration of the content of erstwhile zoo- 
spores of Phy so derma zeae-may dis through narrow bore tubes 
into the epidermal cells of corn. Among certain higher Phy- 
comycetes, such as Peronospora tabacina, entrance may be effected 
with apparently equal facility either by direct penetration or by 
entrance through stomata. 

Among hyperparasites the hvphae of the one species may pene- 
trate the walls of a second and grow within them, and others 
merely entwine themselves closely around the host species. An 
unusual type of hyperparasite and of direct penetration is pre- 
sented in Parasitella (M/tcor) parasiticus and in Chaetocladium, 
both of which are parasitic upon other Alucoraceae. Burgeff 
( 1924) described this relationship, pointing out that the sequence 
of events is as follows: The hypha tip of the parasitic species, 
after contact with the host hypha, cuts off a buffer cell. The 
wall between this buffer cell and the host is then dissolved, effect- 
ing a direct connection between them. The buffer cell then 
enlarges, and hypha branches are developed from it. Meanwhile 



DIRECT PENETRATION 



241 



host and parasite nuclei and protoplasm freely intermingle with 
each other. 

The stimulus causing penetration. The observations of 
Pfeffer that chemotropic stimuli are responsible for the migra- 
tion of antherozoids of mosses and ferns to the archegones fur- 
nished the stimulus for similar studies involving the proximate 
cause of penetration by fungi. Miyoshi, working under PfefTer's 
direction, published two reports (1894, 1895), in which he con- 




Fig. 42. Stages in penetration by the hyperparasite Parasitella (Mucor) para- 
siticus into the hypha of Mucor. The buffer cell is cut off in B. In C the 
wall between buffer cell and host has been dissolved, permitting direct 
protoolasmic contact. In D branches are developing from the buffer cell. 

(After Burgeff.) 

eludes that membranes are penetrated by germ tubes or hyphae 
only when a certain concentration of an attractive substance 
(chemical attractant) is present on the opposite side. According 
to this theory, concentration of the chemical must exceed a spe- 
cific threshold value if it is to attract; but, if a certain maximum 
concentration is employed, the result is repellant action. He 
dealt with penetration of collodion films, parchment, gold leaf, 
cellulose films, and the epidermis of onion scales. The spores of 
the various species used were placed in films of agar that were 
separated from the chemical to be tested by perforated sheets of 
mica. He also injected with water leaves of Begonia and Trades- 
cantia to which spores were applied, but no penetration resulted, 
whereas active penetration followed injection with cane sugar. 
Admittedly his conclusion that chemotropic factors are funda- 
mental in determining whether germ tubes oenetrate is supported 
by quite convincing evidence. 



242 HOST PEXETRATION 

A different point of view, however, results from the experi- 
ments of Fulton (1906). He followed the same techniques as 
Mivoshi, using among others the following fungi: Botrytis vul- 
garis, Penicillium glaucum, Sterigmatocystis nigra, Mucor vm- 
cedo, Monilia sitophila, M. fructigena, and Sphaeropsis malorwn. 
Fulton postulated a negative chemotropism, resulting from meta- 
bolic staling products produced by the fungus itself. The germ 
tubes showed quite as much turning toward pure water and non- 
nutrient solutions as toward substances that were presumed to 
act as attractants. 

Graves (1916) reinvestigated the problem of chemotropism, 
usin^ reactions of germinating spores of Rhizopns nigricans and 
Botrytis cinerea. Me too employed the perforated mica-plate 
technique. His evidence inclined him toward the negative- 
chemotropism hvpothesis of Fulton for these reasons: (a) the 
germ tubes and hvphae turn away from the layer on the opposite 
side of the mica plate if it is already well occupied by hyphae 
or already contains their own staling products; (b) the germ tubes 
and hyphae turn toward the layer on the opposite side of the mica 
plate if it is free of hvphae and staling products, unless it con- 
tains some other substance capable of evoking a negative chemo- 
tropic reaction; (c) the germ tubes and hyphae, when present in 
equal amounts on both sides of the mica plate, exhibit no turning 
from one side to the other. Nevertheless, Graves found justifica- 
tion also for the views of Miyoshi. In his general conclusion he 
took the position that positive chemotropism is to be regarded 
as one of the factors that govern penetration, but that negative 
chemotropism is the major factor. 

It becomes of interest to follow the implications that logically 
follow the acceptance of these conclusions. Susceptibility could 
be attributed to the possession by the host tissues of substances 
that attract and, conversely, resistance to substances that repel. 
A specialized pathogen, then, is one which would react to one 
particular substance onlv, whereas a generalized pathogen would 
react to a variety of substances. That such is not the situation is 
shown by the work of Johnson (1932) in his studies with Col- 
letotrichum circinans. He found that this organism is capable of 
penetrating such widely unrelated species as buckwheat, bean, 
cotton, tomato, cucumber, tobacco, cabbage, castor bean, and 
morning glory. It was unable to produce lesions, to be sure, but 



DIRECT PENETRATION 243 

the organism could be isolated from the interior of these host 
species several days after inoculation. Similarly Young (1926), 
using Diplodia zeae y Cephalosporin?;? acremormum, Colletotrich- 
ium nigrum, Hefomnthosporium gra???i??eu???, and other fungi, was 
able to produce lesions or callosities on many kinds of plants not 
normally infected by these fungi. Since the spores of a multitude 
of different fungi must find lodgment at the surface of every 
green plant, it is reasonable to expect that their hyphae may gain 
entrance yet be unable to establish a pathogenic relationship. 
That this occurs is, moreover, attested by the experiences of 
everyone who has attempted to isolate fungi by using bits of host 
tissues as inocula. It is not surprising, therefore, to find many ad- 
herents to the viewpoint voiced by Brown (1934) that neither 
positive chemotropism nor negative chemotropism plays any sig- 
nificant role in penetration. 

Further examination of the perforated mica-plate method as 
a working model to represent host tissue with its natural openings 
appears pertinent. Without the arguments being followed out, 
this analogy would appear to be in the same category as the sub- 
stitution of glass slides sprayed with fungicides for the surface of 
leaves and fruits in tests to determine the value of fungicides. 
Studies on the toxicity of Bordeaux by Yarwood (1943) show 
that it is more active on bean leaves against the urediniospores of 
Uromyces phase oli than it is on glass slides. These results serve 
to bring into sharp perspective the differences between in vitro 
and i?i vivo tests for the toxicity of fungicides. 

Brown and Harvey (1927) got ready penetration by germ tubes 
of Botrytis cinerea of epidermis from onion scales and of Eucharis 
leaves, either inward or outward, if the membranes were washed 
to remove diffusible substances. Similar results followed the sub- 
stitution of membranes made of paraffin. These results and a 
mass of similar ones by other workers show that penetration may 
be independent of any chemotropic stimulus. 

Failure to establish response to chemotropic stimuli as a satis- 
factory explanation for penetration has focussed attention on the 
role of the stimulus of contact, haptotropism. It may be recalled 
that certain species of Botrytis, Sclerotinia, Colletotrichum, Gloeo- 
sporium, and Marssonia show their reaction to contact by form- 
ing attachment organs. The best evidence in favor of hapto- 
tropism as a factor in penetration comes from experiments with 



244 



HOST PENETRATION 



species that produce appressoria or other means of attachment 
that function in the same manner as appressoria. Here again evi- 
dence and opinion are divided, since some workers maintain that 
the penetration tube enters only in conjunction with the dissolu- 
tion of the cuticle to prepare the way, and others that the cuticle 
is pierced by a mechanical thrust. Brown (1915) made extracts 




Fig. 43. Haustorial tvpes. A. Branched haustorium of Peronospora calo- 
thecae. (After de Barv.) B. Haustoria of Puccinia adoxae. A sheath partly 
invests the haustorium. (After Guttenberg.) C. A bulbous haustorium of 
Erysiphe communis. (After Smith.) D. Digitate, sheathed haustorium of 

Erysiphe graminis. (After Smith.) 

of Botrytis cinerea, as has been stated, which were capable of di- 
gesting tissues very rapidly when injected into them, but which, 
if placed at the surface of delicate rose petals, caused no injury 
within a period of approximately 24 hours. His lack of evidence 
of solvent effect to aid penetration is substantiated by the results 
of Boyle ( 1921 ), Waterhouse (1921), Dey (1919, 1933) and many 
others. Instead they adhere to the mechanical theory of penetra- 
tion. In the experiments of Brown and Harvey (1927) cells of 
Eucharis and other plants were readily penetrated if they had 
previously been plasmolyzed, but no penetration took place if the 
cells were turgid. It becomes difficult to understand how rigid- 
ity of the host cells could inhibit the production of wall-dissolving 
enzymes. 



DIRECT PENETRATION 245 

The work of Link and his associates (1929) involving the in- 
hibitory activity of specific chemicals is of special interest. They 
noted that white onions are subject to attack by Collet otrichum 
(Vermicalaria) circinans, which causes the disease known as 
smudge, and that pigmented onions are disease-free. From these 
onions they isolated protocatechuic acid, which was found to 
inhibit the growth of the pathogen. This organic complex, fur- 
thermore, does not occur in white-scaled onions. Inhibition of 
penetration by the smudge fungus and disease resistance, there- 
fore, are caused by protocatechuic acid. Presumably this is the 
first chemical substance isolated that has been demonstrated to 
render plants immune from infection. 

In connection with mechanical penetration it mav be recalled 
that a mucilaginous matrix aids in sticking the spore, appressorium. 
or germ tube to the cuticle, thus providing anchorage against 
the force of the thrust required to pierce the cuticle. The small 
diameter of the infection hypha minimizes this required force, 
which attempts have been made to measure by mechanical de- 
vices. Hawkins and Harvey (1919) studied penetration of potato 
by the rot-producing fungus, Fythium de baryamnn. They em- 
ployed a modified Joly balance with a needle having a point of 
definite area to test resistance of potato tissues to puncture. Po- 
tatoes of the McCormick variety, resistant to attack by this 
organism, were found to require more pressure to puncture than 
was required for Bliss Triumph or Green Mountain, varieties sus- 
ceptible to decay. Rosenbaum and Sando (1920), using the same 
appliance, correlated resistance of tomatoes to puncture with re- 
sistance to penetration by Meter ospor'mm tomato. Certain of their 
data are presented in Table 23. These data show that, as tomato 
fruits increase in age, they also increase in ability to inhibit pene- 
tration and consequent infection bv this fungus. Thickness of 
the cuticular layer also increases with the age of the tomato fruit, 
but, as Rosenbaum and Sando point out, these results do not prove 
that inhibition of penetration is purely a matter of resistance to 
mechanical pressure. 

Epidermal resistance of barberry to puncture was measured 
with a mechanical device by Melander and Craigie (1927), and 
they correlated their measurements with resistance to penetration 
by germinating basidiospores of Puccinia grcmrinis. They con- 



246 



HOST PENETRATION 



TABLE 23 

Relation of Resistance of Tomato Fruits to Puncture and to Penetration 

by Macros porium tomato 





Diameter 


Average Pressure 


Infection 


e of Fruit 


of Fruit 


Xccessary to 


of Fruit 


{days) 


{centimeters) 


Puncture {grams) 


{percentage) 


7 


0.70 


0.97 


100 


14 


2.30 


2.99 


100 


21 


5.18 


4.21 


85 


28 


5.40 


4.90 


49 


35 


5.46 


5.08 


23.3 


41 


6.55 


5.96 





48 


6.92 


6.74 





55 




5.56 






eluded that species of Berberis which are resistant to puncture are 
usually resistant to rust, but the converse is not usually true. 

Pioneer work on the correlation of structure of plant tissues and 
inhibition of penetration by fungi into plant tissues was instituted 
by Yalleau (1915). Thickness of the skin of plums was found 
correlated with resistance to the brown-rot fundus. Yalleau also 
found that the cells lining substomatal cavities possessed corky 
walls and that the stomata were very commonly completely 
occluded with corky cells. By and laroe, Curtis (1928) verified 
Valleau's findings but believed that cuticular resistance to pene- 
tration by the brown-rot pathogen was equally as important as 
the presence of corky tissue in natural openings, if not more im- 
portant. It might be expected that varieties of stone fruits lack- 
ing stomata or lenticels would be immune. Curtis did not find 
this to be true, however, since in the varieties which he investi- 
gated the germ tubes entered through the stomata in plums, 
through the cuticle in cherries, and down the hair sockets in 
peaches, and penetrated cither through the cuticle or the stomata 
in apricots. 

In varieties of tobacco resistant to invasion by the black root-rot 
fungus, Thielaviopsis basicola, Conant (1927) found that resist- 
ance to infection is correlated with the ability of the host rapidly 
to develop a corky layer to inhibit the spread of the pathogen. 

The short period of time required for penetration of the cell 
wall by Pythiitui de baryamtm [Hawkins and Harvev (1919)] 
also constitutes evidence of mechanical puncture. They observed 



DIRECT PENETRATION 



241 



penetration to be accomplished within approximately 5 minutes. 
They also found the hvphae to possess an osmotic pressure suffi- 
cient to penetrate turgid potato cells. Few measurements of 
osmotic pressure in fungi have been made; thev might be found 
valuable in an interpretation of factors concerned in penetration. 
Studies of this type were conducted bv Thatcher (1939, 1942). 
He used the plasmolvtic method in osmotic pressure and permea- 
bility determinations and was able to show that certain parasitic 
funs^i increase the permeability of the plasma membrane of the 
host cells. His measurements of the osmotic pressure of parasite 
and host are shown in Table 24. In each fungus the osmotic 





TABLE 24 






rs of Measurements 


of Osmotic P 


ressure in Parasite and H 




Average 




Average 




Osmotic 




Osmotic 




Pressure 




Pressure 


Parasite 


{atmospheres) 


Host 


{atmospheres') 


Uromyces jabae 




Pis urn sativum 




germ tubes 


44.25 


leaf 


9.15 


haustoria 


21.90 


petiole 


10.10 


Uromyces caryophyllinus 




Dianthus 




haustoria 


18.6 


leaf base 


11.2 


Puccinia graminis 




Mindum wheat 




haustoria 


18.9 


leaf 


9.4 


Erysiphe polygoni 




Brassica 




hvphae 


18.0 


leaf 


10.6 


Botrytis cinerea 




Apium graveolens 




hvphae 


29.8 


petiole 


8.3 


Sclerotinia sclerotiorum 




Apium graveolens 




hvphae 


23.5 


petiole 


13.4 


Phoma lingam 




Brassica 




hvphae 


41.3 


root 


11.3 



pressure of the parasite is greater than that of its host. Moreover, 
Thatcher was able to demonstrate an increased permeability in 
diseased tissues over healthy tissues, indicating that the parasite 
causes certain substances to leach from the host cells and thus to 
lower their osmotic pressure. 

Although these data as a whole have a bearing on the problem 
of penetration and might be taken to prove that penetration is 
the result of mechanical pressure in certain species of fungi, it 
does not necessarily follow that all species which effect their own 



248 HOST PEXETRATIOX 

entrance do so by the same means. Aronescu ( 1934) concluded 
that both chemical action and mechanical pressure are necessary 
for penetration bv the fungus causing black spot of roses. There 
may exist only the two general means of penetration that have 
been discussed, but perhaps each pathogen has made such modifi- 
cations and adaptations as are suited to its own requirements. 

STOMATAL PEXETRATIOX 

Stomata constitute normal portals for entrance by a large num- 
ber of pathogenic species. Observations on penetration through 
stomata have been recorded for many different fungi. Such ob- 
servations may be made by one of three methods: (a) epidermal 
stripping, (b) sections of fixed, embedded material, and (c) use 
of a stomatoscope. Certain advantages and disadvantages attend 
the use of each. 

If spores are sown in drops of water on leaves and chalk is added 
to indicate the site of the drops, it is not difficult to strip off epi- 
dermis in the areas marked by deposits of chalk, mount it in water 
with the exterior surface upward, and examine it under the micro- 
scope. With a little practice the investigator can learn to tear the 
leaf and thus strip off the epidermis, or to cut it off by holding 
the leaf taut over the end of the finger and slicing parallel to the 
leaf surface. Bv this method many examinations can be made in a 
comparatively short time, and the time interval involved in pene- 
tration can thus be determined. Mounting specimens in cotton 
blue * instead of water may aid in differentiating the hyphae and 
in clearing the host tissues. 

The merit of fixing, at known intervals after inoculation, tissues 
which have had spores applied to their surfaces has the feature of 
permanency to recommend its use. These tissues may be em- 
bedded, sectioned, stained, and examined whenever time is avail- 
able and may be kept indefinitely. This method, however, is 
obviously both laborious and time-consuming. 

Direct examination with an apparatus known as the stomato- 
scope requires familiarity with the operation of an apparatus that 
has been available to only a few investigators. Pool and McKay 
(1916) used such an appliance in their studies of penetration of 

'Use 0.1% cotton blue in lactophcnol, which contains equal parts of 
phenol, lactic acid, glycerin, and distilled water. 



STOMATAL PENETRATION 



249 




Fig. 44. Types of appressoria. A. Appressorium of urediniospore at stomatal 
aperture. B, C, and D. Appressoria formed by one of the anthracnoses, as 
an early stage in germination. B. In culture a hypha arises from the appres- 
sorium. C. In the host the appressorium anchors the organism, and the 
slender penetration tube arises from beneath the appressorium. E and F. 
Appressoria formed by Diplocarpon rosae. (After Aronescu.) 



250 HOST PENETRATION 

sugar-beet leaves by germ tubes of Cercospora beticola. They 
observed that this organism is unable to penetrate at night when 
stomata are closed but can do so during daytime when the open- 
ing of the stomata permits the entrance of germ tubes into sub- 
stomatal cavities. No doubt many fungi among those that pene- 
trate through natural openings are able to do so only during day- 
light hours. This factor must be borne in mind in tests involving 
the pathogenicity of a given fungus. 

The germ tubes of aeciospores and urediniospores of rusts very 
commonly enter through stomata, although the germ tubes of the 
basidiospores of these same species may penetrate directly. Pady 
( 1935) noted that germinating aeciospores of Gymnoconia inter- 
stitialis enter blackberry leaves not through the stomata but by 
direct penetration. The urediniospores of many species are 
known to produce a special appressorium, which functions in the 
mechanism of entrance. The sequence of events in penetration 
is as follows: When the tip of the germ tube comes to lie imme- 
diately above a stoma, the protoplasm accumulates in the tip. 
This apical region then swells, and the end cell is delimited by a 
cross-septum to become the appressorium. By nuclear division 
two or more pairs of nuclei form within the appressorium. Then 
a hypha develops from the lower side of the appressorium and 
forces its way between the guard cells into the substomatal cavity; 
once inside, its tip swells to form a vesicle into which the proto- 
plast of the appressorium migrates. Meanwhile more conjugate 
nuclear divisions occur, and hvphae, whose cells contain paired 
nuclei, grow radiately from this substomatal vesicle. These hy- 
phae course between the host cells and establish intimate contact 
with them by forming haustoria. 

Opinions differ as to whether the appressoria of rusts adhere 
by means of a mucilaginous matrix. Rice (1927) saw no evidence 
of such a matrix in Fuccinia sorghi. 

Study of penetration by Fuccinia graminis tritici into resistant 
Khapli emmer by Allen (1926) indicates that the appressoria se- 
crete a toxin upon the guard cells. This observation led her to 
opine that ". . . the appressorial secretion is a survival from an 
earlier period in the evolution of the fungus when it did dissolve 
its way into the host." 

The time required for penetration is correlated with tempera- 
ture, as has been capably shown by Peltier in studies with Fuccinia 



PENETRATION THROUGH WOUNDS 251 

graminis tritici. When he inoculated 7-dav-old wheat seedlings 
with urediniospores within the range optimum for germination, 
he found that maximum infection required at least 36 hours | Pel- 
tier (1925)]. This period was determined by use of a series of 
plants inoculated at the same time by a suspension of uredinio- 
spores. After definite intervals the surfaces of some of the plants 
were permitted to become dry. An arbitrary scale to show 
severity of infection was then employed as a basis of comparison. 
Leaves on which 5 or fewer rust pustules developed were regarded 
as in class 1; those with 6 to 10, in class 2; and those with 1 1 to 25, 
in class 3. Certain of Peltier's data are shown in Table 25. Ap- 

TABLE 25 

Time Required for Infection by Puccina graminis tritici 

Definite Intervals Plants Severity of 

after Which Leaves Infected Infection 

Dried {hours) {percentage) {class) 

2 

3 1.7 1 
6 17.0 1 

12 28.0 1 

16 33.0 1 

20 59.0 1 

24 89.0 2 

30 98.0 2 

36 100.0 3 

parentlv the minimum time required by the wheat stem-rust 

fungus for actual entrance through the stomatal aoerture is he- 
ir ^ 

tween 2 and 3 hours. 

PENETRATION THROUGH WOUNDS 

There is a large group of facultative parasites that lack ability 
to produce disease or decay in^ intact tissues but can establish 
themselves in wounds and thence spread to normal tissues. The 
heartwood and sapwood rots of trees are notable in this respect. 
Many of these fungi gain entrance through branch stubs or scars 
left in pruning, through fire scars, through abrasions from contact 
of limbs, or through injuries by other fungi, insects, or rodents. 

Little is known about the fundamental differences between these 
so-called facultative parasites and true parasites or about actual 



252 HOST PENETRATION 

changes in aggressiveness or pathogenicity which they may un- 
dergo as the'result of growth on wounded tissues. This matter 
has'been the subject of experimentation and speculation by many 
students of fungi. Salmon ( 1905) made the observation that races 
of Erysiphe graminis occurring on various genera of grasses are 
morphologically indistinguishable yet cannot be made to infect 
reciprocally when cross-inoculated from one genus to another. 
If, however, he wounded the leaf by cutting away a small piece of 
tissue or by applying a hot needle to its surface and then placed 
the spores on the surface opposite the wound, ready infection 

resulted. 

Many have concerned themselves with what may be a closely 
related problem in trving to account for the inability among het- 
eroecious rusts of basidiospores to infect the telial host. The mere 
oeneralization that aggressiveness is enhanced or rejuvenated by 
sexuality does not appear to constitute a satisfactory explanation. 

HAUSTORIA AND THEIR SIGNIFICANCE 

Penetration of tissues by fungi is also concerned with host- 
parasite relations after the pathogen has pierced the cuticle or 
epidermis, the first line of defence. Some species remain entirely 
intercellular; others are intercellular but possess intracellular haus- 
toria; and in others the mycelium itself courses intracellularly from 
cell to cell. Our immediate interest is in the haustorium-forming 
species. This group includes such obligate parasites as the downy 
mildews, powderv mildews, rusts, and smuts but is not confined 
to obligate parasites, since haustoria have been observed in Coc- 
ci >mvces, Diplocarpon, and other genera. Among the rusts, in- 
tracellular mycelium has been observed in one species only, 
namely, the short-cycled form of Gymnoconia interstitialis. In 
this species, which is systemic, Pady (1935) described peculiar 
intracellular elements which functioned to establish the fungus 
in the host. They were therefore interpreted as being haustoria! 
in nature. An elaborate account by Rice (1927), dealing espe- 
cially with haustoria of rusts, contains much of value regarding 
the structure and function of haustoria in general. 

Haustoria vary in form among the different species of fungi, 
bein^ spherical in the simplest forms and variously branched and 
tabulate in the most complex ones. Their size indicates conform- 



PENETRATION BY ECTOPARASITES 253 

ity to that necessary to maintain a delicate nutritional balance. 
Haustoria mav be uninucleate, may contain a pair of nuclei, or 
may be multinucleate. They possess a conspicuous sheath that is 
deposited by and is continuous with the host-cell wall, as generally 
believed. In Diplocarpon rosae the sheath does not extend com- 
pletely around the haustorium [Aronescu (1934)1, as has been 
described for many parasitic fungi. In Erysiphaceae, however, 
staining reactions indicate that haustorial sheaths are chitinous. 
More should be known regarding the chemical nature of the 
sheath as an aid in understanding how the sheath modifies absorp- 
tion and passage of food. 

Haustoria are always connected with the intercellular hyphae 
by narrow tubes of a length slightly in excess of the thickness 
of the host-cell membrane. These constrictions facilitate pene- 
tration. Presumably both mechanical pressure and dissolution of 
the wall are involved in haustorial penetration. Allen (1923) 
found no evidence of enzyme secretion in connection with haus- 
torial penetration by Puccinia graminla tritici, but the walls be- 
neath the appressoria appeared to be altered during initiation of 
infection. 

PENETRATION BY ECTOPARASITES 

Several distinct types of host-parasite relationships occur among 
fungi possessing mycelium which remains wholly external to the 
infected plant organs. One type is represented by the powdery 
mildews, all of which, except Fhyllactinia cor y lea and Leveillula 
(Erysiphe) taurica, are ectophytic. In P. cor y lea both internal 
and external mycelium is produced; in L. taurica the mycelium is 
wholly internal, an adaptation to xerophytic environment. All 
parts of all other powdery mildews, except the haustoria, are 
borne externally. 

A very unusual type of ectoparasitism is exhibited by Cerco- 
sporella rubi, the cause of rosette and double blossom of blackber- 
ries and dewberries. Plakidas (1937) found that the mycelium 
of this fungus occurs in buds between the embryonic leaves. If 
the buds are opened at any time during summer, fall, winter, or 
early spring, a delicate arachnoid weft will be observed to be pres- 
ent between the young leaves. The fungus at no time actually 
penetrates the young branch buds and flower buds but absorbs 
its nourishment directly through the thin walls of the embryonic 



254 HOST PENETRATION 

cells. Transfer of food from host to parasite does not require, in 
this species, the production of specialized organs of penetration. 

A third type of ectoparasitism is exhibited by the Meliolaceae 
and Capnodiaceae, which apply themselves to the surface of plant 
tissues by means of hyphopodia. Graff (1932) found that, al- 
though some species of Meliola form haustorial vesicles within the 
epidermal cells, M. civ ductus is entirely superficial. Its cell walls 
are in intimate contact with the host epidermis and are thinner 
wherever contact is maintained. The epidermal cell walls are 
more or less corroded at these points of contact, and evidence of 
defeneration products was noted within them. 

Internal mycelium is wanting or scanty in many Microthy- 
riaceae and Hemisphaeriaceae. Luttrell (1940) concluded that 
Morejioclla quercina, one of the Microthyriaceae, absorbs its nu- 
trients through the intact host cuticle at first, and later certain 
hyphae penetrate only to the extent of becoming subcuticular. 

IMPLICATIONS 

Problems of host penetration remain of utmost importance in 
spite of the large number of studies that have been devoted to this 
phenomenon and in spite of the conflict among observations and 
the interpretations of them. They should continue to receive un- 
stinted attention because of their bearing on matters of tolerance 
or resistance to disease, on studies involving the causes of natural 
immunity, and on production of races of disease-resistant crop 
plants. 

LITERATURE CITED 

Vi.i.en, Ruth F., "A cvtolomcal study of infection of Baart and Kanrcd 
wheats bv Puccinia graminis tritici," J. Agr. Research, 23: 131-151, 1925. 
"Cvtological studies of forms IX, XXI, XXVII of Puccinia grarmnis tritici 
on Khalpi emmer," /. Agr. Research, 52:701-725, 1926. 

Arones< u, Ai ice, "Diplocarpon rosae: from spore germination to haustorium 
formation," Bull. Torrey Botan. Club, 57:291-329, 1934. 

Barn. A. i>i . "(her einige Sclerotinicn und Sclerotienkrankheitcn," Botan. 
Z., 44: 377-387, 393-404, 409-126, 433-441, 449-461, 465-474, 1886. 

Blackman, V. H., "Physiological aspects of parasitism," Proc. Brit. Assoc. 
Bot. Toronto, 23 3-246, 1924. 

Bi..\( KMAN, y. H., and I". J. Wl lsford, "Studies in the physiology of para- 
sitism. II. Infection by Botrytis ciuerea" Ann. Botany, 50:389-398, 
1916. 



LITERATURE CITED 255 

Boyle, C, "Studies in the physiology of parisitism. VI. Infection by Sclero- 

tinia libertiana" Ann. Botany, 35:117-147, 1921. 
Brown, W., "Studies on the physiology of parasitism. I. The action of 
Botrytis cinerea" Ann. Botany, 22:313-348, 1915. 
VIII. "On the exosmosis of nutrient substances from the host tissue into 

the infection drop," Ann. Botany, 36: 285-300, 1922. 
"Mechanism of disease resistance in plants," Trans. Brit. Mycol. Soc, 

19: 11-33, 1934. 

"The physiology of host-parasite relation," Botan. Rev., 2:236-281, 1936. 

Brown, W., and C. C. Harvey, "Studies in the physiology of parasitism. 

X. On the entrance of parasite fungi into the host plant," Ann. Botany, 

41:641-662, 1927. 

Burgeff, H., "Untersuchungen liber Sexualitat und Parasitismus bei den 

Mucorineen," Botan. Abhandl. (Herausgeg. von Goebel), 4: 1-135, 1924. 

Busgen, M., "Uber einige Eigenschaften der Keimlinge parasitischer Pilze," 

Botan. Z., 51:51-72, 1893. 
Conant, G. H., "Histological studies of resistance in tobacco to Thielavia 

basicola," Am. J. Botany, 14: 457-480, 1927. 
Curtis, K. M., "The life history and cytology of Synchytrium endobio- 
ticnm," Phil. Trans. Roy. Soc. London, Ser. B, 270:409-478, 1921. 
"The morphological aspect of resistance to brown rot in stone fruit," 
Ann. Botany, 42: 39-68, 1928. 
Dey, P. K., "Studies in the physiology of parasitism. V. Infection by Col- 
let otrichum lindemiithianwn" Ann. Botany, 53:305-312, 1919. 
"Studies in the physiology of the appressorium of Collet otrichum 
gloeosporioides" Ann. Botany, 47:305-312, 1933. 
Frank, A. B., "Uber einige neue und weniger bekannte Pflanzenkrankhei- 

ten," Ber. dent, botan. Ges., 7:29-34, 58-63, 1883. 
Fulton, H. R., "Chemotropism of fungi," Botan. Gaz., 47:81-108, 1906. 
Graff, P. W., "The morphological and cytological development of Meliola 

circinans," Bull. Torrey Botan. Club, 59: 241-266, 1932. 
Graves, A. H., "Chemotropism in Rhizopus nigricans" Botan. Gaz., 62: 337— 

369, 1916. 
Hasselbring, H., "The appressoria of the anthracnoses," Botan. Gaz., 42: 135- 

142, 1906. 
Hawkins, L. A., and R. B. Harvey, "Physiological study of the parasitism of 
Yythium de Baryanum Hesse on the potato tuber," /. Agr. Research, 
18: 275-297, 1919. 
Higgins, B. B., "Physiology and parasitism of Sclerotimn rolfsii," Phyto- 
pathology, 77:417-448, 1927. 
Johnson, Burt, "Specificity to penetration of the epidermis of a plant by 

the hyphae of a pathogenic fungus," Am. J. Botany, 19: 12-31, 1932. 
Link, K. P., H. R. Angell, and J. C. Walker, "The isolation of proto- 
catechuic acid from pigmented onion scales and its significance in rela- 
tion to disease resistance in onion," /. Biol. Cheni., SI: 369-375, 1929. 
Luttrell, E. S., u Moroenoella quercina, cause of leaf spot of oaks," Mycol., 
32:652-666, 1940. 



256 HOST PENETRATION 

Melander, L. W., and J. H. Craigie, "Nature of resistance of Berberis spp. 

to Puccinia graminis," Phytopathology, 77:95-114, 1927. 
Miyoshi, M., "Uber Chemotropismus der Pilze," Botan. Z., 52: 1-28, 1894. 
"Die Durchbohrung von Membranen durch Pilzfaden," Jahrb. iviss. Botan., 
28: 269-289, 1895. 
Pady, S. M., "Aeciospore infection in Gymnoconia interstitialis by penetra- 
tion of the cuticle," Phytopathology, 25:453-474, 1935. 
"The role of intracellular mycelium in systemic infections of Rubus with 
the orange rust," Mycol., 27:618-637, 1935a. 
Peltier, G. L., "A consideration of the physiology and life history of a 
parasitic Botrvtis on pepper and lettuce," Mo. Botan. Garden Rept., 
25:41-74, 1912. 
"A studv of the environmental conditions influencing the development 
of stem rust of wheat in the absence of an alternate host," Nebr. Agr. 
Expt. Sta. Research Bull, 35. 11 pp. 1925. 
Plakidas, A. G., "The rosette disease of blackberries and dewberries," /. Agr. 

Research, 54: 275-303, 1937. 
Pool, V. W., and M. B. McKay, "Relation of stomatal movement to infec- 
tion bv Cercospora beticola," J. Agr. Research, 5: 1011-1038, 1916. 
Rice, Mabel A., "The haustoria of certain rusts and the relation between host 

and pathogene," Bull. Torrey Botan. Club, 54: 63-153, 1927. 
Rosenuaum, J., and C. E. Saxdo, "Correlation between size of the fruit and 
the resistance of the tomato skin to puncture and its relation to infection 
with Macrosporiwn tomato Cooke," Am. J. Botany, 7: 78-82, 1920. 
Salmon, E. S., "Cultural experiments with biologic forms of the Ery- 

siphaceae," Phil. Trans. Roy. Soc, Ser. B, 191: 107-122, 1905. 
Smith, R. E., "The parasitism of Botrytis cinerea," Botan. Gaz., 25:421-436, 

1902. 
Thatcher, F. S., "Osmotic and permeability relations in the nutrition of 
fungus parasites," Am. J. Botany, 26: 849-858, 1939. 
"Further studies of osmotic and permeability relations in parasitism," 
Can. J. Research, 20:283-311, 1942. 
Tisdale, \Y. H., "Physoderma disease of corn," /. Agr. Research, 16: 137- 

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Valleau, W. D., "Varietal resistance of plums to brown rot," /. Agr. Re- 
search, 5:365-396, 1915. 
Ward, H. Marshall, "A lily disease," Ann. Botany, 2: 319-382, 1888. 
Waterhouse, W. L., "Studies in the physiology of parasitism. VII. Infec- 
tion of Berberis vulgaris by sporidia of Puccinia graminis" Ann. 
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Yarwood, C. E., "The function of lime and host leaves in the action of 

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Young, P. A., "Penetration phenomena and facultative parasitism in Alter- 
naria, Diplodia, and other fungi," Botan. Gaz., SI: 258-279, 1926. 



Chapter 11 

PHYSIOLOGIC SPECIALIZATION AND VARIATION 

AMONG FUNGI 

The concept that physiological differences exist between the 
members that together constitute a given species of fungus prob- 
ably has its origin in bacteriology. In the early years of bacteri- 
ology there were two opposing schools of thought, one of which 
held to the monomorphic hypothesis and the other to the poly- 
morphic or pleomorphic hypothesis. Adherents of the mono- 
morphic hypothesis believed in fixity and immutability of species; 
adherents of the polymorphic hypothesis, in variability in mor- 
phological and physiological characteristics. Billroth (1874), 
representing an extreme of the polymorphic group, for example, 
believed that only one species of bacteria, "Coccobacteria 
septica" existed. 

To the person who compares a considerable number of isolates 
of any one fungus, especially when grown on artificial media, it 
quickly becomes apparent that the species is variable and that 
differences exist between the several isolates. Although these dif- 
ferences may be so minute as to be morphologically indistinguish- 
able, they are none the less real and of tremendous importance, 
especially as they concern pathogenic species. In fact, problems 
of virulence of species, of their aggressiveness, of the outbreak 
of epidemics, and of the breeding of crop plants that are resistant 
or immune to attack, all hinge upon the fact that these differences 
are meaningful and must be taken into account. Questions con- 
cerning the origin of these differences have been regarded too 
largely as of only academic interest. Actually no useful purpose 
is served by assigning them to the academician. 

Definition of terms. The concept embodied in the term 
physiologic species has changed since Schroeter (1879) first sug- 
gested that physiologic specialization in fungi exists. He observed 
that Paccinia graminis, growing on wheat, failed to produce in- 

257 



258 PHYSIOLOGIC SPECIALIZATION AND VARIATION 

f ection if inoculated onto other grass hosts, such as rye, oats, timo- 
thv, and blue grass, whereas under the same environmental condi- 
tions wheat readily became infected. Similarly Fuccinia graminis 
from any of the other grasses produced infection on the host 
species from which the inoculum was taken, but reciprocal inocu- 
lations always failed to cause infection. Further work of the same 
nature by Eriksson (1894) led to the division of Puccinia graminis 
into the following groups, which he called "formae speciales": 
Fuccinia graminis tritici, P. graminis secalis, P. graminis avenae, 
P. graminis phlei-pratensis, P. graminis agrostidis, and P. graminis 
poae. He also showed that subdivisions can similarly be made of 
P. ghimavum, P. dispersa, and P. coronata. He recognized five 
specialized forms, tritici, secalis, elymi, agropyri, and hordei, of 
P. ghimavum. Four specialized forms, secalis, agropyri, bromi and 
tritici, comprise P. dispersa; and P. coronata consists of six, avenae, 
alopecnri, festucae, lolii, calamagrostis, and vielicae. To these 
groupings within the species the terms biologic forms, biologic 
races, physiologic forms, biologic species, physiologic species, 
physiologic races, parasitic strains, sister species, and specialized 
varieties have been applied. They are now generally regarded 
as varieties, and many workers designate them as of varietal rank. 
Their pathogenic behavior thus serves as the basis for the varietal 
separations. Within the past 25 years it has been found that many 
parasitic strains or biotypes comprise a given variety and that 
some of these strains can be isolated by their pathological effects 
on appropriate suscept species, and others by cultural character- 
istics. It is to these strain groupings that the term physiologic 
specialization is properly applied. 

Some mycologists maintain that it is impossible to establish 
varieties among pathogenic fungi, as in Fuccinia graminis, on the 
basis of morphologic differences. If this be true, there is little 
justification for the use of varietal names. Minute yet recogniz- 
able differences are indicated by others to exist, and they there- 
fore find it convenient to employ varietal names. Without the 
threadbare problem of what constitutes a variety or physiologic 
species being raised again, it is clearly established by students 
of rusts and smuts that secondary groupings within the variety 
may be made on the basis of pathogenicity on selected suscepts. 
These secondary groupings are called physiologic strains. They 



WHAT FUNGI HAVE PHYSIOLOGIC SPECIALIZATION? 259 

are not sufficiently distinct morphologically to entitle them to 
specific rank but must be distinguished from each other by 
pathogenic reactions. 

If the worker is dealing with fungi that can be cultivated on 
artificial media, he may employ differences in cultural character- 
istics, for example, in color of mycelial mat, shape of colonies, 
surface markings, size of colonies, branching of hyphae, and 
abundance of conidia, to distinguish physiologic forms. This 
situation is typified by the cultural differences noted by Chris- 
tiansen (1932) in the 15 races of Pestalozzia fwierea that he iso- 
lated from needles of longleaf pine. These races differed in 
abundance, color, and zonation of surface and aerial mycelium, in 
abundance, distribution, and size of acervuli, and in size, shape, and 
color of spores. 

Several other terms, including variation, mutation, saltation, and 
dissociation, have been more or less loosely used in connection 
with the phenomenon of differences among the members that 
comprise a given species of fungi. For clarity these terms may 
at this point be defined. Variation is applied to divergences, 
whether morphological or physiological, from the observed char- 
acteristics of the usual or normal condition. They are regarded 
as non-hereditary. Variation is usually regarded as svnomvmous 
with dissociation. Mutation, as originally employed by de Vries, 
refers to sudden variations, the offspring differing from the par- 
ents in one or more clearly defined characteristics. Mutation is 
to be distinguished from gradual variation, such as may occur 
during the course of countless generations. Furthermore muta- 
tions are hereditary, since once they appear, they can be trans- 
mitted to the progeny. Saltation may be defined as a type of mu- 
tation that appears in artificial cultures. Saltations may be main- 
tained indefinitely in subcultures if conidia or hyphae are used in 
transplantation. Sports, as the term is applied to seed plants that 
can be propagated by cutting or other vegetative structures, cor- 
respond to saltants among fungi. 

In what fungi has physiologic specialization been observed? 
Numerous species of pathogenic fungi are known to consist of 
many physiologic forms. Presumably all do. At least, it would 
be scientific news if after extensive study one was found that was 
not comprised of numerous physiologic forms. 



260 PHYSIOLOGIC SPECIALIZATION AND VARIATION 



TABLE 26 
Fungi Reported to Exhibit Physiologic Specialization 



Name of Fungi 

Rhizopus nigricans 
Albugo Candida 
Albugo ipomoeae-panduranae 
. llbugo tragopogonis 
Phvtophthora parasitica var. rhei 
Pcronospora spp. 
Erysiphe communis 
Erysiphe graminis 
Erysiphe graminis hordei 
Erysiphe graminis tritici 
Erysiphe horridula 
Phyllactinia guttata 
Sphaerotheca hamuli 
Claviceps purpurea 
Pleospora spp. 
Plonrightia morbosa 
Puccinia a no mala 
Puccinia coronata avenae 
Puccinia glumarum 

Puccinia graminis avenae 



Puccinia graminis secalis 
Puccinia graminis tritici 



Puccinia rubigo-vera tritici 



Puccinia sorghi 
Sphacelotheca sorghi 

Sorosporium reilianum 
Tilletia laevis, T. tritici 

Ustilago avenae, U. levis 



Vstilago hordei 
Ustilago tritici 
Ustilago violacea 
Ustilago zeae 

:oria >pp. 
Pestalozzia guepini 
Pestalozzia fit nerea 
Co lletotrich u m li ndem uth ia n u m 

Helminthosporium gramineum 

Helminthos porium sativum 
Polyspora lini 
Fusarium lini 

Rhizoctonia solani 



Authority for Report 

Harter and Weimer (1923) 

Toeashi and Shibasaki (1934) 

Citerri (1928) 

Pfister (192") 

Leonian (1926) 

Gaumann (1923) 

Hammarlund (1925) 

Reed (1918), Salmon (1904), Marchal (1903) 

Mains and Dietz (1930) 

Mains (1933) 

Blumer (1922) 

Hammarlund (1925) 

Steiner (1908) 

Stager (1903), Stakman (1926) 

Diedicke (1902) 

Gilbert (1913) 

Mains (1933a), Hev (1931) 

Hoerner (1919), Peturson (1930), Frenzel (1930) 

Eriksson (1894), Allison and Isenbeck (1930), 

Wilhelm (1931) 
Eriksson (1894), Stakman, Levine, and Bailey 
(1923), Bailey (1925), Waterhouse (1929), 
Gordon (1933) 
Cotter and Levine (1932) 

Eriksson (1894), Stakman and Piemeisel (1917), 
Stakman and Levine (1922), Waterhouse 
(1929), Stakman, Levine, and Hines (1934), 
Newton and Johnson (1927) 
Mains and Jackson (1926), Waterhouse (1929), 
Johnson and Mains (1932), Mains (1933), Ra- 
dulescu (1932) 
Stakman et al. (1928) 
Tisdale, Melchers, and Clemmer (1927), Melch- 

ers, Fricke, and Johnston (1932) 
Reed, Swabev, and Kolk (1927), Stakman (1926) 
Rodenheiser and Stakman (1927), Reed (1928), 
Gaines (1928), Holton (1931), Bressman (1931) 
Reed (1924, 1927, 1929, 1940), Reed and 
Stanton (1932), Aamodt (1931), Flor (1933), 
Melchers (1934) 
Faris (1924) 

Stakman (1926), Grevel (1930) 
Zillig (1921), Goldschmidt (1928) 
Christiansen and Stakman (1926), Stakman et al. 

(1929) 
Beach (1919) 

LaRue and Bartlett (1922) 
Christiansen (1932) 

Barrus (1918), Burkholder (1923), Leach (1922), 
Budde (1928), Penser (1931), Schreiber (1932) 
Christiansen and Graham (1934) 
Christiansen (1922) 
Stakman (1926) 
Stakman (1926) 
Matsumoto (1921), Briton-Jones (1924) 



PATHOGENICITY TESTS 



261 



Although the list in Table 26 is by no means complete, it indi- 
cates that physiologic specialization occurs among all the major 
groups of fungi. A survey of accounts from which this list was 
compiled shows that in the recognition of physiologic forms four 
criteria were employed: (a) pathogenicity on special hosts, (b) 
differences in artificial culture, (c) minor morphological differ- 



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Length of spores (microns) 



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Fig. 45. Variation in length of spores of six strains of Pestalozzia guepini, 
plotted as length of spores in microns against percentage of the total 

number measured. (After LaRue.) 

ences, and (d) physico-chemical reactions. Each will be given 
further consideration. 

Pathogenicity tests. Concerning the pathogenic potentialities 
of disease-producing fungi, two diametrically opposed theories 
have been advanced. One is that disease-producing potentialities 
are inherent in the fungus itself and therefore are hereditary. 
The other is that the pathogens become adapted, modified, or 
"educated" under the influence of the host or of other environ- 
mental factors. According to the first view, the physiologic 
forms are true-breeding entities that maintain a uniform potential- 
ity to produce disease throughout many generations or over a 
long period of years. The weight of evidence in recent years 
favors this viewpoint, since physiologic characteristics appear to 



262 PHYSIOLOGIC SPECIALIZATION AND VARIATION 

be governed bv genetic factors in the same way as morphologic 
characteristics are governed. Nevertheless the idea of adaptation 
is maintained to be operative, and it has the support of a certain 
oroup who believe in evolution through adaptative modifications. 
Attention was directed to this point of view bv the results of 
Ward (1903) on Puccinia dispersa on brome grasses and of Sal- 
mon (1904) on various powdery mildews, especially Erysiphe 
graminis on grasses. Ward maintained that this rust could acquire 
the ability to attack a resistant host if it were first transferred to 
another host with lesser resistance. After repeated transfers on 
this less resistant host, it acquired the ability to attack the resist- 
ant one. The less resistant variety, therefore, served as a "bridge." 
Similarly, Salmon maintained that E. graminis from barley could 
not infect wheat unless the leaves were injured. When grown 
on injured wheat leaves for several transfers, it acquired the ability 
to infect intact ones. From this type of results he concluded, 
"The restriction in power of infection characteristic of biologic 
forms breaks down if the vitality of the leaf on which the conidia 
are sown is interfered with in certain ways." He also noted that 
the powdery mildew from Brovnis racemosus did not infect B. 
comrnutatus in 12 trials, but that it infected B. hordeaceus in each 
of 36 trials. Furthermore failure of infection resulted in 36 at- 
tempts when conidia from B. comrnutatus were applied to B. race- 
7/iosus, and infection occurred in 40 out of 49 trials in which 
conidia from B. hordeaceus were applied to B. comrnutatus. From 
these experiments B. hordeaceus was concluded to act as a bridg- 
ing species for powdery mildews on B. racemosus and B. com- 
rnutatus. 

Hammarlund (1925) attempted to repeat Salmon's work, using 
Erysiphe comimniis tritici cultivated for 37 generations on 
wounded leaves of Hordeum europaeuvu with the result that 
there was no increasing tendency to become adapted to barley. 
He also employed E. graminis tritici cultivated on wounded leaves 
of Hordeum vulgare for 128 generations. In this experiment like- 
wise there was no evidence at the end that the powdery mildew 
had acquired the ability to infect intact barley leaves. 

Stakman and his associates (1926) attempted to adapt Erysiphe 
graminis tritici to grow on barley, rye, and oats. They subjected 
the plants to "every conceivable form of torture," but all refused 
to become infected. 



DIFFERENCES IN ARTIFICIAL CULTURE 



263 



The existence of adaptation and "bridging" among pathogens 
remains questionable in the light of these experiments. That the 
pathogenicity of specialized races is hereditary and therefore con- 
stant, on the other hand, has volumes of evidence in its support. 
Those who have studied the rusts over a period of years, as have 



H. hordeaceus 



H. secalinus 



H. arduennensis 




H. interruptus 



H. commutatus 



H. racemosus 

Fig. 46. Reciprocal inoculation of species of Hordeum with conidia of 
powdery mildew. Solid lines show successful transfer with resultant in- 
fection in direction indicated bv arrow; broken lines, failure of infection 
after transfer of conidia. The numerator indicates the number of successful 
trials; the denominator, the number of attempts made to secure infection. 
The species hordeaceus is a "bridging species" for Erysiphe gram'inis. 

(After Salmon.) 

Stakman and his associates, or the smuts, as have Reed and his 
associates, are able to isolate the same physiologic forms year 
after year. These identical forms may range widely in one area 
or country or may even be found in different continents. 

Differences in artificial culture. Macroscopic or micro- 
scopic differences between strains isolated from monosporous cul- 
tures have been reported for numerous species of fungi. From 
our knowledge of genetics these strain differences may have arisen 
either through hybridization or through mutation. 

From the voluminous literature on plus and minus strains within 



264 PHYSIOLOGIC SPECIALIZATION AND VARIATION 

a s;iven species, it would be anticipated that new forms are being 
produced constantly. There is abundant evidence that this is the 
situation, especially among rusts and smuts [Christiansen and Stak- 
man (1926), Newton and Johnson (1927), Christiansen (1929), 
Flor (1932)]. 

Sectoring. Sometimes differences appear as sectors in the col- 
onies on Petri dishes. These sectors may appear as wedge-shaped 
areas of different color, of different compactness of mycelial 
growth, with less profuse sporulation, or of some other very 
marked difference. Christiansen and Stakman (1926) and Stak- 
man, Tyler, and Hafstad ( 1933) noted sectoring in Ustilago zeae. 
In their report Stakman, Tyler, and Hafstad (1933) recorded the 
isolation of 14 variant lines of U. zeae from a single monosporidial 
cell that sectored in culture. Each line maintained distinctive 
cultural characteristics for 5 years. Evidently each was a distinct 
biotype, and each arose as a mutation. 

Dod^e ( 1931) isolated an albino strain of Nenrospora sitophila 
that produced few conidia. Johnson and Yalleau (1935) isolated 
from a sector an albino strain of Thielaviopsis basicola. Leonian 
(1930) observed sectoring in Fusarium moniltforvie, and it appears 
to be of rather common occurrence among Fusaria in the section 
Elegans. Hansen and Smith (1932) recorded sectoring in Botrytis 
cinerea, and Wolf and Wolf (1939) in Botryosphaeria ribis. Pes- 
talozzia funerea sectored, giving rise to conidia with only a single 
seta [Christiansen (1932)], which is characteristic of the genus 
Monochaetia. 

The occurrence of dissociation with the production of albinistic 
mutants has been noted in Brachysporhnn trifolii [Bonar (1922)] 
and among sclerotia in Botrytis cinerea. 

Brierlev (1920) and Christiansen (1922) secured evidence that 
some of the mutations of Helminthosporiwn sativum were more 
virulent, and others less virulent, than their parents. Ustilago 
zeae from purplish sectors was more virulent than that from tan 
sectors [Christiansen and Stakman (1926)]. Newton and John- 
son (1927) isolated a bright orange and a greyish strain of Pnc- 
cinia graminis tritici from form species 9. Both seemed identical 
in pathogenecity, however, with the normal form. 

Sectoring among fungi has been compared with "bud sporting" 
among seed plants. The causes of sectoring are not understood, 
although certain factors are known to exert an influence. These 



SECTORING 265 

include kind and amount of nutrients, temperature, light and other 
radiations, pH, staling products, and the addition of certain salts 
and toxic substances, a subject brief! v summarized by Christian- 
sen (1940). 

Sectoring could be expected to take place among fungi in which 
hvphal fusions occur or in those with multinucleate spores, as in 
Botrytis cinerea. Hansen and Smith (1932) have shown that the 
propagative elements of this fungus are heterocaryotic, resulting 
from anastomoses that permit the migration of nuclei from one 
cell to another. In Botryosphaeria ribis, which has multinucleate 
ascospores and conidia, however, all the nuclei within anv asco- 
spore or conidium have the same origin and hence are homo- 
caryotic [Wolf and Wolf (1939)]. The causes of sectoring in 
this species are unknown, and the phenomenon may be wholly 
spontaneous. 

Normally when a culture originates from a single conidium it 
is regarded as clonal and is presumed to be genetically pure. 
Variations occur in the colonies from these clones, as has been 
shown by LaRue (1922) in Festalozzia guepiiii, by Christiansen 
(1932) in Festalozzia funerea, and by Leonian (1929) in many spe- 
cies and varieties of Fusarium. Sometimes the variants in subcul- 
tures of Fusarium remained different from the parent type and 
that of the variant biotype that arose by sectoring. Leonian 
(1929) concluded, "The presence of distinct strains and variants 
within the same species and their decidedly different reactions (to 
various acids and toxic substances) seem to indicate that the con- 
cept of the species must not be that of a single organism but that 
of a group of many organisms having in general the same principal 
characters." 

Brierley (1929) summarized his observations on variation of 
fungi in cultures, especially of Botrytis cinerea, by stating that his 
distinct monosporial isolates of B. cinerea remained stable for long 
periods of time when cultivated under different nutritional and 
environmental conditions, both in vitro and in vivo. When the 
different isolates were then brought back to the common stand- 
ardized environment, all immediately reverted in their conidial 
dimensions to a common original condition. This phenomenon 
shows genotypic fixity within the species, which has been re- 
peatedly demonstrated to occur in other organisms. 



266 PHYSIOLOGIC SPECIALIZATION AND VARIATION 

Four variant strains of Hypomyces ip07?weae—purp\e, alba, con- 
voluta, and reverta— were isolated by Dimock (1939). They 
originated by gene mutations. None of them appeared to be 
capable of perpetuating themselves in competition with the 
normal type, because all had growth rates slower than the normal, 
produced conidia less abundantly, and were quite incapable of 
inbreeding. 

The causes underlying these variational phenomena are un- 
doubtedly diverse. In some cases they have been shown to be 
frenetic, but in others a different explanation must be sought. 
Dodge (1942) noted increased vigor of growth and production of 
conidia in Neurospora tetrasperma when he crossed a dwarf 
race of this fungus with a normal one of opposite sex or else 
with one of the same sex with resultant mycelial fusions. The 
cells of these mvcelia, containing nuclei of both races, grew two 
or three times as fast as those of normal ones. He ascribed in- 
creased vigor to synthesis of vitamins by the heterocaryotic my- 
celium. 

Hybridization. Stakman, Levine, and Cotter (1930) crossed 
Fuccinia gr avium tritici form 36 with Puccinia graminis agrostidis. 
When segregation occurred in the progeny, 3 new form species 
were isolated that had previously not been encountered among 
the numerous form species of tritici.. Stakman, Levine, Cotter, 
and Hines (1934) segregated over 20 different races of wheat- 
stem rust from aecial collections and 80 from uredinial collections. 
In the Mississippi Valley, where barberry occurs, there is ample 
opportunity for hybridization to occur, whereas in Australia, 
where barberry is absent, as was pointed out by Waterhouse 
(1929), there are few races of wheat-stem rust. In consequence, 
an abundance of races can exist, and new ones can continue to 
arise naturally by segregation and recombination of factors for 
differences in pathogenicity wherever the barberry host thrives. 
Emphasis was also placed on hybridization as a means of securing 
new races of stem rust by Craigie (1940) in his summary of 
studies conducted at the Dominion Rust Research Laboratory, 
Winnepeg, Canada. 

Similarly Tisdale, Alelchers, and Clemmer (1927) found in 
Kansas, New .Mexico, and Texas a new kernel smut that infects 
milo :md hegari but is non-infectious to feteretia, and presented 
evidence that it arose as a hybrid between Sphacelotheca sorghi 



MORPHOLOGICAL DIFFERENCES 261 

and 5". cruerita. The phenotypes obtained had characters common 
to both parents. Other interspecific smut hybrids, such as those 
between Ustilago avenae and U. levis, and between U. hordei and 
U. medians, have been produced that are intermediate in the Fi 
generation but segregate in the F 2 generation. The status of 
present knowledge of genetic factors as applied to hybridization 
in smuts, and especially to the origin of physiologic races by this 
means, is summarized by Rodenheiser (1940). New specialized 
races of smuts are known to arise in nature, presumably by hy- 
bridization. Reed (1935) isolated from a collection of loose smut 
of oats from Texas two distinct new races, one capable of infect- 
ing Red Rustproof oat and the other Fulgum oat. 

It should be recalled that in some species of smuts infection and 
production of chlamydospores occur only if there has been fusion 
of lines of opposite sex. In such species hybridization between 
biotypes undoubtedly is of common occurrence. Moreover, inter- 
specific hybrids between Ustilago hordei and U. medians, U. levis 
and U. avenae, Tilletia levis and T. tritici, and Sphacelotheca 
omenta and S. sorghi have been produced. Certain intergeneric 
crosses, as between Sorosporiwn reilianum X Sphacelotheca 
sorghi, and Sorosporhtm reilianum X Sphacelotheca omenta, have 
also been effected [Tyler and Shumway (1935), Christiansen and 
Rodenheiser (1940)]. 

Morphological differences between physiologic species. It 
has been pointed out that minor morphologic differences have 
been noted between urediniospores of varieties of Pnccinia granii- 
nis. This observation has led to a search for distinctive differences 
by means of which to separate specialized races of this rust as it 
occurs on wheat. The outstanding of these attempts is that of 
Levine (1928), who by the aid of biometrical methods was able 
to show minor differences in size and shape of spores between 
the several physiologic forms. Newton and Johnson (1927) 
were able to show that a bright orange form species and a greyish 
brown one can be distinguished from the normal form 9 of P. 
graminis tritici. Similar segregation of species followed from 
monographic studies on Peronospora by Gaumann (1923). By 
making numerous measurements of the lengths and widths of 
sporangia of Peronospora parasitica and then plotting these data 
as population curves, he was able to separate the species into a 
number of distinct groups. 



268 PHYSIOLOGIC SPECIALIZATION AND VARIATION 

Savulescu and Ravss (1930) found minor differences in the 
sporangia of Albugo Candida and used them as a basis to divide it 
into 8 form species. Togashi and Shibasaki (1934) were able, by- 
means of a large series of measurements of sporangia, to divide this 
species into 2 varieties, microspora and macrospora, and then to 
separate microspora into 3 form species and macrospora into 2 
form species. 

Leonian (1925) isolated from Phytophthora parasitica rhei 5 
tvpes of colonies that were so different no one would regard them 
as members of the same species. For a long time mycologists have 
placed great emphasis upon the host species as an aid in identifying 
rusts, smuts, downy mildews, powdery mildews, and other obli- 
gate parasites. Undoubtedly some so-called species are in reality 
only form species. The converse may, of course, be found to be 
equally true, with changes in concepts of what constitutes the 
species. 

Physico-chemical differences among specialized races. In 
the light of statements already made, it would seem possible to 
isolate races that possessed more marked ability than other isolates 
to produce a given by-product as the result of their metabolic 
activity. This is true in the case of the groups, baker's yeasts 
and brewer's yeasts, that have been selected from the complex 
known as Sac char omyces cerevisiae. Growth of the baker's 
yeasts is inhibited in wort in which the alcohol content has ac- 
cumulated to a concentration of 4 to 5 0/ , and of the brewer's 
yeast, at an alcohol concentration of 14 to 17%. Similarly races 
of molds, especially of Penicillium, Rhizopus, and Aspergillus, 
differing in fermentative ability in the formation of oxalic acid, 
acetic acid, lactic acid, and other products, have been isolated. It 
would seem that these races are merely selections within the 
species. This interpretation has direct bearing on the "species 
concept." The degree of difference requisite in separating species 
and varieties, and sometimes genera, of funsri is not fixed. Alor- 
phology is agreed to be the primary basis of specific distinctions. 
In some genera, such as Botrytis, Fusarium, and Phytophthora, 
morphologic differences are either minute or non-existent and 
hence a source of confusion. Physiologic differences amongr 
them are, therefore, employed as a convenient basis for specific 
taxonomic units. If physiologic differences were employed 
among rusts, precise means of cleaving species exist that are more 



INFLUENCE OF ENVIRONMENTAL FACTORS 269 

distinctive than morphologic differences between accepted spe- 
cies in certain other genera. Similarly constant physico-chemical 
differences amonsj funoi can be demonstrated to exist. Their 
taxonomic value, like that of other bases, however, remains a 
matter of dispute. 

Influence of environmental factors on physiologic species. 
The pathogenic potentialities of fungi are modified by environ- 
mental factors, as has been demonstrated to the satisfaction of 
everyone who has worked with plant pathogens. There is evi- 
dence also that the specialized races that together constitute a 
species respond differently to a single factor. For example, 
Waterhouse (1929) has shown that certain physiologic forms of 
Fiiccinia graminis tritici, P. gramims avenue, P. triticina, and P. 
simplex are pathogenic to a particular host variety in summer but 
not in winter. Waterhouse reported that P. simplex was capable 
of infecting 14 varieties of barley equally potently in winter and 
in summer, but 8 varieties were resistant under winter weather 
conditions and susceptible in summer. A similar response to 
weather was noted by Peturson (1930) in P. coronata avenae. At 
57° F Red Rust-proof oats were resistant, but at 70° F or higher 
this variety was susceptible. Ruakura oats were resistant within 
the range 57° to 77° F, whereas the varieties Green Mountain, 
White Tartar, and Green Russian were susceptible within this 
range. Susceptibility to form species 21 of P. gramims tritici was 
dominant at high temperature in the cross between Marquillo and 
Marquis wheat, but at low temperature resistance was dominant 
[Harrington (1931)]. Presumably these effects of temperature, 
representative of similar observations on other pathogenic fungi, 
involve the metabolic activities of both interacting organisms and 
are to be regarded as quantitative rather than qualitative. 

It is well known that certain diseases involve only mature plants 
or plant parts, whereas others are limited to seedlings or to young 
tissues. Fomes pini, for example, causes disease of mature conifers 
and becomes a very important cause of decay in overmature stands. 
Again, the leaves and fruits of grapefruit and orange are subject 
to melanose, caused by Diaporthe citri, and to scab, caused by 
Sphaceloma faivcetti, in the period of 4 to 6 weeks after the petals 
have fallen but become highly resistant thereafter. The funda- 
mental causes of differences between young and old tissues in 
susceptibility to infection by fungi are little understood. Further- 



270 PHYSIOLOGIC SPECIALIZATION AND VARIATION 

more, that such differences may be correlated with the existence 
of physiologic races of the particular species is shown by the 
experiments of Goulden, Newton, and Brown (1930). Among 
the 16 form species of Puccinia graminis tritici that they used 
some were more pathogenic on wheat in the seedling stage than on 
the same variety in the mature condition. 

Environmental factors and variation among saprophytes. 
Variations among saprophytic fungi, in relation to their produc- 
tion by such factors as temperature, chemicals, kind and amount 
of food, and effects of radiations, have also been given due con- 
sideration. An appreciation of the influence of environmental 
factors is shown in the report by Barnes (1936). In it he states 
that Hansen, in his work with yeasts in 1883, was the first to in- 
duce variation in a fungus. He secured an anascosporous yeast 
by use of high temperature. In Barnes' own studies, involving 
Eurotium herbariorum, Botrytis cinerea, and Thamnidium elegans, 
he secured variants by exposure to temperatures just insufficient 
to kill. These variations were manifest by reduced fertility or 
less vigorous vegetative development. Barnes judiciously indicates 
the need for distinguishing between modifications that are tempo- 
rary in nature and variants characterized by permanency. Both 
modificatory types appeared in his own experiments and in those 
of certain others. Barnes' (1936) discussion involves the possibil- 
ity that wounding which results from breaking the hyphae while 
making transfer from one medium to another may induce varia- 
tion. He would not attribute all variation to nuclear changes, 
since physiological processes might conceivably be deranged 
without nuclear derangement. Barnes concludes by saying, 
"Variants are damaged versions of the normal stocks . . . and the 
evolutionary process may depend in part on the running down 
of the biological machine." 

Evidence presented by Barnes (1928, 1930) shows that variation 
can be induced in Eurotium herbariorum and Botrytis cinerea by 
subjecting the spores to high temperatures. In E. herbariorum 
these variations are manifest by differences in amount of aerial 
mycelium, density of growth, color of conidia, and abundance 
of perithecial formation; in B. cinerea, by the change in color 
and density of the mycelium and by abnormalities in abundance 
of conidia and sclerotia. Certain variants that arose by high-tem- 
perature treatment seemed capable of retaining these characteristic 



PHYSIOLOGIC SPECIALIZATION AND VARIATION 211 

differences even after repeated transfer, whereas others reverted 
to the normal. 

Importance of physiologic specialization and variation. 
Thus far emphasis has been placed upon the fact that specialized 
races exist, and their possible origin has been considered. The 
significance and practical application of these facts and theories 
cannot have been kept from mind during the perusal of this dis- 
cussion. Their importance in the field of plant pathologv is not 
believed to be properly appreciated; indeed, it can scarcely be 
overestimated. For a period of years these problems have engaged 
the attention of many students of the rusts and smuts, especially 
Stakman and his associates. In a report Stakman (1936) has sum- 
marized them in their application to the need for plant quaran- 
tines and to the breeding of varieties resistant to disease. 

Investigations at the Minnesota Agricultural Experiment Sta- 
tion, at, the Dominion Rust Research Laboratory, and in Australia 
[Waterhouse (1929)] are in accord in showing the relationship 
between the presence of barberry and the existence of numerous 
races of Puccinia graminis tritici. Race 34 seems to be the only 
one present in quantity in Australia, whereas in the wheat-grow- 
ing belt of North America approximately 150 races are known 
to exist. In addition, new races are continuously being developed 
as the result of hybridization on the barberry. The unrestricted 
introduction of the North American races into Australia or other 
continents might easily result in epidemics of rust on varieties of 
wheat that are highly resistant to races of rusts already present in 
these countries. This supposition is supported by Stakman's 
(1936) observations on the rust epidemic on the varieties Ceres 
and Thatcher in 1935. Both had previously rather uniformly re- 
sisted rust for a term of years. It should be added that in 1935 
no races of rust capable of infecting Vernal emmer were isolated 
from uredinial collections made in reoions where barberries are 
absent, whereas three capable of infecting this variety of emmer 
were isolated from barberries or from grain growing near them. 
Several of the races isolated that year from barberries were new 
biotypes, showing that hybridization and segregation in rusts are 
taking place in nature. Experiences of this sort should convince 
the most hard-headed unbeliever that barberries should be 
eradicated. 



272 PHYSIOLOGIC SPECIALIZATION AND VARIATION 

The recent work of Reed (1940) serves to emphasize the im- 
portance of physiologic races in the breeding of oats resistant to 
smuts. He differentiated 29 races of Ustilago avenae and 14 of 
U. levis by their pathogenic behavior on strains and varieties of 
9 species of Avena. Avena bar bat a was susceptible to all races of 
smuts. The variety Canadian was susceptible to all physiologic 
races of these smuts except one of each. The varieties Markton, 
Victoria, and Navarro proved to be highly resistant to many races 
of both loose and covered smuts. 

Literature on plant pathology contains many accounts of varie- 
ties of crop plants that are resistant to a specific pathogen when 
grown in one region but are susceptible when grown in another 
region. Of course this apparent breakdown of resistance cannot 
be attributed to one cause in every case, but the existence of dif- 
ferent specialized races in different regions is no doubt frequently 
the primary cause. For example, it is a common observation that 
durum wheats in the United States are more resistant to stinking 
smut than are vulgare wheats. The opposite situation has been 
observed in Palestine. Abundant evidence is now at hand that 
these conflicting observations can be explained by the existence 
of different physiologic races of Tilletia tvitici and T. levis in these 
two regions. It is to be expected that hybridization is less im- 
portant in breeding crops resistant to smuts if the smuts belong 
to the group in which the promycelium or its branches directly 
penetrate the host tissues. Even in these species fusions between 
different promvcelia could occur, and new races could be formed. 

From the numerous examples of interracial and interspecific 
hybridization and of variation by sectoring that have been oh- 
served, it is apparent, as has been indicated, that new forms are 
continuously being produced in nature. The plant pathologist 
must therefore first know the pathogen thoroughly, if the breed- 
ing or selecting ot resistant host varieties is to be successful. 

LITERATURE CITED 

A \.\iodt, O. S., "Varietal trials, physiologic specialization, and breeding 
spring wheats for resistance to Tilletia tvitici and T. levis" Can. J. Re- 
search, 5: 501-528, 1931. 

Allison, C. C, and K. [senbeck, "Biologisches Spccialisicrung von Puccinia 
glumarum tritici Eriksson and Henning," Phytopath. Z., 2:87-98, 1930. 



LITERATURE CITED 213 

Bailey, D. L., "Physiologic specialization in Puccinia graminis avenae Erikss. 
and Henn.," Minn. Agr. Expt. Sta. Tech. Bull., 35: 36 pp. 1925. 

Barnes, B., "Variations in Eurotium herbariorum (Wigg) Link, induced by 
the action of high temperatures," Ann. Botany, 42:783-812, 1928. 
"Variations in Botrytis cinerea Pers., induced by the action of high tem- 
peratures," Ann. Botany, 44: 825-858, 1930. 
"Induced variation," Trans. Brit. Mycol. Soc, 20: 17-32, 1936. 

Barrus, M. F., "Varietal susceptibility of beans to strains of Colletotrichum 
lindemuthianum (Sacc. et Magn.) B. and C," Phytopathology, 8: 589- 
614, 1918. 

Beach, W. S., "Biologic specialization in the genus Septoria," Am. J. Botany, 
6: 1-33, 1919. 

Billroth, T., Untersuchungen iiber die V egetationsformen von Cocco-bak- 
teria septic a. Berlin. 1874. 

Blumer, B., "Beitrage zur Specialisation der Erysiphe horriduhx Lev. auf 
Boraginaceen," Zentr. Bakt., Parasitenk., II Abt., 55:480-506, 1922. 

Bonar, L., "An albino mutation of the dematiaceous fungus Brachysporium 
trifolii," Science, 56:226-221, 1922. 

Bressman, E. N., "Varietal resistance, phvsiologic specialization, and inheri- 
tance studies in bunt of wheat," Ore. Agr. Expt. Sta. Bull., 281: 1-44, 
1931. 

Brierley, W. B., "On a form of Botrytis cinerea with colorless sclerotia," 
Phil. Trans. Roy. Soc. London, Ser. B, 220: 83-114, 1920. 
"Variation in fungi and bacteria," Proc. Intern. Congr. Plant Sci. Ithaca, 

2: 1629-1654, 1929. 
"Biological races in fungi and their significance in evolution," Ann. Appl. 
Biol, 18: 420-434, 1931. 

Briton-Jones, H. R., "Strains of Rhizoctonia solani (Corticium vaginn Berk 
and Curt.)," Trans. Brit. Mycol. Soc, 9: 200-210, 1924. 

Budde, A., "Uber Rasenbildung parasitischer Pilze unter besonderer Beriick- 
sichtigung von Colletotrichum lindemuthianum (Sacc. et Magn.) Bri. 
et Cav. in Deutschland," Forsch. Gebiete Pflanzenkr. Immunitat im 
Pflanzenreich, 5: 115-147, 1928. 

Burkholder, W. H., "The gamma strain of Colletotrichum lindemuthianum 
(Sacc. and Magn.) B. and C," Phytopathology, 13: 316-323, 1923. 

Christiansen, C, "Cultural races and the production of variants in Pesta- 
lozzia funerea," Bull. Torrey Botan. Club, 59: 525-544, 1932. 

Christiansen, J. J., "Studies on the parasitism of H ' elminthosporium sati- 
vum," Minn. Agr. Expt. Sta. Tech: Bull., 11: 3—42, 1922. 
"Mutation and hvbridization in Ustilago zeae. Part II. Hvbridization," 

Minn. Agr. Expt. Sta. Tech. Bull., 65: 85-108, 1929. 
"Studies on the genetics of Ustilago zeae," Phytopath. Z., 4: 129-188, 1931. 
"The origin of parasitic races of phvtopathogenic fungi through muta- 
tion," Genetics of Pathogenic Organisms, Am. Assoc. Adv. Sci., 77-82, 
1940. 

Christiansen, J. J., and T. W. Graham, "Phvsiologic specialization and vari- 
ation in H elminthosporhnn graminenm Rab.," Minn. Agr. Expt. Sta. 
Tech. Bull., 95: 1-10, 1934. 



214 PHYSIOLOGIC SPECIALIZATION AND VARIATION 

Christiansen, J. J., and H. A. Rodenheiser, "Physiologic specialization and 

genetics of the smut fungi," Botan. Rev., 6: 389-425, 1940. 
Christiansen, J. J., and E. C. Stakman, "Physiologic specialization and mu- 
tation in Ustilago zeae" Phytopathology, 16: 979-999, 1926. 
Ciferri, R., "Osservasioni sulla specializzazione dell Albugo ipomoeae-pan- 

duranae (Schw.) S\v., Nuov. giorn. ltd., 35: 112-134, 1928. 
Cotter, R. U., and M. N. Levine, "Physiologic specialization in Puccinia 

graminis secalis," J. Agr. Research, 45:297-315, 1932. 
Craic;ie, J. H., "The origin of physiologic races of rust fungi through hy- 
bridization," Genetics of Pathogenic organisms, Am. Assoc. Adv. Sci., 

66-72, 1940. 
Diedicke, H., "Uber den Zusammengehang zwischen Pleospora und Helmin- 

thosporium-artcn," Zentr. Bakt., Parasitenk., II Abt., 9: 317-329, 1902. 
Dimock, A. W., "Studies of ascospore variants of Hypomyces ipomoeae," 

My col., 57:709-727, 1939. 
Dodge, B. O., "Inheritance of the albinistic non-conidial character in inter- 
specific hybrids in Neurospora," My col., 23: 1-50, 1931. 
"Heterocarvotic vigor in Neurospora," Bull. Torrey Botan. Club, 69: 75- 

91, 1942. ' 
Eriksson, J., "Uber die Specialisierung des Parasitismus bei den Getreiderost- 

pilzen," Ber. dent, botan. Ges., 12: 292-331, 1894. 
Faris, J. A., "Physiologic specialization of Ustilago hordei" Phytopathology , 

1 4: 537-557, 1 924. 
"Factors influencing infection of Hordeum sativum by Ustilago hordei" 

Am. J. Botany, 11: 189-214, 1924a. 
Flor, H. H., "Heterothallism and hybridization in Tilletia tritici and T. 

levis" J. Agr. Research, 44: 49-58, 1932. 
"Studies on physiologic specialization in Tilletia tritici and T. levis in the 

Pacific Northwest," /. Agr. Research, 41: 193-213, 1933. 
Frenzel, H., "Beitrage zur Spezialisierung des Haferkronenrostes Puccinia 

coroiiifera, f. sp., avenae Kleb," Arb. biol. Reichs. Land-u. Forstw. Berlin- 

Dahlem, 18: 153-176, 1930. 
Gaines, E. F., "New physiological forms of Tilletia levis and T. tritici,'"' 

Phytopathology, 18: 579-588, 1928. 
Gaumann, E., "Beitrage zu einer Monographic der Gattung Peronospora 

Corda," Beitr. Kryptogainenflora, 5: 1-360, 1923. 
Gilbert, E. AI., "Biologic forms of black knot," Phytopathology, 5:246-247, 

1913. 
Goldschmidt, V., "Vererbungsversuche mit den biologischcn Arten der 

Antherenbrandes (Ustilago violacea Pers.)," Z. Botan., 21: 1-90, 192s. 
Gordon, \V. L., "Effect of temperature on host reaction to physiologic forms 

of Puccinia graminis avenae Erikss. and Henn.," Sci. Agr., 11: 95-103, 

1930. 
' \ suulv of the relation of environment to the development of the 

uredinial and telial stages of the physiologic forms of Puccinia graminis 

avenae Erikss. and Henn.," Sci. Agr.. 14: 184-237, 1933. 
Goulden, C. H., M. Newton, and A. AI. Brown, "The reaction of wheat 



LITERATURE CITED 215 

varieties at two stages of maturity to sixteen physiologic forms of 
Puccinia graminis tritici" Sci. Agr., 11:9-25, 1930. 

Grevel, F. K m "Untersuchungen iiber das Vorhandensein biologischer Ras- 
sen des Flugbrandes des Weizens (Ustilago tritici) ," Phytopath. Z., 
2:209-234, 1930. 

Hammarlund, C, "Zur Genetik, Biologie, und Physiologie einiger Erysiph- 
aceen, Hereditas, 6: 1-126, 1925. 

Hansen, H. N., and R. E. Smith, "The mechanism of variation in imperfect 
fungi: Botrytis cinerea" Phytopathology, 22:953-964, 1932. 

Harrington, J. B., "The effect of temperature on the expression of factors 
governing rust reaction in a cross between two varieties of Triticum 
vulgare," Can. J. Research, 5:200-207, 1931. 

Harter, L. L., and J. L. Weimer, "Some physiological variations in strains 
of Rhizopus nigricans" J. Agr. Research, 25:363-371, 1923. 

Hey, A., "Beitrage zur Spezialisierung des Gerstenzwergrostes, Puccinia 
simplex Eriksson et Henning," Arb. biol. Reichs. Land-u. Forstiv. Ber- 
lin-Dahlem, 19:227-261, 1931. 

Hoerner, G. R., "Biologic forms of Puccinia corojiata on oats," Phyto- 
pathology, .9:309-314, 1919. 

Holton, C. S., "The relation of physiologic specialization in Tilletia to re- 
cent epiphvtotics of bunt in Durum and Marquis wheats," Phyto- 
pathology, 21: 687-694, 1931. 

Johnson, C. O., "An aberrant physiologic form of Puccinia triticina Erikss.," 
Phytopathology, 20: 609-620,' 1930. 

Johnson, C. O., and E. B. Mains, "Studies on physiologic specialization in 
Puccinia triticina;' U. S. Dept. Agr. Tech. Bull., 313: 1-23, 1932. 

Johnson, E. M., and W. D Valleau, "Cultural variations of Thielaviopsis 
basicola," Phytopathology, 25:1011-1018, 1935. 

LaRue, C. D., "The results of selection within pure lines of Pestalozzia 
guepini" Genetics, 7: 142-183, 1922. 

LaRue, C. D., and H. H. Bartlett, "A demonstration of numerous distinct 
strains within the nominal species Pestalozzia guepini Desm.," Am. J. 
Botany, 9: 79-92, 1922. 

Leach, J. G., "The parasitism of Colletotrichum lindemuthiammt" Minn. 
Agr. Expt. Sta. Tech. Bull., 14: 39 pp. 1922. 

Leontan, L. H., "Physiological studies on the genus Phytophthora," Am. J. 
Botany, 12: 444-498, 1925. 
"The morphology and pathogenicity of some Phytophthora mutations," 

Phytopathology, 16:723-731, 1926. " 
"Studies on the variability and dissociation in the genus Fusarium," 

Phytopathology, 19: 753-868, 1929. 
"Attempts to induce 'mixochimaera' in Fusarium monilifonne;'' Phyto- 
pathology, 20:895-901, 1930. 

Levine, M. N., "Biometrical studies on the variation of physiologic forms 
of Puccinia graminis tritici and the effects of ecological factors on the 
susceptibility of wheat varieties," Phytopathology, 18: 7-126, 1928. 

Mains, E. B., "Host specialization of barley rust, Puccinia anomala" Phyto- 
pathology, 20: 875-882, 1930. 



216 PHYSIOLOGIC SPECIALIZATION AND VARIATION 

.Mains, E. B., "Host specialization of Erysiphe graviinis tritici" Proc. Nat. 
Acad. Sci., 1 9: 49-53, 1933. 
"Host specialization in the leaf rust of grasses, Puccinia rubigo-vera" 
Mich. Acad. Sci. Papers, 77:289-394, 1933a. 
.Mains, E. B., and S. .M. Dietz, '"Physiologic forms of barley mildew, Ery- 
siphe graminis hordei Marchal," Phytopathology, 20:229-239, 1930. 
.Mains, E. B., and H. S. Jackson, "Physiologic specialization in the leaf rust 
of wheat, Puccinia graviinis Erikss.," Phytopathology, 16: 89-120, 1926. 
Marchal, E., "De la specialization du parasitisme chez V Erysiphe graminis 

DC," Comp. rend., 755:210-212, 1902; 136: 1280-1281, 1903. 
Matsumoto, T., "Studies in the physiology of fungi. XII. Physiological 
specialization in Rhizoctonia solani Kiihn," Ann. Mo. Botan. Garden, 
8: 1-62, 1921. 
.Melchers, L. E., "Investigations on physiologic specialization of Tilletia 

laevis in Kansas," Phytopathology, 24: 1203-1226, 1934. 
.Melchers, L. E., C. H. Fricke, and C. O. Johnston, "A study of physiologic 
forms of kernel smut (Sphacelotheca sorghi) of sorghum," /. Agr. Re- 
search, 44: 1-11, 1932. 
Newton, .M., and T. Johnson, "Color mutations in Puccinia graviinis tritici 
(Pers.) Erikss. and Henn.," Phytopathology, 77:711-725, 1927. 
"Specialization and hybridization of wheat-stem rust, Puccinia graviinis 
tritici, in Canada," Dom. Canada Dept. Agr. Bull., 160: 1-60, 1932. 
Newton, .M., T. Johnson, and A. .M. Brown, "A preliminary study of the 
hybridization of physiologic forms of Puccinia graviinis tritici" Sci. 
Agr., 70:721-731, 1930. 
Penser, H., "Fortgesetzte Untersuchungen iiber das Vorkommen biolo- 
gischer Rassen von Colletrotrichuvi lindevmthianuvi (Sacc. et .Magn.) 
Bri. et Cav.," Phytopath. Z., 4: 83-112, 1931. 
Peturson, B., "Effect of temperature on host reactions to physiologic forms 

of Puccinia corovata avenae," Sci. Agr., 11: 104-110, 1930. 
Pfister, R., "Zur Biologie von Cystopus tragopogonis Pers," Zentr. Bakt., 

Parasitenk., II Abt., 77: 312-313, 1927. 
Radulescu, E., "Zur phvsiologischen Spezialisierung des Weizenbraunrostes 

{Puccinia triticina Erikss.)," Kiihn- Arch., 33: 195-205, 1932. 
Reed, G. M., "Physiological specialization of parasitic fungi," Brooklyn 
Botan. Garden Mevi~., 7:348-409, 1918. 
"Physiologic races of oat smuts," Am. J. Botany, 77:483-492, 1924. 
"Further evidence of physiologic races of oat smuts," My col., 79:21-28, 

1927. 
"Physiologic races of bunt of wheat," Am. J. Botany, 15: 157-170, 1928. 
"New physiologic races of oat smuts," Bull. Torrey Botan. Club, 56: 449- 

470, 1929. 
"Physiologic specialization of the parasitic fungi," Botan. Rev., 1: 119-137, 

1935. 
"Physiologic races of oat smuts," Am. J. Botany, 21: 135-143, 1940. 
Reed, G. .M., and T. R. Stanton, "Physiologic races of Ustilago levis and 
U. avenae on red oats," /. Agr. Research, 44: 147-153, 1932. 



LITERATURE CITED 211 

Reed, G. M., Marjorie Swabey, and Laura A. Kolk, "Experimental studies 

of head smut of corn and sorghum," Bull. Torrey Botan. Club, 54: 295- 

310, 1927. 
Rodenheiser, H. A., "Physiologic specialization in some cereal smuts," 

Phytopathology, i#: 955-1003, 1928. 
"The origin of physiologic races in the smut fungi by hybridization," 

Genetics of Pathogenic Organisms, Am. Assoc. Adv. Sci., 73-76, 1940. 
Rodenheiser, H. A., and E. C. Stakman, "Physiologic specialization in 

Tilletia levis and Tilletia tritici," Phytopathology, 11: 247-253, 1927. 
Salmon, E. S., "On Erysiphe graminis DC, and its adaptive parasitism within 

the genus Bromus," Ann. My col., 2:255-267, 307-343, 1904. 
"Cultural experiments with biologic forms of the Erysiphaceae," Phil. 

Trans. Roy. Soc. Lo?idon, Ser. B., 191: 107-122, 1904a. 
"Recent researches on the specialization of parasitism in the Erysiphaceae," 

New Phytopathologist, 3: 55-60, 1904. 
Savulescu, T., and T. Rayss, "Contribution a la connaissance des Pero- 

nosporacees de Roumaine," Ann. Mycol., 28: 297-320, 1930. 
Schreiber, F., "Resistenzziichtung bei Phaseolus vidgaris," Phytopath. Z., 

4:415-454, 1932. 
Schroeter, J., "Entwickelung einiger Rostpilze," Beitr. Biol. Pflanzen, 3: 69- 

70, 1879. 
Stager, R., "Infectionsversuche mit Gramineen-bewohnenden Claviceps- 

arten," Botan. Z., 61: 111-158, 1903. 
Stakman, E. C, "Physiologic specialization in pathogenic fungi," Proc. 

Intern. Congr. Plant Sci. Ithaca, 2: 1312-1330, 1926. 
"The problem of specialization and variation in phytopathogenic fungi," 

Genetica, 18: 372-389, 1936. 
Stakman, E. C, J. J. Christiansen, and H. E. Brewbaker, "Physiologic 

specialization in Puccinia sorghi," Phytopathology, 18: 345-354, 1928. 
Stakman, E. C, J. J. Christiansen, C. J. Eide, and B. Peturson, "Mutation 

and hybridization in Ustilago zeae," Minn. Agr. Expt. Sta. Tech. Bull., 

65: 108 pp. 1929. 
Stakman, E. C, and M. N. Levine, "The determination of biologic forms 

of Puccinia graminis on Triticum spp.," Minn. Agr. Expt. Sta. Tech. 

Bull., 8: 1-10, 1922. 
Stakman, E. C, M. N. Levine, and D. L. Bailey, "Biologic forms of 

Puccinia graminis on varieties of Avena spp.," /. Agr. Research, 24: 1013- 

1018, 1923. 
Stakman, E. C, M. N. Levine, and R. U. Cotter, "Origin of physiologic 

forms of Puccinia graminis through hybridization and mutation," Set. 

Agr., 10: 707-720, 1930. 
Stakman, E. C, M. N. Levine, R. U. Cotter, and L. Hines, "Relation of 

barberry to the origin and persistence of physiologic forms of Puccinia 

graminis? J. Agr. Research, 48:953-969, 1934. 
Stakman, E. C, M. N. Levine, and L. Hines, "Relation of barberry to the 

origin and persistence of physiologic forms of Puccinia graminis? J. 

Agr. Research, 48:951-969, 1934. 



218 PHYSIOLOGIC SPECIALIZATION AND VARIATION 

Stakman, E. C, and F. J. Piemeisel, "A new strain of Pucc'inia graminis" 
Phytopathology, 7: 73, 1917. 

Stakman, E. C, L. J. Tyler, and G. E. Hafstad, "The constancy of cul- 
tural characters and pathogenicity in variant lines of Ustilago zeae" 
Bull. Torrey Botan. Club, 60: 565-572, 1933. 

Steiner, J. A., "Die Specialization der Alchenillen-bewohnenden Sphaero- 
theca humuli (DC) Burrill," Zentr. Bakt., Parasitenk., II Abt., 21: 677- 

736, 1908. 
Tisdale, W. H., L. E. Melchers, and H. J. Clemmer, "Strains of kernel 

smuts of sorghum, Sphacelotheca sorghi and S. cruenta," J. Agr. Re- 
search, 34: 825-838, 1927. 
Togashi, K., and Y. Shibasaki, "Biometrical and biological studies of Albugo 

Candida (Pers.) O. Kuntze, in connection with its specialization," Imp. 

Coll. Agr. Forestry, Morioka, Bull, IS: 1-88, 1934. 
Tyler, L. J., and C. P. Shumway, "Hybridization between Sphacelotheca 

cruenta and Sorosporiwn reiliammi" Phytopathology, 25: 375-376, 1935. 
Ward, H. .Marshall, "Further observations on the brown rust of bromes, 

Puccinia dispersa (Erikss.), and its adaptive parasitism," Ann. My col., 

1: 132-151, 1903. 
Waterhouse, W. L., "A preliminary account of the origin of two new 

Australian phvsiologic forms of Puccinia graminis tritici," Proc. Lin- 

nean Soc. N.S. Wales, 54:96-106, 1929. 
"Australian rust studies. I," Proc. Linnean Soc. N.S. Wales, 54: 615-680, 

1929a. 
Wilhelm, P., "Studien zur Spezialisierung-weise des Weizengelbrostes, 

Puccinia glumarum, f. sp., tritici (Schmidt) Eriksson et Henning und 

zur Keimungsphvsiologie seiner Uredosporen," Arb. biol. Reichs. 

Land-u. Forstw. Berlin-Dahlem, 19: 95-133, 1931. 
Wolf, F. T., and F. A. Wolf, "A study of Botryosphaeria ribis on willow," 

My col., 37:217-227, 1939. 
Zillig, H., "Uber spczializierte Formen beim Antherbrand Ustilago vio- 

lacea (Pers.) Fckl.," Zentr. Bakt., Parasitenk., II Abt., 53:33-74, 1921. 






Chapter 12 
ASSOCIATIVE EFFECTS AMONG FUNGI 

In this chapter it is proposed to consider those phenomena 
manifest as a result of different species of fungi living together 
in close proximity. It is essentially an ecological study of fungi 
and corresponds in some measure to a consideration of associations 
among seed plants. Of course, a great deal has been learned re- 
garding the influence of one species of seed plant upon another 
growing in close juxtaposition. Our knowledge of similar asso- 
ciative relationships among fungi is strikingly much more meager 
and fragmentary, and the data are rather widely dispersed in the 
literature. Such facts regarding fungi appear none the less im- 
portant, however, and they may be found to possess interesting 
applications and economic potentialities. 

For convenience, the effects of interaction of fungi, one upon 
the other, may be divided into the following categories: antibiotic, 
symbiotic, and synergetic. The associative relationships have 
been designated antagonism, symbiosis, and synergism, respec- 
tively. In antibiotic effects are included those antagonistic, com- 
petitive, or harmful effects that result to organisms from their 
growth in close proximity. Effects resulting from parasitism are 
among those included in this classification. In symbiotic effects 
are included mutualistic advantages that result from the living to- 
gether of two or more species. In synergetic effects are included 
those in which two or more species through their combined action 
produce effects or changes that neither could produce alone. 

It becomes apparent immediately that these categories are arbi- 
trary, and that evidence might be found to show that they inter- 
grade. Indeed, such evidence is at hand. Among the factors 
studied that have to do with inter£radation and with associative 
effects generally are competition for food, modification of food 
supply by the metabolism of one or the other of the associated 
species, relative availability of food constituents, changes in re- 

219 



280 ASSOCIATIVE EFFECTS AMONG FUNGI 

action of the medium, production of inhibitory toxic products, 
production of stimulatory or growth-controlling substances, such 
as vitamins, auxins and hormones, and variation in temperature 
and in 2 tension. These matters, as they apply to associative 
effects, have been capablv reviewed bv Waksman (1937), Porter 
and Carter (1938), Weindling (1938), D'Aeth (1939), and 
Waksman (1941), each of whose summaries should be carefully 
perused. 

ANTAGONISM 

In our social organization the human race may be spoken of as 
constituted of two groups, producers and consumers, and if this 
analogy is applied to fungi, all of them, by virtue of their lack of 
chlorophyll, are perforce in the consumer grouping. They not 
only are largely dependent upon other organisms, living or dead, 
as sources of food, but also the "struggle for existence" is just as 
acute among them, with resultant "survival of the fittest," as it is 
among a nv other tvpe of organism. This associative interaction 
exists not only among the fungi themselves but between bacteria 
and fungi, slime molds and fungi, actinomycetes and fungi, proto- 
zoa and fungi, and also various other organisms and fungi. In fact, 
it is doubtful if anv chlorophyll-bearing species of plant is free 
from attack bv fungi, and, moreover, records of hyperparasitism 
among fungi are not infrequent. 

Evidence of antagonism from cultures. One of the essential 
techniques in the study of fungi is their isolation in pure culture. 
These procedures are based upon the use of semisolid media, first 
utilized by Koch to isolate bacteria in pure culture. All mycolo- 
gists have come to place enormous importance upon the use of 
pure cultures, although they know full well that in nature pure 
cultures are either non-existent or else occur as miraculous oddi- 
ties. In consequence of insistence upon use of pure cultures, too 
little attention has been given to studies of known mixtures of 
fungi [Fawcett (1931)]. 

It has long been known that microorganisms in culture produce 
substances that limit their own period of growth. As evidence 
the production of alcohol by yeasts, of citric acid by Aspergillus 
niger, and of lactic acid by Rhizopus sp. may be cited. These 
growth-inhibiting substances have been regarded as aids in the 
struggle for existence of microorganisms. 



ANTAGONISM 281 

All who have studied microorganisms on artificial media have 
noted evidence of this antagonism between the colonies of differ- 
ent species. An explanation for this phenomenon was first sought 
by Raulin in 1869 [D'Aeth (1939)1 in experiments involving the 
growth of A. niger on liquid synthetic media. He removed the 
mycelial mat by filtration at intervals of 3 days and determined the 
amount of growth during each successive 3 -day period. Most 
growth occurred in the first period, with less in each period there- 
after. From these results it was concluded that growth-affecting 
substances are excreted by A. niger and that they remain in the 
filtrate. The reciprocal influence of the simultaneous production 
of such substances upon paired organisms in the same culture was 
first studied by Reinhart in 1892 [D'Aeth (1939)]. Since then 
similar studies have been made by, among others, Zeller and 
Schmitz (1919), Porter (1924), Sanford and Broadfoot (1931), 
Endo (1931, 1932, 1932a), Weindling (1932), Broadfoot (1933), 
and Arrillaga (1935). 

Porter (1924) used 80 species of fungi and bacteria grown in 
pairs on corn-meal agar. The fungi employed included Penicil- 
lium glaucum, P. italicum, Rhizopus nigricans, Fnsariiim lini, F. 
culmonem, F. coeruleum, Gloeosporinm piperatum, Colleto- 
trichum nigrum, C. lindemiithianum, and Helminthosporinm 
sativum. He classified their interactions into five groups, four of 
which are antagonistic, showing differences in degree of inhibitory 
action as follows: 

1. One species overgrows and inhibits the other. 

2. Each member of the pair exerts a slight mutual inhibition. 

3. One of the pair grows close to but around the other. 

4. Mutual inhibition is exhibited at a considerable distance, and 
the two remain separate. 

Endo (1931, 1932, 1932a) found that Hypochnns centrifugus, 
H. sasakii, and Sclerotium oryzae-sativae, causing root-rot diseases 
of rice, are indifferent to certain other fungi, and antagonism was 
exhibited in other combinations. 

Broadfoot (1933) studied the interaction of 66 species of micro- 
organisms, many of them bacteria, with special consideration to 
their antagonisms toward Ophiobolns graminis. Among the fungi 
that he found to be antagonistic to O. graminis are Ascochyta 
graminis, Botrytis cinerea, Helminthosporium sativum, Lepto- 



282 ASSOCIATIVE EFFECTS AMONG FUNGI 

sphaeria herpotrichoides, Flenodomus 7/ieliloti y and Wofnoimda 
grandnis. 

Arrillaga (1935) made all possible combinations on potato dex- 
trose agar of 12 species of fungi associated with disease or decay 
of Citrus fruits, with the result that Diaporthe cirri checked the 
growth, especially of Phytophthora parasitica and P. citrophthora. 

Causes of antagonism. It is apparent from the reports of 
these studies that the range of interaction between fungi extends 
from complete indifference of both members of the pair on the 
one extreme to very active inhibition on the other. Since these 
effects are manifest indifferently between members of all classes 
of fungi, it appears improbable that one and the same proximate 
cause is responsible for all. Instead a variety of causes has been 
suggested, and evidence in their support has been submitted. Some 
of these causes are exhaustion of nutrients, modification of their 
balance or concentration, differential in optimal pH, which may 
be the result of metabolic products formed by one of the species, 
differential in optimal temperature, production of excretory prod- 
ucts, which cause staling, production of toxic substances, and 
aversion. Not all need be considered in this discussion, nor need 
evidence in their support be reviewed. 

The term staling is applied to the well-known phenomenon in 
which the growth rate of a fungus on an artificial medium grad- 
ually decreases and eventually ceases. This phenomenon is not 
the result of an exhaustion of nutriment but of the presence of a 
progressive increase in amount of products of metabolism. Niki- 
tinsky ( 1904) grew on liquid media repeated crops of Pemcillhim 
glaucum, P. griseim?, Mucor stolonifer, Aspergillus flavzis, Sac- 
charomyces cerevisiae, and S. rosaceus. At intervals the mycelial 
mat was removed by filtration, dried, and weighed. The medium 
was then sown with the same or a different species, and the mat 
was again removed. This procedure was repeated until the me- 
dium would no longer support growth. He observed that, when 
ammonium chloride was used as the source of nitrogen, the inhibi- 
tion set in quickly, and the medium became increasingly more 
acid. To such media he then added alkali, and the media again 
supported good growth. When he employed ammonium tartrate 
as the source of nitrogen, the medium became stale less quickly, 
the XH S being used as the source of nitrogen and the tartrate radi- 
cal as the source of carbon. When peptone was used, the media 



ANTAGONISM 283 

quickly became alkaline, and good growth could again be pro- 
moted by the addition of acid. 

From similar studies with Aspergillus niger, Botrytis cinerea, 
Cladosporhim herbarum, Fusarium solani, Mucor mncedo, Peni- 
cillium glaucwn, and Rhizopus nigricans Lutz (1909) concluded 
that a variety of materials cause staling. Although he was unable 
to indentify any of them, he determined that some filtrates were 
free from growth-inhibiting substances after passage through a 
porcelain filter, whereas in others the staling products passed read- 
ily through such filters. In some instances, moreover, heating to 
80° C destroyed the inhibitory properties, indicating a relation- 
ship to enzymes. Even after dilution with 20 volumes of water 
the filtrates still greatly inhibited growth. 

Boyle (1924) grew Botrytis cinerea and Fusarium sp., isolated 
from apple, on Richards' solution, potato extract, and apple ex- 
tract. On each medium these organisms caused increased alka- 
linity, which if eliminated in slightly stale media by addition of 
acid, caused growth to be restored. At a later stage of staling, 
however, adjustment of reaction did not correct conditions. He 
concluded from these results that change in reaction is not per se 
the limiting factor but that it accompanies the accumulation of 
other inhibitory metabolic products. Filtration through a col- 
lodion membrane removed part of the inhibitory properties. 
Boiling of the staled medium also resulted in improved growth 
but indicated that both thermolabile and thermostable products 
were present. 

Pratt (1924, 1924a), using a species of Fusarium that rapidly 
staled Richards' solution and Botrytis cinerea, which had little 
staling properties, noted that hydrogen peroxide added to the 
staled medium removes staleness, as does charcoal also, provided 
that the alkalinity is first removed. Her chemical tests of media 
staled by Fusarium indicate that ammonia, alcohol, and salts of 
acetic, propionic, butyric, valeric, and lactic acids are produced. 
Her general conclusion is that alkaline staling is caused by the pro- 
duction of bicarbonates from the carbon dioxide of respiration 
whenever basic radicals are set free. 

From the foregoing accounts it is clear that a variety of inhibi- 
tory staling products are elaborated and that different species of 
fungi may produce different products. Some of them may be 
either simple or complex, some either heat-labile or heat-stable, 



284 ASSOCIATIVE EFFECTS AMONG FUNGI 

some either filterable or non-filterable. There may also exist 
inter^radations between slight inhibition of growth and marked 
toxic action, and in consequence it becomes practically impossible 
to separate staling products from toxic products. Much has 
been written regarding these toxins, since they have been em- 
ployed to explain the proximate cause of wilting by pathogenic, 
vascular-tissue-invading species of Fusarium. The chemical con- 
stitution of many definitely toxic products has been determined. 
Some appreciation of the extent of our knowledge on this matter 
may be gained from the excellent summaries of Raistrick (1932, 
1938). Clutterbuck, Lowell, and Raistrick (1932) isolated one 
such toxic substance, a yellow pigment, chrysogenin, with the 
empirical formula C 8 Ho 2 b (1 . It is formed by one of the Penicil- 
lium chrysogemnn group on a svnthetic medium containing glu- 
cose. Tests showed it to possess very powerful antibacterial prop- 
erties, especially against the pyogenic cocci and the diphtheria 
group, but it was ineffective against the colon-typhoid organisms. 
Weindlinc; and Emerson (1936) isolated a proteinaceous toxin 
with the formula C14H1CN2S2O4 from Gliocladium fimbriatum, 
whereas Dutcher (1941) determined its formula to be Ci 3 Hi 4 - 
4 N 2 S 2 . In concentration of 2.5 mg per milliliter it was bac- 
tericidal to Staphylococcus albus, and of 1.0 mg per milliliter to 
5. aureus and Streptococcus viridans. 

Recently Abraham and associates (1941) isolated penicillin, 
presumably from Penicillium uotatum, finding that it was very 
potent against several species of bacteria pathogenic to man. Peni- 
cillin appears to have therapeutic value when used in place of sul- 
fonamides, as is indicated in Chapter 4. In some cases the toxic 
principles appear, from their extractability by ether or chloroform, 
to be lipoidal in nature. 

A very different type of antagonism, in which the cause is asso- 
ciated with sex, has been encountered among all the principal 
groups of fungi. It has been widely studied in connection with 
the phenomenon of heterothallism, which need not be discussed 
at this time. Suffice it to say that, when the mycelia of mono- 
sporic cultures are grown in the same Petri-dish culture, mutual 
aversion may be manifest by sexual incompatibility. Cayley 
(1923, 1931) has given special consideration to aversion, pri- 
marily as it concerns Diaporthe permc'wsa, the cause of wilt of 
plums in Europe. In cultures of this organism, the isolates may 



ANTAGONISM 285 

exhibit mutual aversion at their line of contact, evidenced by kill- 
ing of the hvphal tips. This property is heritable but is not 
influenced by sex. Hoppe (1936) noted a similar aversion in the 
conidial fungus, Diplodia zeae, pathogenic to maize, and the prop- 
erty remained fixed as shown by repeated inoculation into the 
living host and reisolation. 

Evidence of antagonism from growth in host tissues. Clear- 
cut evidence of antagonism between microorganisms when associ- 
ated within green host plants is lacking or meager. Bamberg 
(1931) found that several species of unidentified bacteria reduced 
the virulence of Ustilago zeae and prevented the formation of smut 
galls when injected into maize coincidentally with smut sporidia 
or even 3 days later. After smut galls % in. in diameter had de- 
veloped, injection of bacteria was followed by disintegration of 
the gall and failure of chlamydospores to form. Johnson (1931) 
found that certain bacteria produced enzymes capable of dissolv- 
ing the cell walls of sporidia of several smuts and that others with 
the same enzymes were unable to attack the sporidia. From these 
results she concluded that the antagonistic principle was not an 
enzyme. 

Savastano and Fawcett (1929) inoculated citrus fruits with 
combinations of various fungi normally associated with decays 
of such fruit. In some combinations the rate of decay was slower 
than that produced by the slower-growing component by itself. 
These investigators conclude that the cause of modification of 
rate of decay is correlated with specific food requirements of the 
respective species and with the competition for these foods that 
must occur. The two common molds, Penicillium italicum and 
P. digitatum, that attack citrus fruits are antagonistic, P. digitatum 
being able to grow with greater rapidity and to surround the area 
decayed by P. italicum. 

Perhaps the best evidence in hand of antagonism between fungi 
is exhibited by the numerous instances of hyperparasitism familiar 
to every mycologist. Among the better known are Cicinnobolus 
cesatii, parasitic on various Ervsiphaceae, Darhica filum on the 
uredinia and telia of rusts, Tuber cidina maxima on the pycnia and 
aecia of various blister rusts, including Cronartium ribicola, Myco- 
gyne pemiciosa on mushrooms, Hypomyces sp. on Russula, Lac- 
tarius, and other Hymenomycetes, and Sclerotinia fructicola on 
hypertrophies induced by Taphrina mirabilis. 



286 ASSOCIATIVE EFFECTS AMONG FUNGI 

Buller (1924) has compiled a list of hyperparasites of more than 
50 species. They include members in the Chytridiaceae, Alucor- 
aceae, Pyrenomycetes, Agaricaeae, Polyporaceae, and Fungi Im- 
perfecti. Little of a fundamental nature is known about the an- 
tagonisms in any of them. 

Evidence of antagonism between fungi in soils. The soil 
constitutes the normal habitat of many species of fungi. Such 
factors as texture, organic content, acidity, moisture, temperature, 
and character of the vegetational cover, are known to influence 
the presence or absence of a particular species and its relative 
abundance. How these interrelated factors influence competi- 
tion between soil fungi remains largely unknown, but undoubt- 
edly the fungus flora is never in equilibrium. The type of ob- 
servations that have been made on these problems is indicated in 
the following discussion. Millard and Taylor (1927) observed 
that potato scab, caused by Actinomyces scabies, was eliminated 
in fields containing large amounts of organic matter resulting from 
green manuring. Under these conditions the development of a 
saprophytic species, A. precox, was favored, and it was able to 
suppress the pathogen. A proximate cause appears from the 
studies by Sanford ( 1926). He noted in cultures that the limiting 
acidity for germination of A. scabies was about pH 5.3 and that 
the optimum reaction was pH 8.5. From these results it may be 
anticipated that acidity from decomposition of green crops that 
have been plowed under would be unfavorable for the scab patho- 
gen but might be favorable for other microorganisms to the ex- 
tent that they would predominate and manifest their antibiosis. 

Fungi causing root rots are known to survive in the soil for 
varying periods in the absence of their host plants. Supposedly 
they live under these conditions as saprophytes. Hence it follows 
that the incorporation of organic matter should increase their 
incidence, but this anticipated result is not invariable. Other soil- 
inhabiting species have been shown to modify prevalence of the 
root-invading pathogens, as the work of Sanford and Broadfoot 
(1931, 1934) on Ophiobolus gr ami iris, Helminthosporiitm sativum, 
and Fusarium culmorum illustrates. Not only were soil-inhabit- 
ing saprophytes able to modify pathogenicity in their pot cul- 
tures but also similar effects were secured by the use of filtrates 
from cultures of the saprophytes. Presumably toxic products 
caused this inhibitory action against the pathogens. Similarly 









STIMULATION BY ASSOCIATIVE INTERACTION 281 

Greaney and Machacek (1935) were able to demonstrate that 
Cephalothecium roseum inhibits Helminthosporiwn sativum. 

Garrett (1936) explains somewhat differently the relative inci- 
dence of Ophiobolus graminis in soils. His observations led him 
to conclude that O. graminis increases in amount only so long as 
there are living host roots, along which it spreads. Its rate of 
spread is hypothesized to be related to the carbon dioxide content 
arising from respiratory processes in the microclimate along the 
root. The presence of alkaline receptors for carbon dioxide in the 
soil stimulates spread of the pathogen. Decline of O. graminis 
occurs in its saprophytic phase at which time the mycelium is be- 
ing decomposed by other soil-inhabiting species. 

Recently Weindling (1932, 1934, 1938) found that Tricho- 
dervia lignorwn and Gliocladium fimbriatum penetrate the hyphae 
of such soil-borne parasites of seed plants as Rhizoctonia solani, 
Sclerothtm rolfsii, and Phytophthora parasitica. Undoubtedly 
antagonisms of this sort are not uncommon in the fungus flora of 
soil, and such relationships are factors in the control of diseases 
of cultivated plants. Evidence in support of this type of antago- 
nistic action by Trichoderma against fungi that cause damping-off 
of cucumber seedlings is derived from the experiments of Allen 
and Haenseler (1935). They applied cultures of Trichoderma 
to the soil with the result that damping-off was apparently 

checked. 

It would appear to be feasible to evaluate the several factors 
previously mentioned that are known to influence the incidence 
of fungi in soil generally. This field of research certainly offers 
many possibilities. As is indicated by the rate at which invasion 
of heat-sterilized soils is accomplished, for example, by Pyronema 
confluens, more attention should be devoted to such problems as 
they relate to culture of plants in cold frames, hotbeds, and 
greenhouses. 

STIMULATION BY ASSOCIATIVE INTERACTION 

Apparently one fungus may be stimulated by the presence of 
another in either of two ways: increased assimilatory or vegetative 
activity or else reproductive activity. The proximate cause of 
these responses need not be the same metabolic product but may 
be different specific entities. Much interest in recent years has 



288 ASSOCIATIVE EFFECTS AMONG FUNGI 

centered around the complex problem of factors regulating 
growth and reproduction in plants and animals. The terms auxins, 
hormones, and vitamins, applied to stimulatory and regulatory 
substances, are commonly used not only by the biologist but also 
by the man in the street. 

Stimulation of vegetative activity. Wildiers (1901) first 
established that Sacchacomyces cerevisiae will not grow in a syn- 
thetic medium consisting of ammonium chloride and sugar unless 
some substance essential for growth is added. This result revived 
an old controversy that existed years before between Pasteur and 
Liebig. Pasteur claimed that yeasts made abundant growth on a 
nutrient medium containing sugar, ammonium salts, and the ashes 

DO 7 

of yeast. Liebig was unable to grow yeast successfully on this 
formula, whereupon Pasteur offered to produce for him "all the 
yeast he could require." Liebig declined the challenge, and in 
consequence Pasteur was considered to have won the scientific 
argument. Wildiers noted that, when he placed a single yeast 
cell or a few cells only in this medium, little or no growth took 
place. If, however, he introduced as many yeast cells as were 
contained in two drops of beer wort from a vat in which yeast 
was being grown, abundant growth resulted. He also induced 
growth by the addition of a few cubic centimeters of boiled yeasts. 
His results were so striking that he assumed some hypothetical sub- 
stance that he called "bios" to be essential for growth. He ex- 
tracted this bios from yeasts by boiling. It was dialyzable from a 
watery extract; it was not present in yeast ashes. Of course, the 
results of Wildiers attracted wide attention and were sharply 
criticized. They were substantiated, however, and with the dis- 
covery of vitamins and the flood of investigation that followed, it 
became apparent that bios and vitamins are similar. In fact, bios 
is now known to be a complex consisting of a number of compo- 
nents identified as vitamin Bi (thiamin), biotin, /-inositol, and 
additional factors [Eastcott (1928)]. 

Conflicting evidence exists regarding the necessity of the addi- 
tion of growth factors to culture media used to grow other fungi. 
Kogl and Fries (1937) have shown that Polystictas adustiis grown 
on a synthetic medium requires the addition of thiamin, and 
Nematospora gossypii requires biotin. Polystictus adustus is 
capable of producing biotin, and N. gossypii thiamin, so that they 
can supply their mutual needs when they are grown in association. 



STIMULATION BY ASSOCIATIVE INTERACTION 289 

These two growth factors appear to be necessary for a large num- 
ber of fungi, as is indicated by rather numerous reports of trials. 

Schopmeyer and Fulmer (1931) indicated that bios is produced 
by Aspergillus niger, A. clavatus, and Trichoderma lignorum, as 
judged by the ability to stimulate the growth of yeast. On the 
other hand, Williams and Honn (1932) have shown distinct stim- 
ulation in growth by the addition of yeast extract to media on 
which Aspergillus niger, Mucor racemosus, Microsporum fulvum, 
Monilia metalondinensis , and M. macedoniensis were grown. 
They called these stimulatory substances "nutrilites." A recent 
summary by Williams (1941) reviews pertinent literature on nu- 
trilites, which have been identified as biotin, inositol, pantothenic 
acid, pyridoxin, and thiamin. 

Leonian and Lilly (1940) have shown that certain thiamin- 
requiring fungi are greatly influenced by specific amino acids and 
by zinc, iron, and other minor elements. 

Extracts from different fungi and from bacteria have been used 
experimentally to stimulate the growth of fungi, but in most cases 
little is known of the nature and properties of the extracted sub- 
stances. Such studies are worth while, but the value of similar 
investigations will be greatly enhanced if, in the future, more 
attention is devoted to analyses to determine the identities of the 
extracted materials. Evidence is given in one study [du Vigneaud 
et al. (1940) ] of the identity of biotin and vitamin H. 

Both growth-stimulating and growth-inhibiting factors would 
be expected to be present in extracts from fungi. Such a situation 
was encountered by Satoh (1931) with Ophiobolus miyabeamis. 
When the liquid on which this fungus had been grown was passed 
through a Chamberland (F) filter, a material stimulatory to 
Aspergillus niger was contained in the filtrate, and one inhibitory 
to the same fungus was retained on the filter. The stimulatory 
component proved to be thermostable and the inhibitory one 
thermolabile. 

Stimulation of reproductive activity. The opinion was 
long ago voiced that some chemical attractant aids in bringing 
together plant sex cells of opposite potentialities. De Bary (1881) 
supposed that this was true of fungi and also that such substances 
were operative in stimulating the production of antheridial and 
oogonial branches among certain Phycomycetes. Ever since the 
discovery of heterothallism the same opinion has been entertained 



290 ASSOCIATIVE EFFECTS AMONG FUNGI 

generally in connection with reproductive activities among hetero- 
thallic fungi. Recently .Moreau and Aloruzi (1931) claimed that 
perithecia of Neurospora are produced if two strains are grown 
in opposite ends of a U-tube, and that this response is the result 
of diffusion of a hypothetical hormone through the medium from 
one arm of the tube to the other. Dodge (1931) attempted to 
repeat their experiments with Neurospora sitophila and N. tetra- 
sperma but did not succeed in obtaining perithecia unless and 
until the hyphae of opposite colonies were in contact. Raper 
(1939, 1939a, 1940) presented evidence that the sexual reactions 
in Achlya bisexualis and A. ambisexualis are controlled by four 
specific substances, two produced by the male mycelia and two by 
the female. Responses are evident when the mycelia are 6 mm 
apart, if mated on agar. Both sex strains are activated when 
grown on opposite sides of a cellophane membrane. Male plants 
form antheridial branches when placed in water in which female 
plants have previously been grown. Furthermore, female plants 
produce oogonial initials when placed in water in which male 
plants have been grown and have formed antheridial branches, 
although there is no such activation in water in which a vegetative 
male has been grown. Of the two hormones produced by the 
female plant, one initiates the formation of antheridial branches, 
and the other, in connection with a thigmotropic response, in- 
duces the delimitation of antheridia. Of the two hormones pro- 
duced by the male plant, one initiates the formation of oogonial 
branches, and the other brings about the delimitation of the 
oogonium. The chemical constitution of none of the hormones 
is yet known. 

The filtrate of old cultures of Aspergillus niger contains a prin- 
ciple that promotes conjugation of Zygosaccharomyces acld'i- 
jaciens [Nickerson and Thimann ( 1943) ]. This principle, on be- 
ing fractionated, appears to consist of an organic acid and a mem- 
ber of the vitamin B complex, neither fraction having much 
activity by itself. Nickerson and Thimann were unable to iden- 
tify these constituents with certainty, but when they imitated the 
principle by a mixture of glutaric acid and riboflavin, conjugation 
was promoted. 

Synergetic reactions. Synergism or svnergetic reaction logi- 
cally appears to be a form of stimulation, the term applying, how- 
ever, only to cooperative phenomena that might not be produced 



STIMULATION BY ASSOCIATIVE INTERACTION 291 

by either of the associated organisms acting alone. Molliard 
(1903) first recorded the influence of one microorganism in stimu- 
lating sporulation by another. He secured apothecia of Ascobolus 
on carrot only when a bacterial contaminant was present. The 
same sort of influence between species of fungi associated in the 
same culture was first described by Heald and Pool ( 1908). They 
secured an abundance of perithecia of Melanospora pampecma 
grown in a mixture with Fasarhim moniliforme, Melanospora 
ciilmorum, or Basispor'mm gallannn. Similar reactions occurred 
if M. pampeana was planted on the media after these fungi had 
grown on them and they had been sterilized. 

McCormick (1925) secured perithecia from monoconidial cul- 
tures of Thielavia basicola, grown mixed with Cladosporhim ful- 
vum, Aspergillus umbrosus, A. glaucus, Eurotium amstelodami, or 
Fusicladmm pirinum. If aqueous extracts of these fungi were 
passed through a Berkefeld filter, the filtrate retained its effective- 
ness in stimulating perithecial production. Asthana and Hawker 
(1936) got active stimulation of fruiting in Melanospora destruens 
and other Ascomycetes by the addition to the culture medium of 
the ether-insoluble fraction of nutrient solutions "staled" by 
Fusarium, Botrytis, or Melanospora itself. Arrillaga (1935) noted 
that the presence of Diaporthe citri stimulated the formation of 
reproductive structures by Phytophthora citrophthora. 

Evidence of synergetic effects is not confined to responses in 
cultures. It appears also to be manifest when a mixture of or- 
ganisms is grown in tissues. Fawcett (1931) employed combina- 
tions of several pathogens of citrus to inoculate into the bark of 
citrus trees, with the result that lesions developed more rapidly 
than when one organism alone comprised the inoculum. Fawcett 
used in these experiments Diplodia natalensis, Colletotrichum 
gloeosporioides, Diaporthe citri, Sphaceloma jawcetti, and Phy- 
tophthora citrophthora. Savastano and Fawcett (1929) found 
that Oospora citri-aurantii accelerated the rate of decay of citrus 
fruits when, as inoculum, it was mixed with other organisms of 
decay. 

More attention should be given to the synergetic reactions in- 
volved in the production of lesions on plant parts. Once a lesion 
has been initiated by the primary organism, it soon becomes in- 
vaded by secondary organisms. These secondary species may 
be found in some instances to play an important role in the pro- 



292 ASSOCIATIVE EFFECTS AMONG FUNGI 

duction of mature lesions. Evidence strengthening this supposi- 
tion is found in the frequent occupancy of lesions by secondary- 
invaders. 

Wolf (1916) found fungi belonging to Gloeosporium, Fu- 
sarium, and Phoma associated with citrus canker, whose primary 
cause is Phytomonas citri. Of these fungi a species of Phoma 
was noted to be capable of secreting cellulase, invertase, diastase, 
and maltase, and from this fact it was concluded that this Phoma 
is actively associated with processes involved in the destruction of 
citrus tissues. 

GENERAL CONSIDERATIONS 

The numerous observations cited in the foregoing account may 
be assumed to prove the obvious fact that fungi interact, but the 
assumption is not warranted that certain combinations are always 
antagonistic or stimulatory, as the case may be, under all condi- 
tions. Combinations that are antagonistic in culture may not be 
so under natural conditions, as Broadfoot's (1933) experiences 
with Ophiobolns gramims and certain other soil-borne organisms 
indicate. Whether an associative interaction is beneficial or in- 
jurious may prove to be a matter of adjustment of climatic, 
edaphic, and biotic factors whose balance is delicately poised. 

The possibility that fungi occur within plants that appear to 
be entirely normal is worthy of consideration, and it is indicated 
that systematic attempts should be made to isolate fungi from 
"normal tissues." If this were done, it should not come as a 
surprise to discover that certain fungi may prove capable under 
some environmental conditions of producing serious diseases, 
under others of being benign, and under still others of inducing 
no evidence of abnormality. No doubt many of the fungus asso- 
ciations in the soil are intricately complex. Whether stability is 
ever attained among soil fungi or whether a condition approxi- 
mating such a vegetational climax as a prairie or a hardwood forest 
ever obtains among fungi is extremely doubtful. 

Attention has been centered in this account on the effect of 
one fungus on another, to the almost complete exclusion of the 
interaction of bacteria, protozoa, and green plants with fungi. 
Much has been learned from studies of these problems, but these 
topics are regarded as outside the scope of the present summary. 
Interactions between parasites and saprophytes on living host 



GENERAL CONSIDERATIONS 293 

plants appear to be less complex than interactions between micro- 
organisms in the soil, primarily because a smaller number of spe- 
cies is involved. Undoubtedly the spores of many species germi- 
nate at the surface of the plant, but only those of the pathogenic 
species succeed in producing infections. Once the lesions are 
formed, however, saprophytes may enter. In some instances 
tissue plantings from young lesions are found to yield pure cul- 
tures of the pathogen, but at a later date the tissues always yield 
a mixture of the pathogen and one or more secondary invaders. 
Still later it may be impossible to isolate the primary fungus, the 
secondary ones may also have been eliminated, and the tissues may 
be completely occupied by tertiary species. It is highly probable 
that successions of this sort do not result simply from exhaustion 
of specific food materials by the several organisms concerned. A 
solution of the problem of these interactions must be based upon 
an understanding of the physiology of each organism concerned, 
especially of their enzyme-producing abilities and the metabolic 
products they form. Only a beginning has as yet been made in 
this field of research. 

The existence of several species of fungi in the same lesion may 
also be interpreted to indicate that the conception of mono- 
etiology of disease in plants, as in animals, is altogether too narrow 
and may actually lead to misinterpretations. 

LITERATURE CITED 

Abraham, E. P., E. Chain, C. M. Fletcher, A. D. Gardner, N. G. Heatley, 

and M. A. Jennings, "Further observations on penicillin," Lancet, 2: 7, 

177-188, 1941. 
Allen, M. C, and C. M. Haenseler, "Antagonistic action of Trichoderma 

on Rhizoctonia and other soil fungi," Phytopathology, 25: 244-252, 

1935. 
Arrillaga, J. G., "The nature of inhibition between certain fungi parasitic 

on citrus," Phytopathology, 25:161-115, 1935. 
Asthana, R. P., and L. E. Hawker, "Influence of certain fungi on the 

sporulation of Melanospora destruens Shear and of some other Ascomy- 

cetes," Ann. Botany, 50: 325-343, 1936. 
Bamberg, R. H., "Bacteria antibiotic to Ustilago zeae" Phytopathology, 

27:881-890, 1931. 
Bary, A. de, Beitrage zur Morphologie und Physiologie der Pilze, 4: 85, 1881. 
Boyle, C, "Studies on the physiology of parasitism. X. The growth re- 
actions of certain fungi to their staling products," Ann. Botany, 38: 113- 

135, 1924. 



294 ASSOCIATIVE EFFECTS AMONG FUNGI 

Broadfoot, W. C, "Studies on foot and root rot of wheat. II. Cultural 
relationships on solid media of certain microorganisms in association 
with Ophiobolus graniinis Sacc," Can..]. Research, 8: 545-552, 1933. 

Buller, A. H. R., Researches on fungi, Vol. 3. Longmans, Green and Co., 
London. 1924. (Cf. 432-473.) 

Calev, D. A I., "The phenomenon of mutual aversion between monospore 
mvcelia of the same fungus (Diaporthe pemiciosa Alarchal) with a dis- 
cussion of sex heterothallism in fungi," /. Genetics, 13: 353-370, 1923. 
"The inheritance of the capacity for showing mutual aversion between 
monospore mvcelia of Diaporthe pemiciosa Alarchal," /. Genetics, 
24: 1-63, 1931.' 

Clutterbuck, P. W., R. Lowell, and H. Raistrick, "Studies in the bio- 
chemistry of microorganisms. XXVI. The formation from glucose by 
members of the Penicillhmi chrysogenum series of a pigment, an 
alkali-soluble protein, and penicillin— the antibiotic substance of Flem- 
ming," Biochem. J., 26: 1901-1918, 1932. 

D'Aeth, H. R. X., "A survey of interaction between fungi," Biol. Rev. 
Cambridge Philos. Soc, 24:103-131, 1939. 

Dodge, B. O., "Heterothallism and hypothetical hormones in Neurospora," 
Bull. Torrey Botan. Club, 5^:517-522, 1931. 

Dutcher, J. D., "The chemical nature of gliotoxin: a microbial compound 
produced by the fungus Gliocladium fimbriatum," J. Bact., 42:815-816, 
1941. 

Eastcott, E. V., "\Yildiers' bios. The isolation and identification of bios. 
I," /. Physiol. Chem., 32: 1093-1111, 1928. 

Endo, S., "Studies on antagonism of microorganisms. I. Growth of Hy- 
pochnus centrifugus (Lev.) Tul. as influenced by the antagonistic action 
of other microorganisms," Miyazaki Coll. Agr. Forestry Bull., 3:95-118, 
1931. 

II. "Growth of Hypochnus sasakii Shirai as influenced by the antagonistic 
action of other microorganisms," Miyazaki Coll. Agr. Forestry Bull., 
4: 133-158, 1932. 

III. "Pathogenicity of Hypochnus centrifugus (Lev.) Tul. and Hypochnus 
sasakii Shirai in the presence of other microorganisms," Miyazaki Coll. 
Agr. Forestry Bidl., 4: 159-184, 1932a. 

Fawcett, H. S m "The importance of investigations on the effects of known 
mixtures of microorganisms," Phytopathology, 27:545-550, 1931. 

Garrett, S. D., "Soil conditions and the take-all disease of wheat," Ann. 
Appl. Biol., 23: 667-699, 1936. 

Greaney, F. J., and J. E. AIachacek, "Studies on the control of root-rot 
disease of cereals caused bv Fusarhnn culmorum (W. G. Smith) Sacc. 
and Helminth osporiwn sativum P. K. and B. II. Pathogenicity of 
Helminth os porium sativum as influenced by Cephalothecium roseinn 
Corda in greenhouse pot tests," Sci. Agr., 75:377-386, 1935. 

Heald, F. D., and V. W. Pool, "The influence of chemical stimulation upon 
the production of perithecia of Melanospora pampeana Spcg," Nebr. 
Agr. Expt. Sta. Ann. Rept., 22: 130-134, 1908. 



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Johnson, Delia, "The antibiosis of certain bacteria to smuts and some other 

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inosit auf das Wachstum verschiedener Pilzarten," Hoppe-Sey lev's Z. 

physiol. Chem., 249: 93-110, 1937. 
Leonian, L. H., and V. G. Lilly, "Studies on the nutrition of fungi. IV. 

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Machacek, J. E., "Studies on the association of certain phytopathogens," 

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McCormick, F. A., "Perithecia of Thielavia basicola Zopf in culture and 

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Appl. Biol., 14:202-216, 1927. 
A'Iolliard, M., "Role des bacteries dans la production des peritheces des 

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Moreau, F., and M. C. Moruzi, "Recherches experimentales sur la formation 

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1931. 
Nickerson, W. J., and K. V. Thimann, "The chemical control of conjuga- 
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Nikitinsky, J., "tiber die Beeinflussung der Entwicklung einiger Schimmel- 
pilze durch ihre Stoffwechselprodukte," Jahrb. wiss. Botan., 40: 1-93, 

1904. 
Porter, C. L., "Concerning the characters of certain fungi as exhibited by 

their growth in the presence of other fungi," Am. J. Botany, 11: 168-188, 

1924. 
Porter, C. L., and J. C. Carter, "Competition among fungi," Botan. Rev., 

4: 165-182, 1938. 
Pratt, Clara A., "The staling of fungal cultures. I. General and chemical 

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fungal spores," Ann. Botany, 38: S99-6X5, 1924a. 
Raistrick, H., "Biochemistry of the lower fungi," Ergeb. Enzymforsch., 

1: 345-363, 1932. 
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Raper, J. R., "Role of hormones in the sexual reaction of heterothallic 

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"Sexual hormones in Achvla. I. Indicative evidence for a hormonal co- 

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296 ASSOCIATIVE EFFECTS AMONG FUNGI 

Raper, J. R., "Sexual hormones in Achyla. II. Distance reactions, conclusive 
evidence for a hormonal coordinating mechanism," Am. J. Botany, 21: 
162-173, 1940. 

Sanford, G. B., "Some factors affecting the pathogenicity of Actinomyces 
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.Mixed cultures," Ann. Mo. Botan. Garden, 6: 183-192, 1919. 



Chapter 13 

MYCORRHIZAE AND MYCOTROPHY 

Our knowledge of the existence of a dual relationship of fungi 
with the roots of green plants begins with the classic work of 
Frank (1885). He regarded mycorrhizae* (literally, fungus 
roots) as compound structures constituted of two components, a 
fungus and a root. These components are associated in a nutri- 
tional or mycotrophic relationship, and the structure produced by 
their association is morphologically distinct, in the same sense that 
a lichen is distinct from the alga and the fungus composing it. 
These findings by Frank immediately stimulated others to under- 
take studies on mycorrhizae, and interest in the problems that 
have arisen has not flagged to the present day. Nevertheless no 
problem involving fungi is so little appreciated and understood bv 
mycologists today, and there does not appear to be any of greater 
significance. Much of the work on mycorrhizae has been done 
by persons with such divergent interests as foresters, silvicultur- 
ists, physiologists, morphologists, pathologists, and cytologists, and 
in consequence an overwhelmingly voluminous literature on my- 
corrhizae has accumulated. An invaluable bibliograohv on the 
subject, covering more than 900 typed pages, was prepared by 
Kelly (1932). As a consequence of the numerous publications 
it might be supposed that mycorrhizae are thoroughly understood, 
but many phases of this subject still remain controversial. The 
conflict of observations, opinions, and conclusions may be at- 
tributed in part to the dangerous habit, even among scientists, of 
making generalizations. Interested students and investigators will 
find the monographs by Rayner (1927), Hatch (1937), and Bjork- 

* Frank (1885, p. 129): "Die ganze Korper ist also weder Baumwurzel 
noch Pilze allein, sondern ahnlich wie die Thallus von Flechten, eine 
Vereinigung zweier verschiedener Wesen zu einem einheitlichen morpho- 
logischen Organ, welches vieleicht passend also Pilzwurzel, Mycorhiza, 
bezeichnet werden kann." 

291 



298 MYCORRHIZAE AND MYCOTROPHY 

man (1942) indispensable in providing a knowledge of the present 
status of studies on mycorrhizae. 

Occurrence of mycorrhizae. Formerly it was generally be- 
lieved that relatively few species of plants possess mycorrhizae. 
Alycorrhizal species were then regarded as objects of scientific 
interest or even of curiosity. It is becoming more and more ap- 
parent from cumulative records, however, that they involve a 
wide variety of plants and that they occur widely throughout the 
world. Mycorrhizae occur on trees, shrubs, and herbs on essen- 
tially all kinds of soils ranging from the Arctic regions to the 
tropics. In 1934 Asai [Burges (1936)] examined members of 134 
families of plants in Japan and found mycorrhizae associated with 
82% of them. McDougall and Glasgow (1929) found mycor- 
rhizae in 28 species of composites. Samuel [Burges (1936)] re- 
corded the occurrence of mycorrhizae in Australia on Euphor- 
biaceae, Geraniaceae, Graminiaceae, Leguminosae, Liliaceae, 
Mvrtaceae, Plantaginaceae, Ranunculaceae, Rosaceae, and Vio- 
laceae. They have also been noted on members of the Burma- 
niaceae, Cunoniaceae, Ericaceae, Epicridaceae, Lauraceae, Or- 
chidaceae, Pyrolaceae, Rutaceae, and Sapindaceae [Burges 
(1936)]. Nearly all species of coniferous and hardwood trees 
examined have proved to be mycotrophic. Moreover, an ever- 
increasing number of crop plants are being found to possess 
mycorrhizae. 

The mycorrhizal habit is not restricted to the seed plants. 
Fungal threads were noticed in the thalli of the liverwort, Preissia, 
nearly 100 years ago. Since then, principally from the observa- 
tions of Nemec in 1899, Galenkin in 1902, and Cavers in 1903 
[Ravner (1927)], intracellular hyphae are of rather common oc- 
currence within the rhizoids and ventral parts of the thalli in both 
Jungermanniaceae and Marchantiaceae. Convincing proof of the 
functional nature of this association, however, is lacking. Several 
investigators have attempted to isolate each constituent in pure 
culture in order to learn of the possible interdependence among 
them. Such experiments have been uniformly unsuccessful, be- 
cause it has been impossible to isolate the fungus on artificial 
media. In consequence the opinion has been expressed that in the 
Hepaticae mycorrhizae may be lacking and the associated fungi 
mav indeed be highly specialized parasites. 



KINDS OF MYCORRHIZAE 299 

Among Bryophyta internal mycelium has been found of com- 
mon occurrence in certain genera, such as Buxbaumia and Tetra- 
plodon, but whether this is a mutualistic relation is a point still 
lacking experimental proof. 

In regard to mycotrophv among Pteridophyta there is also a 
conflict of opinion. A Pythium-like fungus has been found in 
the prothalli of several species of Lycopodium. Alycorrhizae 
have been described as occurring in the root cortex of Ophio- 
glossum and Botrychium. The endophytic mycelium from cer- 
tain marattiaceous ferns is claimed to sporulate when isolated in 
pure culture, an indication that the fungus may not be the true 
symbiont. Rayner (1927) records the presence in Pferidium 
aquilinium, a true fern, of typical endophytic mycorrhizae, with 
arbuscules and sporangioles occurring within the root cortex. 
The evidence that ferns are absolutely dependent upon the fungal 
associate, in any case, may fairly be said to be not too convincing. 

Kinds of mycorrhizae. Various characteristics have been em- 
ployed as bases of distinction in efforts to classify mycorrhizae. 
Most commonly mycorrhizae are spoken of as either ectotrophic 
or endotrophic. The ectotrophic group comprises those in which 
the fungus remains in large part as a mantle over the exterior of 
the roots, whereas the endotrophic group comprises those in 
which the hyphae are within the host cells. In Frank's original 
descriptions (1885) he directed attention to intercellular hyphae 
beneath the mantle. These intercellular hyphae invest the cor- 
tical cells and have been called the "Harti^-net." As mi^ht be 
expected, forms intermediate between the true ectotrophic and 
the true endotrophic exist. Such forms have been termed ectend- 
otrophic. Hatch and Doak (1933), however, include the ec- 
tendotrophic forms among the ectotrophic as transitional stages 
between endotrophic and ectotrophic. 

Melin (1925) described three types of mycorrhizae on Scots 
pine, the external form constituting the basis of separation. He 
designated them as follows: (a) "Gabelmykorrhiza" (forked), 
(b) "Knollenmykorrhiza" (knotted), and (c) "einfach Alykor- 
rhiza" (simple). The first type is most common in nature, espe- 
cially in woodland soils having an abundant layer of raw humus. 
It is characterized by the possession of short, dichotomously 
branched roots invested with mantles of various colors, the color 
being determined by the species of fungus involved in its pro- 



300 



MYCORRH1ZAE AND MYCOTROPHY 



duction. The second type he noted to occur under the same con- 
ditions as the first, but the fusion of mantles merged clusters of 
forked roots and thereby produced knots or tuber-like growths. 
The third type is constituted of long, thin, unbranched structures, 
which occur upon the roots of heath-inhabiting species and are 
believed to be conditioned by decreased "virulence" of the fun- 




Fig. 47. Diagram of root in cross-sections, one-half being normal, the other 
mycorrhizal. The cells of the cortex are hypertrophic, and all are en- 
veloped by fungus filaments, forming a mvcorrhiza of the ectotrophic 

type. (After Hatch and Doak.) 

gus. Decreased virulence, in turn, is the result of growth in the 
more acid soils whose mineral content is relatively unavailable. 
Melin's observations, involving mycorrhizae on pine, spruce, and 
larch, led him to conclude that mycorrhizal types represent phases 
or stages in development. The endotrophic condition is transi- 
tional to the ectendotrophic, which finally becomes transformed 
into a typical ectotrophic type. During this transition the invad- 
ing hyphae are gradually digested and eliminated by the host 
cells. 

No doubt a great deal of the confusion in understanding the 
structure and function of mycorrhizal associations arises from 



KINDS OF MYCORRH1ZAE 301 

failure to distinguish between "long roots" and "short roots," as 
pointed out by Hatch and Doak (1933). Frank (1885) and most 
European investigators, including Alelin (1925), are in accord 
that "short roots" are invariably mycorrhizal roots. Observations 
by Noelle (1910) on the anatomical differences between long 
roots (Bereicherungswurzel) and short roots (Ernahrungswurzel) 
of pine, confirmed by Hatch and Doak (1933), are summarized 
as follows: 

Long Roots Short Roots 

Root cap present. Root cap absent. 

Diarch or polyarch. Monarch. 

Have secondary growth. Lack secondary growth. 

Root hairs arise from second or Root hairs arise from epidermal cells. 

third layer of cortical cells. Branch dichotomously. 

Branch racemosely. Ratio of stele diameter to total di- 

Ratio of stele diameter to total di- ameter low. 

ameter high. 

These anatomical differences support the theory that mycor- 
rhizae are distinct morphological structures, as Frank (1885) first 
maintained. They also indicate that long roots are permanent 
structures, whereas short ones are ephemeral, lasting at most 
throughout one season. 

Hatch and Doak (1933), like earlier workers, distinguish three 
kinds of short roots: (1) uninfected short roots, (2) pseudo- 
mycorrhizal roots, and ( 3 ) mycorrhizal roots. 

The first kind is exceedingly rare and is characterized by the 
following features: (a) formation of root hairs from epidermal 
cells, (b) continuous slow elongation, (c) no hypertrophy of 
cortical cells, (d) complete lack of fungal hyphae, and (e) dichot- 
omous branching. 

The second type, called pseudomycorrhizae by Alelin, may be 
differentiated by these characteristics: (a) absence of root hairs, 
(b) early cessation of elongation, (c) complete absence of hyper- 
trophy of root cortex, (d) lack of intercellular fungal net, (<?) 
occasional dichotomous branching, and (f) intracellular invasion 
by soil-inhabiting fungi. 

Endotrophic mycorrhizae, such as occur among Ericaceae and 
Orchidaceae, are not to be confounded with the intracellular 
hyphae in pseudomycorrhizal roots. These hyphae do not occur 
in masses but may penetrate the cells in small numbers, involving 



302 MYCORRHIZAE AND MYCOTROPHY 

even the parenchyma cells of the central cylinder. One or more 
distinct species may be involved in one and the same pseudo- 
mycorrhiza. 

In the third kind the following features are noteworthy: (a) 
presence of intercellular weft or Hartig net, (b) presence of a 
fungal mantle, (c) hypertrophy of cortical cells, (d) occasional 
intracellular hyphae in cortical cells, (e) profuse dichotomous 
branching, and (f) continued elongation during one growing 
season. 

Fungi involved in mycorrhizal formation. Numerous spe- 
cies of funm have been found to be associated with mycorrhizae, 
and it is not the present purpose to list all of them but merely to 
indicate their number and taxonomic diversity. In short, mem- 
bers of each of the three large classes, Phycomycetes, Ascomv- 
cetes, and Basidiomycetes, enter into mycorrhizal formation. The 
most extensive lists assembled appear to be those of the Italian 
mycologist Peyronel [Rayner (1927)], who designates the fol- 
lowing species: 

1. On Fagus sylvatica, Cortinarius proteus, C. bivelus, Boletus 
cyanescens, B. chrysenteron, Hypochmis cyanescens, Scleroderma 
vulgare, Amanita rubescens, Lactavius subdulcis, L. blennius, 
Russula emetica, and R. nigricans. 

2. On Cory his avellana, Lactarins coryli, L. subdulcis, Boletus 
cbrysenteron, Strobilomyces strobilaceus, Hypochmis cyanescens, 
Amanita rube sc ens, Rhodopaxillus nudus, Cortinarius proteus, C. 
multiformis, C. violaceus, and Hydmnn repandum. 

3. On Betula alba, Amanita muse aria, Amanitopsis vaginata, 
Lactavius necator, L. torminosus, Boletus scaber, Scleroderma 
vulgare, Russula rhodoxantha, and Trichoderma flavobrunneum. 

4. On Larix decidua, Amanita muscaria, Russula laricina, Hy- 
grophorus bresadolae, H. lucorum, Scleroderma vulgare, Lacta- 
rins rufus, Gomphidius gracilis, and Paxillus lateralis. 

5. On Popolus tremella, Cortinarius collinitus. 

6. On Quercus robur, Amanita citrina, Lactarius subdulcis, and 
Russula cyanoxantha. 

7. On Cast an ea vesca, Amanita rube sc ens, Russula lepida, R. 
rubra, and Scleroderma vulgare. 

.Mycologists have long known that certain Hymenomycetes are 
restricted to the area beneath particular species of trees and are 
never collected under other kinds of trees. In Sweden Boletus 



MYCORRHIZAL FORMATION 303 

luteus, for example, occurs constantly on the litter under Finns 
montana. Elsewhere in Europe it has been found under P. mon- 
tana, P. austriaca, and P. sylvestris and is presumed to be respon- 
sible for mycorrhizae. Similarly Boletus elegans occurs under 
Larix and is supposed to be restricted to larches. Noack (1889) 
observed that Geaster fimbriatus, G. fornicatus, and Cortinarius 
calisteus form mycorrhizae on pine, and Tricholoma terreus on 
spruce. Masui (1926) observed Cantharellus floccosits as the 
mycorrhizal associate on Abies firma in Japan. Aielin (1925) 
regarded Boletus luteus, B. gramriatus, B. variegatus, and B. badius 
as responsible for the production of "Knollenmykorrhiza" on 
Finns sylvestris in Sweden. His "Gabelmykorrhiza" on pine, 
fungal components of which he identified as Mycelium radicis 
sylvestris f$, and M. radicis sylvestris y, have features resembling 
the mycelia of species of Tricholoma and Cortinarius, respec- 
tively. 

Certain hypogean Ascomycetes, including Elaphomyces gran- 
ulatus, Terfezia leonis, and Tuber sp., form mycorrhizae on hard- 
woods. The evidence that species of Penicillium can produce 
mycorrhizae appears to be unconvincing. 

The mycorrhizal associates in liverworts and in many herba- 
ceous seed plants are Pythium-like or Phytophthora-like in aspect. 

A Rhizoctonia type of fungus is the common endophyte of 
orchids. Organisms of similar appearance have been isolated from 
the roots of wheat, corn, barley, potatoes, tobacco, carrots, and 
other flowering plants. 

Peyronel has attributed the cause of confusion in the identity 
of the fungal constituent of mycorrhiza to the coincidental inva- 
sion of the roots by two distinct fungi, one a Phycomycete, the 
other a Rhizoctonia-like species. The Phycomycete produces 
vesicles and arbuscles that may eventually be digested by the 
host cells, and it is overgrown by the second species. In 1924 
Peyronel published a list of species, distributed among 37 families, 
that possessed this dual type of invasion [Rayner (1927)]. It 
may be inferred from his observations that the presence of two 
mycorrhizal associates in one and the same host root occurs widely 
among seed plants. 

Several endophvtes of orchids have been specifically identified 
by Bernard. From Cattleya and Cypripedium he isolated Rhi- 



304 MYCORRH1ZAE AND MYCOTROPHY 

zoctonia repens; from Phalaenopsis and Yanda, R. mucoroides; 
from Odontoglossum, R. lanuginosa. 

Fundamental knowledge regarding endotrophic mvcorrhizae 
in Ericaceae comes from the studies of Ternetz published in 1907 
[Ravner (1927)]. Ternetz became interested in the possibility 
of nitrogen fixation by the endophytes that she invariably found 
in Ericaceae growing in peaty soils. From 5 ericaceous species 
she isolated pycnidium-forming fungi, to which she gave the 
names Phovia radicis oxy cocci, P. radicis andromedae, P. radicis 
vaccinii, P. radicis tetralicis, and P. radicis ericae. She was able 
to show that each, when grown in a liquid nitrogen-free medium, 
was capable of fixing appreciable quantities of nitrogen. From 
these results, obligatory symbiosis among Ericaceae has been in- 
ferred to exist, as Ravner has claimed, in a series of studies involv- 
ing Calluna vulgaris. In this species the endophyte occurs within 
the seed and permeates the entire plant. Ravner also found (1929) 
that the endophytic mycelium ramifies throughout the stem tis- 
sues of Vaccinium oxy coccus and V. macro car pon and that ova- 
rian infection occurs in V . vitisidaea, V. myrtilhis, V. pennsyha- 
nicttm, V. ovatnm, V. vacillans, and V. corymbosam. Ravner's 
claims, however, have been disputed. There exists a body of evi- 
dence that ericaceous species, notably cranberries, have been 
grown successfully for a term of years, apart from the endophyte. 
Significance must be attached, however, to the fact that in nature 
certain species always possess endotrophic mvcorrhizae and that 
vigorous growth is promoted by the presence of the fungus. Re- 
cent studies by Barrows (1941) show that an endophyte occurs 
within the roots, stems, flowers, ovules, and fruits of trailing ar- 
butus, Epigaea repens. 

A most unusual kind of mvcorrhizal relationship exists between 
the tuberous, non-chlorophyllous orchid, Gastroidea elata, and 
Arviillaria viellea [Kusano (1911)1. The rhizomorphs of this 
fungus, which is widely known because of its ability to destroy 
forest trees, attack the tubers in such a way that the outer layers 
contain a dense mass of thick-walled hyphae; beneath it occurs a 
region containing thin-walled hyphae, and the innermost layer 
contains a few slender hyphae. Tubers associated with rhizo- 
morphs produce offsets which remain dormant during the winter 
and develop flowers in the following summer. If mvcorrhizae 
are not formed, flowers are not developed. 



FUNCTION OF MYCORRH1ZAE 305 

Another endotrophic mycorrhizal relationship, which is of un- 
usual interest and has been studied rather extensively, involves 
the grasses, Lolhtm perenne and L. temulentum. The fungus in- 
vades the growing point, penetrates the carpels, and has been 
demonstrated to occupy the ovules and embryo. Sampson (1935) 
called attention to the fact that fungus-free seed can be made to 
produce fungus-free plants that set seed. On the other hand, seed 
containing the endophyte may produce plants that again are 
mycorrhizal. This fungus is not identified, but there are reasons 
for believing it may be a smut. 

Function of mycorrhizae. Although knowledge of the exist- 
ence of mycorrhizae dates back at least to the fourth century b.c. 
[Kelly (1932)], definite information concerning their true struc- 
tural nature may be said to begin with Frank's observations in 
1885. In the years that followed, conjecture as to their function 
was rife, and from the publication of Frank's classical studies to 
the present, numerous theories on this subject have been advanced. 
Of these only two have been accorded general acceptance. In 
one theory mycorrhizae are regarded as pathological structures 
induced by the parasitic action of the fungus upon the root tis- 
sues. The other theory is that mycorrhizae are symbiotic struc- 
tures that facilitate the absorption and utilization of organic ma- 
terials, especially of organic nitrogen, contained in humus. It 
appears that evidence in support of these theories may be best pre- 
sented by a brief review of a few of the numerous publications 
on mycorrhizae. 

Over 100 years ago the mode of nutrition of Monotropa hypo- 
pitys, a flowering plant lacking chlorophyll, attracted the atten- 
tion of botanists. This curious plant, classed as a saprophyte in 
modern botanical textbooks, grows with its roots intermingled 
with those of beeches, spruces, and other species of trees. In 
consequence some workers regarded the Monotropa as a root 
parasite, and thev noted that its roots were covered with "a whit- 
ish, silky, somewhat fibrous material, connected with the decaying 
leaves." The fungal nature of this material was first recognized 
in 1832 by Elias Fries. Several early workers demonstrated that 
Monotropa is not a root parasite by the simple expedient of trans- 
planting and maintaining it apart from tree roots. In spite of this 
fact, final settlement of the mode of its nutrition was deferred 
until 1881, when Kamiensky (1881, 1882) again showed that 



306 



MYCORRHIZAE AND MYCOTROPHY 



Alonotropa will grow independently of tree roots and that the 
roots of both the tree and the Alonotropa are invested with a 
similar fungus mantle. Furthermore this mantle is organically 
connected with hvphae that course between the cells of the root 
cortex. In criticism of Kamiensky's work, however, it may be 
indicated that he did not present experimental evidence that either 
the trees or the Alonotropa are dependent upon the fungus. 




mill 



^pvjni 




Fig. 48. Diagram of young root, the upper side mvcorrhizal, the lower 
normal. The root-hair zone, K (in black), occurs near the root tip, M. 
The increased absorbing surface in A, B, C, and D (in black) is to be 
compared with that in E, F, G, and H. The surface area active in ab- 
sorption in mvcorrhizal roots must also take account of the surface area 
of the fungus filaments. (After Hatch.) 

From about 1840 the association of truffles, especially with oak, 
beech, and hornbeam, attracted botanical attention. Certain early 
students of this problem definitely established that the mycelia 
of truffles are connected with the roots of these trees, but the 
relationship was supposed to be parasitic. At first Frank's interest 
in the matter was centered upon the possibility of cultivating 
truffles and other hvpogeous fungi, especially Elaphomyces {rranu- 
latus, in the forest. As an outgrowth of this interest he established 
the fact that the roots of certain trees, especially members of the 
Cupulifereae, are invariably invaded by fungi. Aloreover, he was 
led to formulate the theory that the relationship is not one of para- 
sitism but of definite beneficial symbiosis, in which the fungal 



FUNCTION OF MYCORRHIZAE 301 

component, in lieu of root hairs, functions to absorb water and 
mineral salts from the soil. Some of Frank's experiments involved 
growing seedlings in culture solutions free from mycorrhizae. He 
found that such trees made entirely satisfactory growth, a result 
that has been repeatedly verified by others. Frank's interpreta- 
tions immediately created a great deal of interest throughout 
Europe, and from the investigations that were undertaken in the 
next few years a barrage of criticism arose. The net result of 
these studies was the general admission that mycorrhizae are of 
widespread occurrence in nature, but many workers questioned 
the mycotrophic relationship. 

Stahl's comprehensive study (1900) of mycorrhizae is a land- 
mark among contributions to the literature of this subject. In it 
he elaborated the thesis that the incidence of mycorrhizal devel- 
opment is correlated inversely with soil fertility. Supporting 
evidence for this thesis rests in part upon the assumption, since 
confirmed by a host of investigators, that in the keen competition 
between vascular plants and soil fungi for essential minerals, the 
fungus mycelium possesses superior mechanism. Presumably the 
basis for this superiority is that the ratio of surface area to volume 
is vastly greater in fungus hyphae than in roots. For this reason 
non-mycorrhizal plants, such as Sambiicus nigra, Cvperaceae, and 
various ferns, are at a disadvantage when growing on infertile soils, 
in competition with mycorrhiza-formers. 

Stahl's observations also bore out his assumption that different 
species of plants differ in the extensiveness of their root systems 
and their rates of transpiration. Species with extensive root sys- 
tems and with the capability of losing water rapidly might be 
expected to be best fitted for competition. Actually Stahl found 
that the reverse is true, for the reason that species possessing ex- 
tensive root systems and being capable of transpiring rapidly tend 
to be autotrophic, whereas those with restricted root systems and 
slow transpiration rates are mycotrophic. 

Rayner (1934) concluded from researches involving pines that 
there is a "direct causal relation between mvcorrhiza development 
and the thrifty growth in seedlings of various species of Pinus." 
Further evidence in support of Stahl's mineral-nutrition theory is 
advanced by Hatch (1937) in an extensive series of experiments. 
He emphasizes that the absorbing surface area of short roots is 
increased through the presence of mycorrhizae by the following: 



308 MYCORRH1ZAE AND MYCOTROPHY 

(a) continued elongation, (b) increased diameter, (c) dichoto- 
mous branching, (d) delay in suberization of cortex, and (e) ac- 
quisition of additional surface area, the composite of that of the 
hvphae. His three interpretations made in conclusion are: (1) 
that mvcotrophic relationship is a symbiotic mechanism to in- 
crease the absorption of soil nutrients; (2) that the extent of the 
surface area of short roots is determined bv the availability of 
minerals, mvcorrhizal roots being rarely formed in fertile soils 
but produced in abundance in infertile soils; and (3) that trees are 
dependent upon symbiotic association with mycorrhizal fungi for 
all their mineral nutrients and therefore for their ability to exist in 
all except the most fertile soils. Experiments by Mitchell, Finn, 
and Rosendahl (1937) on mycorrhizae as related to mineral ab- 
sorption by coniferous seedlings led them to arrive at conclusions 
similar in all essentials to those of Hatch. Bjorkman (1942) found 
that light, nitrogen, and phosphorus are each decisive factors gov- 
erning the formation of mycorrhizae. 

Buries (1936) postulated that the higher plants benefit from 
association with fungi by absorbing the nutrients made soluble as 
a result of decomposition by the soil fungi. He does not believe 
that there is any mutualistic relationship between tree roots and 
fungi but that mycorrhizae represent a controlled parasitic at- 
tack. Some support of this idea appears from Rayner's (1934, 
1936) experiments, in which, after inoculation with mycorrhizal 
fungi, she noted markedly improved growth of pine seedlings at 
a period in advance of the actual formation of mycorrhizae. She 
attributed this stimulation to the elaboration of growth-promoting 
substances by the fungus and to the nutrients liberated to the 
seedlings by the activity of the fundus. In view of the body of 
evidence that is being accumulated on the elaboration of auxins 
by fungi, these substances may well be important factors in in- 
creasing the growth of plants possessing mycorrhizae. 

Much has been written to indicate that mycorrhizal fungi are 
parasitic and that the balance may be easily tipped toward one or 
the other partner in the relationship. The observations of Masui 
(1926, 1927) in Japan and of McDougall (1914) in this country 
inclined them to regard the association as one of parasitism by 
the fungus. 

Bernard's experiments (1909), summarized in her "L'evolution 
dans la svmbiose des Orchidees," are fundamental to an apprecia- 



FUNCTION OF MYCORRHIZAE 



309 



tion of the nature of endotrophic mycorrhizae. In an earlv re- 
port * she concluded that the fungal component is a benign para- 
site causing chronic pathogenesis. She became interested in this 
problem because of the difficulty that orchid growers were experi- 
encing in germinating seed and raising seedlings. In the green- 




Fig. 49. A. Short, lateral roots dichotomously branched, typical of mycor- 
rhizae on pine. B. Somewhat enlarged mycorrhizae on pine. (After 
Hatch.) C. Sketch of ectotrophic mycorrhiza on pine, showing mantle 
and "Hartig net." (After Doak.) D. Locus of endotrophic mycorrhiza 
(stippled area) in germinating orchid seed, Odontoglossum. E. Cell from 
stippled area showing hvphae of Rhizoctoma lanuginosa. (After Bernard.) 
F. Ectendotrophic mycorrhiza in cell of strawberry root. (After O'Brien 

and McNaughton.) 

houses of successful growers she noted that certain fungi were 
present in the soil around the roots and that endotrophic mycelium 
occurred within the tissues. If seed were sown near parent plants, 
germination resulted. If the seed were grown aseptically, ger- 
mination failed. These observations led her to believe that the 
presence of the endophvte was essential for germination and 

* She considered orchids, ''comme les plantes atteintes d'une maladie 
parasitaire chronique qui commence a la germination et persiste en general 
jusqu'a l'etat adulte; maladie benigne en un certain sens. . . ." 



310 MYCORRH1ZAE AND MYCOTROPHY 

growth. She isolated the endophvte from several genera, getting 
organisms that were morphologically similar but were dissimilar 
in action when used reciprocally to inoculate seedlings. If, for 
example, she inoculated seed of Phalaenopsis with the fungus iso- 
lated from the same host, normal germination followed, and the 
mycelium was kept in bounds by the digestive activity of the cells 
of the embryo. If instead she used the fungus isolated from 
Odontoglossum, germination stopped short at an early stage, and 
intracellular digestion of the fungus was excessive. In certain 
other orchids, such as Bletilla hyacinthia, the seed germinated even 
when the fungus was absent, but the seedlings did not survive 
beyond the first leaf stage. 

Bernard was able to grow seedlings to a size suitable for trans- 
planting in the absence of the endophvte, if she supplied sugar 
solutions and salep of varying concentrations. She interpreted 
this ability to germinate in the absence of the endophvte to be 
caused by a physico-chemical stimulus of the sugar and not to be 
produced by the sugar as a food. 

More recently Knudson (1929) summarized a series of studies 
(1922, 1925) on the food relationship in these non-symbiotic ger- 
minations. He found that the embryos of Cymbidium, Vanda, 
Ophrvs, and Epipactis lack chlorophyll for the first 5 or 6 weeks. 
They must, therefore, obtain soluble food from the substratum in 
which they are grown. When Knudson supplied Cattleya em- 
bryos with sugar for the period of a month and then removed 
them to a medium lacking sugar, the seedlings continued to make 
good growth for 5 or more years. Sugar concentrations in pure 
cultures as low as 0.02% yielded good (Terminations. Knudson 
was also able, in PfefTer's solution fortified with a mixture of peat 
and sphagnum and adjusted to pH 4.6, to germinate embryos just 
as rapidly as occurs in the presence of the endophvte. .Moreover, 
he was able successfully to substitute a species of Phytophthora 
isolated from lilies for the true endophytic Rhizoctonia. He con- 
cluded from these extensive studies that the unusual requirements 
of orchid seed for germination must be explained by their inability 
to synthesize food. The embryos must therefore be regarded as 
saprotrophic in early development and the associated fungus as 
mildly pathogenic, pathogenicity being controlled by the physio- 
logical condition of the orchid. In criticism it may be noted that 
the universal occurrence of the endophvte in orchid roots in 



IMPORTANCE OF MYCORRH1ZAE TO FORESTRY 311 

nature is not satisfactorily explained by these experiments of 
Knudson. 

Importance of mycorrhizae to forestry. Evidence from ob- 
servations extending over a period of years has been accumulating 
which tends to show that mycorrhizae plav an important role in 
reforestation and afforestation. As long ago as 1917 [Hatch 
(1937)] Aielin noticed that seedlings of pine and spruce, started 
from wind-distributed seed in recently drained peat bogs, ex- 
hibited nitrogen starvation and eventually died unless they be- 

O J J 

came invaded with mycorrhizal funoi. Seedlings in entire nur- 
series in Australia, southern Rhodesia, the Netherlands East Indies, 
and the Philippines remained unthrifty when the nurseries were 
located outside the natural range of the species being grown 
[Hatch (1936)]. When soil from established nurseries or from 
sites where the species was endemic was incorporated as inoculum 
in these seed beds, however, the seedlings recovered and grew 
normally. Similarly, non-mycorrhizal seedlings made poor 
growth in plantations until they were inoculated with small quan- 
tities of soil containing mycorrhizal fungi. 

More convincing evidence of the beneficial nature of mycor- 
rhizae has been supplied by Rayner (1934, 1936, 1939), Hatch 
(1936, 1937), and Young (1940). Rayner reported experiments 
in which she applied pure cultures of mycorrhizal fungi to soils 
in which seedlings were making poor growth and in which my- 
corrhizae were infrequent. As a result of such inoculations, seed- 
ling growth was markedly stimulated, and correlated mycorrhizal 
formation was abundant. 

Since transplants in the Prairie States grew poorly, Hatch 
planted Finns strobus seed in pots of prairie soil in an effort to 
determine the cause. The seedlings grew poorly, and mycor- 
rhizae were lacking. He then inoculated a portion of the pots 
with pure cultures of mycorrhizal fungi. The inoculated seed- 
liners responded by increased growth over the uninoculated ones 
to the extent that, after two months, analyses showed the inocu- 
lated seedlings had 75% more potassium, 86% more nitrogen, and 
234% more phosphorus than the uninoculated. Young grew 1600 
Pinus caribaea in soil that had never grown pines. He mixed 
manure and pine needles with the soil to supply organic materials. 
As inoculum he used 7 mycorrhizal fungi. In all cases the un- 
inoculated controls made the poorest growth, with best growth 



312 MYCORRHIZAE AND MYCOTROPHY 

in those inoculated with Boletus viscidus. The differences would 
no doubt have been greater if he had not used manure. Australian 
mvcorrhizal fungi might also be expected to have been better 
suited as inoculum. 

These observations and experiments, although not numerous, 
indicate several very obvious conclusions. In the first place, at- 
tempts to exclude diseases and pests from prairie or other treeless 
regions by starting nurseries from seed are doomed to failure 
unless suitable mvcorrhizae producers are introduced. Second, 
mvcorrhizal fungi perish in areas that have been long denuded, 
and thev must be reintroduced if the areas are to be reforested. 
Third, planting failure can result if the environment of the plant- 
ing site is unfavorable for the growth of the fungal component. 
This conclusion is supported by the experiments of Romell 
[Hatch (1937)], who found that mycorrhizal fungi may be more 
exacting in their site requirements than are the trees with which 
they became established in this mycorrhizal relationship. 

In the light of these studies on mycorrhizae, the fleshy fungi 
growing on the forest floor have uses aside from supplying the 
mycologist with objects with which he may occupy his time, or 
the layman with victims against which he may employ his toe to 
vent his pent-up emotions. 

For use in silviculture further knowledge should be sought by 
attempting to synthesize mycorrhizae from fleshy fungi and for- 
est trees. Studies of this sort are still too limited in number and 
scope. The value of such work is indicated by Alodess (1941). 
He employed pure cultures of Hymenomycetes and Gastromy- 
cetes with pines and spruce, finding that Scots pine developed 
mycorrhizae with Amanita mappa, A. musctiria, A. pantherina, 
Boletus flavidus, Entoloma rhodopoliuvh Lactarius helvus, Paxil- 
lus primulas, Rhizopogon luteolus, R. roseolus, Scleroderma au- 
rantium, Tricholoma albobrumieum, T. imbricatum, T. pessunda- 
tinih and T. vacciniwm, and that Picea abies synthesized mycor- 
rhizae with each of the species of Amanita, Boletus, Lactarius, 
Tricholoma, and Scleroderma that has been mentioned. 

Tuberizatiox. From time to time evidence has been presented 
which indicates that tuberization in certain plants may be induced 
by mvcorrhizal fungi. In this connection the work of Bernard 
on the group of tuberous orchids is of especial significance. She 
found that the seeds of Bletilla, sown aseptically, develop into 



TUBER1ZATION SIS 

seedlings with slender stems and with leaves borne at distinct in- 
ternodes, whereas in the presence of the endophyte the axes of the 
seedlings are thick, the internodes short, and the leaves crowded. 
These observations led her to conclude that a causal relationship 
between invasion by the endophyte and tuberization exists and 
also induced her to explore the possibility that fungi are the cause 
of tuberization in Ranunculus ficariae and Solamim tuberosum 
[ Bernard (1911), Bernard and Magrou (1911)1. After Bernard's 
untimely death the experiments were continued by Magrou 
(1921, 1924). He observed mycorrhizae in Solanum magia, pre- 
sumably an ancestor of the cultivated potato, and 5. dulcamara 
and named the associated fungus Mucor solani. He reported that 
this fungus was capable of infecting Solanum tuberosum raised 
from sterilized seed. His observations and conclusions have not 
remained unchallenged, however, and in fairness may be said to 
require confirmation by the performance of a series of synthetic 
experiments. Only on the basis of such experiments can his find- 
ings be accepted or discarded. There still remains the interesting 
possibility that the endophyte may provide the stimulus that ini- 
tiates tuberization, and that it may be entirely digested by the host 
cells by the time the tuber is mature. 

Constantin (1922) summarized his studies of tuberization as 
follows: "The association of perennial species of plants with soil 
fungi has brought about a permanent symbiosis— a condition 
which does not occur with annual species. Since the perennial 
character in plants is due to the low temperatures of high altitudes 
and latitudes, cold climates may be considered as favorable to the 
establishment of symbiosis. Cultivated potatoes have lost the 
mycorrhizal relations of the primitive forms to which tuberization 
was due, and in order to produce tubers without this relationship 
they must be grown in cold climates." 

The recent report of Lutmari (1945) directs attention to Ac- 
tinomycetes within tubers of potato and the roots of artichoke, 
parsnip, carrot, and beet. The filaments, demonstrable by special 
stains, pass between the cells and are intimately applied to them. 
Their role remains unknown, but Lutman concludes, "The effects 
of actinomyces filaments surrounding every cell cannot, at this 
time, be even estimated, but the materials which they withdraw 
from the cells and the products which they excrete and which 



314 MYCORRHIZAE AND MYCOTROPHY 

must be absorbed bv the cells cannot fail to change the charac- 
teristics of the cells." 

Implications. The classical interpretations of the mechanisms 
involved in absorption of water and mineral nutrients by seed 
plants must be modified somewhat or must, in some measure, be 
supplanted by a more complicated system in the light of the fore- 
going consideration of mvcorrhizae. Certain seed plants, espe- 
ciallv trees, and certain fungi have been demonstrated to be nu- 
tritionallv interdependent. This interdependence is a partnership, 
and, as in anv partnership, both members may profit mutually or 
one member may exploit the other. Apparently the advantages 
that accrue to each member of the partnership outweigh the dis- 
advantages when environmental factors are normal, but this bal- 
ance may become upset in times of stress. The mycologist may 
speculate to the satisfaction of his scientific soul on how and why 
such a relationship between totally unrelated organisms ever be- 
came established, only to arrive eventually at the unsatisfactory 
conclusion that living things are interdependent. 

LITERATURE CITED 

Barrows, Florence L., "Propagation of Epigaea repens. II. The endophytic 

fungus," Contrib. Boyce Thompson Inst., 77:431^40, 1941. 
Bernard, X., "L'evolution dans la symbiose des Orchidees et leur champig- 
nons commensaux," Ann. sci. not. Botan., 9: 1-96, 1909. 
"Les mycorhizes des Solanum," Ann. sci. not. Botan., 14: 235-252, 1911. 
Bernard, N., and J. Magrou, "Sur les mycorhizes des Pomme de terre 

sauvage," Ann. sci. not. Botan., 9 me. ser., 14: 252-258, 1911. 
Bjorkman, Erik, "Uber die Bedingungcn dcr Mykorrhizabildung bei 

Kiefer und Fichte," Symbolae Botan. Upsalensis, 6, No. 2, 1942. 
Bur(;es, A., "On the significance of mycorrhiza," Neiv Phytol., 35: 117-131, 

1936. 
Constantin, J., "Sur l'heredite acquise," Comp. rend., 114: 1659-1662, 1922. 
Frank, A. B., "Uber die auf Wurzelsymbiose beruhende Ernahrung gewisser 

Baume durch unterirdi'sche Pilze," Ber. dent, botan. Ges., 3: 128-145, 

1885. 
Hatch, A. B., "The role of mvcorrhizae in afforestation," /. Forestry. 

34: 22-29, 1936. 
"The physical basis of mycotrophy in Pinus," Black Rock Forest Bull.. 

6. 168pp. 1937. 
Hatch, A. B., and K. D. Doak, "Mycorrhizal and other features of the root 

systems of Pinus," Arnold Arboretum /., 14: 85-99, 1933. 
Kamienskv, F., "Die Ycgetationsorganender Mouotropa hypopitys L.," 

Botan. Z., 5^:458-461, 1881. 



LITERATURE CITED 315 

Kamiensky, F., "Les organes vegetatifs du Monotropa hypopitys L.," Ext. 

Mem. soc. nationale sci. not. math. Cherbourg, 24: 5-40, 1882. 
Kelly, A. P., "The literature of mycorrhizae" (manuscript 948 pp.), Library, 

U. S. Dept. Agr., Washington, D. C. 1932. 
Kxudson, L., "Nonsymbiotic germination of orchid seeds," Botan. Gaz., 

13: 1-25, 1922. 
"Physiological study of the symbiotic germination of orchid seeds," 

Botan. Gaz., 19: 345-379, 1925.' 
"Physiological investigations on orchid-seed germination," Proc. Intern. 

Congr. Plant Sci. Ithaca, 2: 1183-1189, 1929. 
Kusano, S., "Gastroidia elata and its symbiotic association with Annillaria 

mellea" J. Coll. Agr., Imp. Univ. Tokyo, 4: 1-66, 1911. 
Lutmax, B. F., "Actinomycetes in various parts of the potato and other 

plants," Vt. Agr. Expi. Sta. Bull., 522. 72 pp. 1945. 
McDougall, W.-B., "On the mycorrhizas of forest trees," Am. J. Botany, 

7:51-74, 1914. 
McDougall, W. B., and O. E. Glasgow, "Mycorrhizas of the Compositae," 

Am. J. Botany, 16: 224-228, 1929. 
iMagrou, J., "Symbiose et tuberization," Ann. sci. not. Botan., 10 me. ser., 

3: 181-273, 1921. 
"Remarques sur les cultures experimentales de pomme de terre avec 

endophyte," Ann. sci. not. Botan., 10 me. ser., 6: 285-288, 1924. 
Masui, Koki, "A study of the mycorrhiza of Abies firma S. et Z., with special 

reference to its mycorrhizal fungus, Cantharellus floccosus Schw.," 

Mem. Coll. Sci., Kyoto Imp. Univ., Ser. B., 2: 16-84, 1926. 
"A study of the ectotrophic mycorrhizas of woody plants," Mem. Coll. 

Sci., Kyoto Imp. Univ., Ser. B, 3: 149-279, 1927. 
Melin, Elias, Untersuchnngen iiber die Bedeutung der Baummy corrhiza, 

eine okologisch-physiologische Studie. 152 pp. G. Fischer, Jena. 1925. 
Mitchell, H. F., R. F. Finn, and R. O. Rosendahl, "The relation between 

mycorrhizae and the growth and nutrient absorption of conifer seed- 
lings in nursery beds," Black Rock Forest Paper, 1: 58-73, 1937. 
Modess, O., "Zur Kenntnis der Mykorrhizabildner von Kiefer und Fichte," 

Symbolae Botan. Upsalienses, 5: 3-147, 1941. 
Noack, R., "Liber Mycorrhizenbildene Pilze," Botan. Z., 47:389-397, 1889. 
Noelle, W., "Studien zur vergleichenden Anatomie und Morphologie der 

Koniferenwurzeln mit Riicksicht auf die Systematik," Botan. Z., 68: 169- 

266, 1910. 
Rayner, M. C, Mycorrhiza, an account of non-pathogenic infection by 

fungi in vascular plants and Bryophytes. 246 pp. Weldon & Wesley, 

Ltd., London. 1927. 
"The biology of fungus infection in the genus Vaccinium," Ann. Botany, 

43: 55-70, 1929. 
"Mycorrhiza in relation to forestry. I. Researches on the genus Pinus, 

with an account of experimental work in a selected area," Forestry, 

8:96-125, 1934. 
"The mycorrhizal habit in relation to forestry. II. Organic composts and 

the growth of trees," Forestry, 10: 1-22, 1936. 



316 MYCORRHIZAE AND MYCOTROPHY 

Rayner, M. C, "The mvcorrhizal habit in relation to forestry. III. Organic 
composts and the growth of young trees," Forestry, 13: 19-35, 1939. 

Sampson, K., "The presence and absence of an endophytic fungus in Lolhim 
temulentum and L. perenne" Trans. Brit. Alycol. Soc, 19: 337-343, 1935. 

Stahl, E., "Der Sinn der Mycorrhizenbildung," Jahrb. whs. Botan., 34: 534- 
688, 1900. 

Young, H. E., "Mycorrhizae and growth of Pinus and Araucaria. The in- 
fluence of different species of mycorrhiza-forming fungi on seedling 
growth," /. Australian Inst. Agr. Sci., 6: 21-25, 1940. 






Chapter 14 

GENETICS OF FUNGI 

The principles upon which the science of genetics rests were 
established by Mendel in 1865 but remained unrecognized until 
the beginning of the present century. He determined from hy- 
bridization experiments with peas that heritable characters behave 
as units. These characters may be allelomorphic, that is, they 
may operate as pairs, one member of which is dominant, the other 
recessive. The characters must therefore be controlled by factors 
or determiners which maintain their individuality throughout the 
developmental cycle and are transmitted from generation to gen- 
eration. Moreover, in the second hybrid generation or later these 
characters segregate or become assorted in definite numerical 

ratios. 

With the rediscovery of Mendelism at the beginning of the 
twentieth century attention turned largely to studies of genetics 
of seed plants and higher animals. The application of Mendelism 
to fungi has constituted a neglected field of inquiry until the past 
few years. Some of the reasons will become apparent in the ac- 
count that follows. Not the least of them is the small size of nuclei 
and chromosomes and their constituents. These facts militate 
seriously against the procurement of microscopic evidence to sub- 
stantiate macroscopic evidence of inheritance. 

SEXUAL AND ASEXUAL STAGES OF FUNGI 

In order to appreciate and properly evaluate genetic studies of 
funo-i it is necessary to recall certain knowledge that is funda- 
mentally axiomatic. In the normal life cycle of fungi generally 
there occur fusions between pairs of gametes. This phase is 
called the sexual stage in contrast to the asexual stage, in which 
vegetative units are capable independently of propagating the 
fungus. The fusion of gametes, called fertilization, produces a 

317 



318 GENETICS OF FUNGI 

one-celled structure called the zygote. The most important part 
of each gamete is the nucleus. Gametes of fungi are not known to 
possess any appreciable or functional cytoplasm, such as is known 
to occur among seed plants. Undoubtedly gametes of fungi 
possess functional cytoplasm, but as yet proof of cytoplasmic in- 
heritance either is not forthcoming or is meager. 

Each gametic nucleus is constituted of chromatic materials, the 
chromosomes. Each zygote contains In chromosomes and is 
therefore diploid. As a rule, however, among the Basidiomycetes 
and Ascomycetes, when the two gametic nuclei are brought into 
juxtaposition, they do not fuse immediately but remain as a pair. 
Then the two divide at one and the same time, the process being 
called conjugate nuclear division and giving rise to two daughter 
pairs. Hundreds or even thousands of successive conjugate nu- 
clear divisions may follow, extending in time over a period of 
weeks or months. Finally two such paired nuclei come to lie in 
special cells (basidia in the Basidiomycetes, teliospores in the 
Uredinales, chlamydospores in the Ustilaginales, young asci, asco- 
gonia, or ascogenous hyphae in the Ascomycetes), where they 
actually fuse. 

The fusion nucleus resulting contains 2/7 chromosomes and is 
thus diploid. Shortly after fertilization the fusion nucleus divides 
twice. The processes involved in these two divisions constitute 
meiosis. In one of the divisions the number of chromosomes is 
reduced to 77, and the other division is homotypic (equational). 
Sex factors are segregated during meiosis. Each of the four nuclei 
resulting from meiosis contains n chromosomes, the haploid num- 
ber. In the Basidiomycetes each of the four nuclei migrates into 
a developing basidiospore, which is a haploid cell. When these 
basidiospores germinate, they produce haploid mycelia; if two 
such mycelia or their equivalents of opposite sex fuse, cells again 
containing a conjugate pair of nuclei arise. In the Ascomycetes 
each of the four haploid nuclei again undergoes a mitotic division, 
whereupon each of the eight haploid nuclei becomes invested with 
a wall and is an ascospore. The germination of ascospores gives 
rise to haploid mycelia, and the nuclei may again become paired 
in preparation for fusion within the ascogonium, young asci, or 
ascogenous hyphae, as the case may be. 



HOMOTHALL1SM AND HETEROTHALLISM 319 

HOMOTHALLISM AND HETEROTHALLISM 

From the above generalizations we may pass on to their sig- 
nificance. For a long time it was held that the spore or the indi- 
vidual derived from any spore is totipotent. This concept, it may 
be interjected, as employed in studies of monosporous cultures, has 
been both a great deterent to progress and a potent factor in pro- 
moting progress in the acquisition of knowledge of fungi. It has 
hindered progress because many workers have regarded a mono- 
sporous culture of a fungus as a whole organism, whereas it may 
be, as we now know, only a "hemi-organism." On the other hand, 
the concept has promoted knowledge because by use of mated 
monosporous cultures it has been possible to learn that each 
individual may not be totipotent but may require another comple- 
mentary culture. In 1904 Blakeslee (1904) first proved, for a 
number of species of Zygomycetes, that zygospores can be ob- 
tained only when mycelia of opposite sex are mated. If he grew 
mycelium from a single conidium, sporangia and conidia were 
formed in abundance, but gametangia and zygospores were not 
produced. To those organisms requiring two thalli of opposite 
sex potentialities for fertilization, he applied the term heterothallic. 
One strain or race he called plus ( + ), and the other minus ( — ). 
Sex in these species is segregated in bipolar fashion at meiosis. On 
the other hand, in Sporodinia grandis mycelia from single conidia 
produce zygospores and are therefore hermaphroditic, and sex 
segregation is entirely lacking. Subsequently both heterothallism 
and homothallism have been found to occur side by side in genera 
in all the larger groups of fungi. 

In Phycomycetes. BurgefT (1928) isolated from Phy corny ces 
blakesleeamis a number of variant or mutant races to which he 
gave such form names as arbusculus, mucoroides, gracilis, and 
pallens. When various crosses between the original P. blake- 
sleeamis and any one of the forms were made, the progeny ap- 
peared like that of the original except in the crosses with mucor- 
oides. The type of progeny, therefore, is determined by a single 
factor that is recessive in the form and dominant in the original; 
this was true in all crosses with mutants except in the form 
mucoroides. Burgeff also noted certain linkages with the factor 
for sex. For example, in his crosses of arbuscula with the normal, 



320 



GENETICS OF FUNGI 



the heterozygous zygospores were (Arb arb + — ). When these 
were germinated, half of them gave four different haploid deriva- 
tives with the respective constitutions (Arb +), (Arb — ), 
{or}) _)_) ? an d (arb — ). The other half yielded only two different 




Fig. 50. Plew-age cmser'ma. A. Normal perithecium, external appearance. 
B. Normal ascus, bearing four ascospores. C. Occasionally asci are found 
bearing five ascospores, two of which are smaller than normal. Mycelium 
from small ones bear ascogonia, D, and spermatia. E. The mycelium from 
normal spores can produce perithecia; the spermatia and ascogonia borne 
on mvcelia from small spores are self-incompatible and hence self-sterile, 
but compatible and fertile if reciprocally crossed. F. Spermatia borne by 
phialide-like lateral branches. G. Trichogync with empty spermatium 

attached. (Adapted from Ames.) 



HOMOTHALLISM AND HETEROTHALLISM 



321 



types, either (Arb -f- ) and (arb — ) or (Arb — ) and (arb -f). 

To explain this condition BurgefT assumed properly that one or 

both factor pairs must segregate at the second division. If both 

pairs of factors had segregated at the first division, there would 

have been only two haploid 

types, either (Arb +) and 

(arb — ) or (Arb — ) and 

(arb +). 

In Ascomycetes. The most 

illuminating grenetical studies 

among Ascomycetes have con- 

cerned Neurospora [Shear 

and Dodge (1927), Dodge 

(1927, 1928, 1930, 1931, 1940), 

Wilcox (1928), Lindegren 

(1929, 1933, 1936, 1939)]. 

The best-known species of 

this genus is N. sitophila, 

known as the pink bakery 

mold, which is cosmopolitan 

in distribution. It has a Mo- 

nilia conidial stage. Some of 

the species, represented by N. 

sitophila and N. crassa, are , . MM „«. n «j rt „ n f 

r . Fig. 51. Schematic representation or 

eight-spored and obligately poten tialities of ascospores of Plenr- 
heterothallic. Each spore is age anserina. Circles represent nuclei 
uninucleate and unisexual, of one sex, and black dots nuclei of 
four spores being of ( + ) sex the °PP osite sex ; Although the asci 
reaction and four of (— ) sex 
reaction. Other species, such 
as N. tetrasperma, are nor- 
mally four-spored, each spore being binucleate and bisexual. Oc- 
casionally in this species one or more of the spores are giants or 
dwarfs, as occurs also in Pleurage anserina, a widely distributed 
dung-fungus [Wolf (1912), Dowding (1931), Ames (1934). 
Usually the giant spore replaces two normal spores. The dwarf 
spores occur in pairs, each containing a single nucleus. In N. 
tetrasperma [Dodge (1927)] all eight of the nuclei may occa- 
sionally occur within one giant spore. 

In order to learn something of sex segregation Shear and Dodge 




always have eight nuclei, the asco- 
spores may be uninucleate, binucleate, 
trinucleate, or quadrinucleate. 



322 



GEXET1CS OF FUNGI 



(1927) and Dodge (1927, 1928) isolated each of the eight asco- 
spores of N. crassa and found that four are (-)-) and the other 
four ( — ). This discovery left unanswered the question of when 
segregation of sex factors occurs. Manifestly it might be possible 




Sterile 



Perithecia 



Sterile 



Perithecia 



_-- r- Sterile 



Perithecia 



^.*? Sterile 



Perithecia 



Fig. 52. Schematic representation of a fivc-spored ascus of Pleurage anserina. 
The small spores are of opposite sex, the large spores of both sexes. If 
planted on agar plates, the mycelium of each bears both ascogonia and 
spermatia. The conditions of fertility and sterility are indicated by the 

matings in each culture. (After Ames.) 

to determine this question if each of the ascospores was isolated 
and it were known what position within the ascus each occupies. 
Colonies from each could then be mated reciprocally with each 
of the others. Accordingly Wilcox (1928) employed N. sito- 
phila in such experiments, finding that (-f-) and ( — ) ascospores 
alternate in pairs in the series of eight. This discovery indicates 
that the sex factors are segregated at the second division of the 
fused nucleus of the primary ascus. 



HOMOTHALL1SM AND HETEROTHALLISM 323 

Another type of evidence has been provided by Dodge (1927) 
from cytological study of N. tetrasperma. In this species the 
spindle of the first nuclear division is longitudinal with respect 
to the ascus, and the two daughter nuclei come to lie one above 
the other in the ascus. At the second division two types of posi- 
tion and orientation of the spindle may occur. The spindles may 
lie approximately parallel, perhaps just slightly oblique to the 
long axis, or else are aqain longitudinal. The spindles of the 
third division are nearly transversely oriented, bringing non-sister 
nuclei into symmetrical arrangement. On delimitation of the 
spores two non-sister nuclei are included in each ascospore, 
whether segregation takes place in the first, second, or third 
division. 

A somewhat different explanation may account for the situation 
in PI enrage cms er in a [Dowding (1931)]. She found that the 
paired dwarf spores are always of opposite sex. This discovery, 
together with the fact that normal spores are always bisexual, 
indicates that the ( + ) and (-) nuclei are arranged alternately 
at time of spore formation. This alternate arrangement might be 
taken as prima facie evidence that sex segregation occurs at the 
third division. Dowding indicates, however, that the ascus 
is so wide that there is opportunity for the nuclei or even the 
young spores to slip by each other, so that the final arrangement 
f (_j_) a nd ( — ) nuclei could permit segregation of sex factors 
at any of the divisions. 

Lindegren ( 1929) found that the ratio of first-division to second- 
division segregation f sex in Nenrospora crassa is 8:15. Later 

CO L 

(1936, 1939) he determined that the gene for sex is linked with 
other factors and, by determining crossing-over percentages, was 
able, for the first time with fungi, to construct chromosome maps. 
These data provide an explanation of the mechanism involved 
and appear to prove that the chromosomes disjoin at the first divi- 
sion and that the factors are segregated at the second division. 
Lindegren also emphasizes that pure lines of fungi must be ob- 
tained by inbreeding as stock for genetical studies. Such stocks 
also serve best for experimentation on interspecific hybrids, one 
of which was secured by Dodge (1928) by crossing the eight- 
spored N. sitophila with the four-spored N. tetrasperma. 

Dimock (1939) hybridized strains of Hypomyces ipomoeae 
obtained by isolating single ascospores. From these isolations 



324 



GENETICS OF FUNGI 





o 



o 



o 
o 



G 



Fig. 53. Diagrams to illustrate sex of ascospores of Neurospora tetraspervia, 
conditioned bv whether sex is segregated at the first division of the primary 
ascus nucleus or at the second nuclear division. Above. Segregation at the 
first division, C, resulting in four free nuclei of one sex after the third 
division, and four nuclei of the other sex, E. Two of the four spores 
formed are therefore of one sex, F, and two of the other. The two upper 
ascospores may both be + (G) with the lower pair, or the pairs may occupy 
the reverse positions. Below. Sex not separated in C at the first division 
of the fusion nucleus, but separated in the second division. When asco- 
spores are delimited, each contains part + and — , F, or else sex may not 
segregate at all, and each nucleus is as shown in G. (Adapted from Dodge.) 



HOMOTHALLISM AND HETEROTHALL1SM 325 

he obtained four strain groups, which he designated purple, alba, 
revohtta, and revecta. When he attempted to inbreed bv mating 
within each of these strains, perithecia were not produced except 
in one purple X purple mating. When back-crossed to normal, 
all had low fertility except the alba strains. The evidence, he be- 
lieves, indicates that these variants in H. ipomoeae arise by gene 
mutation. 

Edgerton and his associates (1945) employed, in crosses, strains 
of Glomerella, isolated from Ipomoea, that differed only in that 
some were (-f-) and the others ( — ). In certain of these crosses 
each ascus contained four ascospores of the ( + ) type and four 
of the ( — ) type. In others all ascospores were of the ( — ) type. 
Some ( + ) strains originating from single ascospores segregated 
into two strains, but no explanation of this phenomenon based on 
nuclear constitution has been forthcoming. 

The synthesis of vitamins and amino acids may be gene con- 
trolled, and loss of such synthetic ability has been induced by 
treatment of Neurospora crassa with X-rays and ultraviolet light. 
Tatum (1944) secured approximately 400 mutant strains from 
60,000 single-spore cultures. Among these mutants were strains 
which required each of the B vitamins except folic acid and ribo- 
flavin. Others required most of the amino acids. These muta- 
tions involved only a single gene. One strain was unable to com- 
plete the synthesis when supplied with /^-alanine and pantoyl-lac- 
tone. It required that pantothenic acid be supplied as such from 
an exogenous source [Tatum (1944)]. From the results of such 
differences among strains of N. crassa the question arises of why 
some are able to synthesize their required vitamins and what the 
mechanism is for loss of such ability by other strains. 

Lindegren (1945) in an extended study of cultivated yeast 
demonstrated ( -f- ) and ( — ) races that must be mated to secure 
ascospores. 

In Basidiomycetes. Two investigators, Bensaude (1918) and 
Kniep (1919), working independently, called attention to the 
fact that heterothallism occurs among Hymenomycetes and that 
it is correlated with the presence of long-known mycelial struc- 
tures called clamp connections. Since then a large number of 
other workers have contributed to our knowledge of sexuality 
and genetics of Basidiomycetes, those dealing mostly with Hy- 
menomycetes having come from Buller and his students and those 



326 



GENETICS OF FUNGI 




• 


o 


• 


o 


• 


o 


• 


o 


o 


• 


o 


• 


o 


• 


o 


t 



H 




• 


• o o 


• 


• o o 


o 


o • • 





o • • 





• o • 


o 


• o • 


• 


o • o 


• 


o • o 



H 



Fig. 54. Above. Schematic representation of sex segregation at the first 
nuclear division in Neurospora sitophila. There result two possible arrange- 
ments of the ascospores with regard to sex, as shown in H. Either the four 
spores at the upper end of the ascus are + and the four at the lower end — , 
or else the fours are in reverse position. (After Dodge.) Below. Schematic 
representation of sex segregation at the second nuclear division in Neuro- 
spora sitophila. There result four possible arrangements of ascospores with 

regard to sex, as shown in H. 



HOMOTHALL1SM AND HETEROTHALLISM 



321 



dealing with rusts and smuts from Stakman and his students. It 
should be kept clearly in mind that the Hymenomycetes do not 
possess sexual organs, although they occur in the Uredinales, nor 




oooo oooo 



tttt 

OOOO 



oooo 



tttt 

oooo 
oooo 



OOtt 
• •00 
OttO 

toot 



oooo 
oott 

MOO 

otto 
toot 



oooo 
oott 
ttoo 
otto 
toot 



oo 

ttoo 

otto 

toot 



Fig. 55. Schematic representation of sex segregation at the third nuclear 
division in Neitrospora sitophila. Each of the pair at C, the first division, 
and each of the pair at F, the second division, is H , but at the third divi- 
sion + and - segregate. There result 16 possible arrangements of the 
ascospores with regard to sex potentialities, as shown by the series of rings 



below. 



(After Dodge.) 



do they produce definitive sex cells. Instead sexual functions are 
carried out by paired nuclei. Nevertheless most of the species 
are heterothallic and exhibit a definite sexual process. The basi- 
diospores, whether of a homothallic or a heterothallic species, are 
haploid; that is, their nuclei contain n chromosomes. When two 
haploid mycelia of opposite sex of a heterothallic species grow 



328 GENETICS OF FUNGI 

in contact, hvphal fusions occur, and the mycelia become, in con- 
sequence, dicarvotic (two-nucleate). The nuclei become associ- 
ated in conjugate pairs of (//) + (7;) chromosomes. Then, as the 
dicarvotic mycelium continues to grow, conjugate nuclear divi- 
sions occur, but with each conjugate division a clamp connection 
separates the two pairs of daughter nuclei. Finally a conjugate 
pair is delimited in each basidium. Here they fuse, whereupon 
meiosis occurs, and each resulting haploid nucleus migrates into a 
developing basidiospore. 

Up to this point observations are quite in accord. Kniep (1919, 
1922) found in Schizophyllum commune and Aleurodiscus poly- 
gonius that sometimes two of the tetrad of basidiospores were of 
one sex and two of the other sex, although each species is normally 
quadrisexual, that is, quadripolar or quadripotential. In explana- 
tion he proposed that, when the abnormal situation obtained, dis- 
junction of sex occurred in the first division. The quadrisexual 
situation he explained by assuming that sex is determined by two 
pairs of allelomorphic factors, which segregate independently of 
each other during the second division. 

In Coprimis rostmpiamis Newton (1926) found only two kinds 
of spores in each basidium, two (A) spores and two (a) spores, in 
which case sex is determined by one set of factors. In C. lagopus 
she (1926) found, however, as had Kniep, that sex is determined 
by two pairs of linked factors, so that the nucleus of the primary 
basidium has the constitution AaBb. The basidiospores then can 
be (1 ) AB, Ab, aB, and ab\ or (2) two AB and two ab\ or (3) two 
Ab and two aB. Similar results have been obtained by others with 
Hypholoma fascicular -e and Colly bia velutipes. Newton analyzed 
42 tetrads, 25 of which were of the first of the 3 types, 9 of the 
second, and 8 of the third. 

Brunswik (1924) analyzed 93 tetrads of Coprimis fimetarius 
(lagopus) with these findings: 37 gave all four types of spores AB, 
Ab, aB and ab\ 29 gave the two types AB and ab; 27 gave the two 
types Ab and aB. 

These data, together with those of other observers [Buller 
(1931)], show that sexuality is both bipolar and tetrapolar among 
Basidiomvcetes. The mechanism of these patterns of behavior, 
as Dodge ( 1940) indicates, is readily explainable if we assume that 
the genotypes of the parental nuclei in the matings made by New- 
ton and by Brunswik were either AB X ab or Ab X aB. This 



HOMOTHALL1SM AND HETEROTHALLISM 32<s 

assumption would account for the presence of two types of bi- 
polar basidia in equal proportions, if reduction (segregation) 
occurred in the first division and there was random segregation 
without genetic linkage. With such genotypes a simple crossing- 
over during meiosis would account for the tetrapolar basidia. 

Further light on this problem was shed by the studies of Sass 
(1929). He found four-spored and two-spored forms of each of 
the three species Coprinus ephemeras, Nancoria semiorbiciilatus, 
and Galera tenera. The two-spored form of each is normally 
homothallic. The four-spored forms are heterothallic and bi- 
sexual, and sex is determined by one pair of Mendelian factors. 

Pamis stiptictis from Europe is non-luminous, but the same spe- 
cies from North America is luminous. Studies by Macrae (1942) 
of both European and North American strains of this fungus show 
that each strain is heterothallic and tetrapolar. When she crossed 
a luminous with a non-luminous one, the haploid mycelium of the 
Fi generation was luminescent. Luminosity is therefore dominant 
and is governed by a single pair of factors. 

hi Ustilaginales. Some of the more important contributions to 
the genetics of smut fungi are those of Stakman and Christiansen 
(1927), Christiansen (1929), Hanna (1929), Dickinson (1931), 
Flor (1932), Allison (1937), Kernkamp (1939), and Schmitt 
(1940). The smuts constitute a group of destructive plant para- 
sites which, in regard to their sexual process, resemble the other 
Basidiomycetes generally in that thev lack sexual organs and 
definitive gametes. Their most distinctive feature, the mature 
chlamydospore or smut spore, is uninculeate. Its nucleus is a 2n 
structure. At germination meiosis occurs, and each haploid nu- 
cleus finds its way into a basidiospore or sporidium. According 
to Hanna (1929), infection of maize by Ustilago zeae is accom- 
plished by haploid mycelia, and the chance meeting of two hap- 
loid mycelia of opposite sex within the host tissues is followed by 
hyphal fusions, whereupon the mycelial cells are dicaryotic. Pre- 
viously Stakman and Christiansen (1927) had failed to obtain 
fusions in artificial cultures between strains of opposite sex but 
had found hyphal fusions and clamp connections in hyphae within 
the maize tissues. Moreover, infections from monosporidial cul- 
tures failed to result in the production of smut galls and of 
chlamydospores. When dicaryotic mycelium eventually becomes 



330 



GENETICS OF FUNGI 



transformed into young chlamydospores, the conjugate nuclear 
condition still obtains, and actual fusion takes place only within 
the maturing chlamydospore. 

Stakman and Christiansen (1927), by isolating the individual 
sporidia, were able to show that U. zeae is heterothallic, and by 



Ustilago hordei 




<b § ^ 



XD CIX 



Ustilago medians 



W (3) W 





F, { Ss - I + . 



^XE) ®C± 



+ 





(§> WZ&. 



F *0000 0000 Fz 




Fig. 56. Schematic representation of hybridization of two species of smuts, 
one smooth-walled and one rough-walled. P represents parents. The sex 
factor, + or — , segregates independently of the chlamydospore-wall char- 
acter. 5 in sporidia represents spiny walls; s in sporidia represents smooth 
walls with S dominant. In the Fi generation all spore walls are spiny. In 
the F-2 generation the ratio of spinv-walled spores to smooth-walled spores 
is 4:0, 3:1, and 2:2, if all possible combinations are made. 

the same techniques Flor (1932) showed that this situation exists 
also in Tilletia tritici and T. levis. 

Working with oat smut, Ustilago levis, Dickinson (1931), con- 
sidered that the two pairs of factors Aa and Bb, representing sex 
and color, were additive in their effect, AB causing brown color, 
ab causing cream color, and either Ab or aB causing velknv color. 
He isolated the four sporidia of known position. It was apparent 
that the haploid parental mating was AB X ab, that is brown X 
cream. Out of this mating came tetrads of sporidia of three 



HOMOTHALLISM AND HETEROTHALL1SM 331 

groups in the proportion of: (1) two AB and two ab, (2) two 
Ab and aB, (3) one each of AB, ab y aB, and Ab. Segregation 
would appear therefore to occur in the fashion described bv 
Newton (1926) and Brunswik (1924) in Coprimts lagopus, with 
the linkage and crossing-over mechanisms as interpreted by Dodge 
(1940). These observations bv Dickinson (1931) are further 
substantiated bv the findings of Schmitt (1940) on Ustilago zeae. 
The segregations of factors for colonv color, sex, and tvpe of 
growth (sporidial or mvcelial) occurred in both the first and the 
second divisions. For each of these characters numerical ratios 
of 1:1, 3:1, and 1:3, were found, with a 4:0 segregation for sex 
in one case amoncr several thousand. 

Smuts constitute especially favorable material for genetical 
studies for the reason that all the sporidia from a promvcelium 
can be isolated and can then be propagated in culture. Each spo- 
ridium by budding can be made to form numerous haploid in- 
dividuals that can be mated under controlled conditions. Gene- 
tic studies of smuts have been concerned with colony characters 
and tendency to sector. Other studies have included such factors 
as color of the peridium of sori, pathogenicity, sex compatibility, 
color of the chlamydospores and the nature of their walls, and 
tendency to be myceloid or to form buds [Christiansen and 
Rodenheiser (1940)']. 

Kernkamp (1942) isolated monosporidial lines of U. zeae to 
study the effects of genetic and environmental factors on types 
of colonial growth. Some isolates were entirely sporidial, some 
entirely mycelial, and some intermediate. Strictly sporidial lines 
could not be mated and could not infect maize. The growth 
types of sporidial or of mycelial lines could not be modified by 
changes in concentration of food or by addition of certain vita- 
mins, poisons, amino acids, or other substances. The growth 
type of intermediate lines, however, could be modified for in- 
creased sporidial production by the presence of dextrose or for 
mycelial production by unfavorable growth conditions. 

Stakman and his associates (1943) found that mutation is un- 
believably common in U. zeae. In this smut mutability and con- 
stancy are governed by genetic factors, as has been determined 
from the results of numerous crosses between monosporidial lines 
of opposite sex. Stakman and his associates conclude, "Ustilago 
zeae definitely comprises an indefinite number of biotypes that 



552 



GEXET1CS OF FUNGI 




Fig. 57. Hybrids between monosporidial lines of Ustllago zeae. Progeny 

of the four primary sporidia from each of three chlamydospores. Note 

recombination of characters. (Courtesy of E. C. Stakman.) 



HOMOTHALL1SM AND HETEROTHALLISM 333 

• 

differ either widely or slightly in every observable character or 
combination of characters. New ones are continually being pro- 
duced as a result of mutation and of recombinations resulting 
from interbiotypic hybridization." 

In Uredinales. The Uredinales, or rust fungi, are a group of 
obligate parasites of enormous economic importance, because 
many of them attack crop plants. Although many studies, from 
which have come a number of fundamentals concepts, have been 
concerned with their sexuality, an understanding of this subject 
was first established by the investigations of Craigie (1927, 1927a, 
1928). He showed that the pycnia are functional structures and 
that the pycniospores are essential in diploidization. 

Undoubtedly many rusts are heterothallic, for Craigie's studies 
have shown that such is the situation in Pucclma graminis, P. 
helianthi, P. coronata, P. pringsheimiana, and Gymno sporangium 
sp. At germination of the teliospore, whose mature cells are uni- 
nucleate, the nucleus divides meiotically within the promycelium, 
the homolomje of the basidium, and the four resultant nuclei are 
haploid. Each migrates through a sterigma into the basidiospore 
that arises at the apex of a sterigma. Craigie found that these 
basidiospores are of either (-+-) or ( — ) potentialities. If mono- 
sporidial inoculations are made, pycnia containing pycniospores 
are developed. However, aecia never develop in association with 
such pycnia unless pycniospores from a pycnium of opposite sex 
are applied. In nature this interchange of pycniospores is accom- 
plished either by insects attracted to the sugary exudate in which 
pycniospores are embedded or by water. Buller (1940) has 
shown that flexuous hyphae extend from the orifices of the pycnia 
and that pycniospores fuse with these flexuous hyphae. The 
pycniospores are thus spermatia, and the flexuous hyphae are re- 
ceptive surfaces comparable to trichogynes. Buller (1940) has 
observed flexuous hyphae in 21 species belonging in Coleospo- 
rium, Cronartium, Gymnoconia, Gymnosporangium, Melampsora, 
Alelampsorella, Alilesia, Phragmidium, Pucciniastrum, Puccinia, 
and Uromyces. 

If spermatization is accomplished, aecia bearing dicaryotic 
aeciospores with conjugate, (n) -f (72), nuclei are developed. 

In full-cycled rusts, not only the aeciospores but also the myce- 
lia arising when they germinate, the urediniospores and the my- 
celia arising from their germination, and the young teliospores 



334 GENETICS OF FUNGI 

are dicarvotic. The thousands of nuclear divisions that occur 
meanwhile are conjugate, and the complete cycle from the mono- 
caryotic to the dicarvotic condition and back again to the mono- 
caryotic may require a period of, at one extreme, only a few days 
to, at the other, 5 to 7 years, as in Cronartium ribicola. 

Inability to grow rusts on artificial media has no doubt inter- 
fered to some extent with genetical studies of them. Nevertheless 
such studies have been energetically pursued, especially by Stak- 
man and his associates at the Minnesota Experiment Station and 
by a ^roup at tne Dominion Cereal Rust Laboratory in Canada. 
The presence of barberry bushes in areas devoted to cereal crops 
permits the development in nature of new races or strains of rusts 
by hybridization. There is abundant evidence that such new- 
hybrid rusts are continuously beino- developed and that their 
presence accounts for the breaking down of resistance in cereal 
varieties that possess a high degree of resistance to old strains of 
rusts. The production of new strains of rusts tends to nullify the 
laborious efforts of plant breeders to develop resistant varieties of 
cereals and to control cereal rusts bv use of these resistant varieties. 

Newton and Johnson (1940) crossed Puccini a graminis tritici 
and P. graminis avenae, finding them completely interfertile. 
These workers were concerned primarily with pathogenic po- 
tentialities. Crosses within the avenae variety showed that the 
less virulent pustule type is dominant over the more virulent type, 
whereas within the tritici variety the less virulent type is dominant 
in some crosses but recessive in others. In reciprocal crosses be- 
tween these two varieties the maternal cytoplasm appears to exer- 
cise the controlling influence. A cross between a variant whose 
urediniospores were orange and a variant whose urediniospores 
were grayish-brown gave all normal red color in the Fi genera- 
tion. When selfed, four different colors-red, orange, grayish- 
brown, and white— appeared in the F 2 generation, with a distribu- 
tion ratio suggesting 9:3:3:1, respectively. 

In experiments with physiologic races of P. graminis tritici, 
Johnson and Newton (1940) found that absence of pustules, 
which they called O type, was dominant over large pustules (4 
type) on Kanred wheat. When this hybrid was selfed, the O type 
was approximately 3 times as abundant in the F 2 generation as 
the 4 type. In this instance pathogenic behavior is governed by 



DOMINANCE AND LETHAL FACTORS 335 

a single factor pair. On Mindum wheat the 4 type was dominant 
over very small pustules ( 1 type) with a 3 : 1 ratio in the F 2 
generation. On the other hand, when the emmer variety, Vernal, 
served as the host, the 1 tvpe was 15 times as abundant as the 4 
tvpe in the F 2 generation. Pathogenic behavior on Vernal emmer 
appears therefore to be governed by duplicate factors. Johnson 
and Newton conclude that the genes in the binucleate uredinio- 
spores function as if they occurred in a single diploid nucleus. 

DOMINANCE AND LETHAL FACTORS 

The existence of dominance and recessiveness among fungi 
would appear to have been amply demonstrated in the studies de- 
scribed, which are representative of experiments among the larger 
groupings of fungi. This Mendelian principle can be demon- 
strated for interested students, however, by hybridizing an eight- 
spored Neurospora with a four-spored Neurospora. All the F x 
progeny will be found to be eight-spored. Similarly, when a 
rough-spored race of smut is crossed with a smooth-spored one, 
all the Fi are rough. 

Lethal factors exist among fungi, just as they are known to 
occur among seed plants. Dodge (1934) reported the results of 
studies on N. tetrasperma, known to possess bisexual ascospores. ■ 
After treatment with X-rays a strain appeared that was practi- 
cally self -sterile, as manifest by ascus abortion. When this strain 
was mated with a normal one, the F x generation gave asci that 
formed spores normally. Further results show r ed that at meiosis 
the lethal factor was segregated, so that each bisexual ascospore 
contained a normal nucleus and one with the lethal factor. This 
situation insured the transmission from generation to generation 
of the lethal factor. 

More recently Fischer (1940) noted a haplo-lethal factor in five 
collections of Ustilago bullata on species of Agropyron, Bromus, 
Elymus, and Festuca. When he germinated the chlamydospores 
and isolated the basidiospores in monosporidial cultures, he found 
that approximately half yielded typical colonies, and in the re- 
mainder the basidiospores budded a few times and then underwent 
complete lysis. Fischer was able to show that the lethal factor 
was sex-linked in four of the five collections. 



336 GENETICS OF FUNGI 



RESUME 

Mendelian principles apply in genetical studies of fungi, just 
as in similar studies involving other living organisms. By means 
of hybridization evidence has been obtained of dominance and re- 
cessiveness, of segregation in predictable numerical ratios, of sex 
linkage, of lethal factors, of mutations, of crossing-over at reduc- 
tion division, and of other genetic features. As Dodge (1940) has 
aptly said, "The fungi in their reproduction and inheritance fol- 
low exactly the same laws that govern these activities in higher 
plants and animals." The practical consideration to be kept in 
mind, a conclusion that follows from these facts, is that new races 
of fungi are continually arising by hybridization. This fact must 
be taken into account in breeding plants for disease resistance. 

LITERATURE CITED 

Allison, C. C, "Studies on the genetics of smuts of barley and oats in rela- 
tion to pathogenicity," Minn. Agr. Expt. Sta. Tech. Bull., 119: 1-34, 1937. 

Ames, L. A I., "Hermaphroditism involving self-sterility and cross-fertility 
in the ascomvcete Plenrage anserina," My col., 26: 392-414, 1934. 

Bensaude, AL, "Recherches sur le cycle evolutif et la sexualite chez la Basi- 
diomvcetes," These (Paris), Nemours. 153 pp. 1918. 

Blakeslee, A. F., "Sexual reproduction in the Alucorineae," Proc. Am. Acad. 
Sci., 40:205-319, 1904. 

Brunswik, H., "Untersuchungen iiber die Geschlechts- und Kern-verhalt- 
nisse bei der Hvmenomvzetengattung Coprinus," Bot. Abhandlung. 
herausg. K. von Goebel, 5: 1-152, 1924. 

Buller, A. H. R., Researches on Fungi, Vol. IV. 329 pp. Longmans, Green 
& Co., London. 1931. 
"The flexuous hvphae of Puccinia graminis and of other rust fungi," Proc. 
3rd Intern. Congr. Microbiol., p. 534, 1930. 

Burgeff, H., "Variabilitat, Vererbung und mutation bei Phycomyces blake- 
slee amis BgrT.," Z. Indukt. Abstain. Vererb., 49: 26-94, 1928. 

Christiansen, J. J., "Alutation and hvbridization in Ustilago zeae. Part II. 
Hybridization," Minn. A^r. Expt. Sta. Tech. Bull., 6): 85-108, 1929. 

Christiansen, J. J., and H. A. Rodenheiser, "Phvsiologic specialization and 
genetics of the smut fungi," Bot an. Rev., 6: 389-425, 1940. 

Crak;ie, J. H., "Experiments on sex in rust fungi," Nature, 120: 116-117, 1927. 
"Discovery of the function of pycnia and aecia in certain rust fungi," 

Nature, 120:765-767, 1927a. 
"On the occurrence of pvcnia and aecia in certain rust fungi," Phyto- 
pathology, 18: 1005-1015, 1928. 



LITERATURE CITED 337 

Dickinson, S., "Experiments on the physiology and genetics of the smut 

fungi. Cultural characters. II. The effect of certain external conditions 

o 

on their segregation," Proc. Roy. Soc. B, 108: 395-423, 1931. 
Dimock, A. W., "Studies on ascospore variants of Hypomyces ipomoeae" 

My col., 31: 709-727, 1939. 
Dodge, B. O., "Nuclear phenomena associated with heterothallism and honio- 
thallism in the ascomycete Neurospora," /. Agr. Research, 35: 289-305, 
1927. 
"Production of fertile hybrids in the ascomycete Neurospora," /. Agr. 

Research, 36: 1-14, 1928. 
"Breeding albinistic strains of the Monilia bread mold," My col., 22: 9-38, 

1930. 
"Inheritance of the albinistic non-conidial characters in inter-specific hy- 
brids in Neurospora," My col., 23: 1-50, 1931. 
"A lethal for ascus abortion in Neurospora," My col., 26: 360-376, 1934. 
"Second-division segregation and crossing-over in the fungi," Bull. Torrey 
Botan. Club, 67:467-476, 1940. 
Dowding, E. S., "The sexuality of the normal, giant, and dwarf spores of 

Fleurage anserina (Ces.) Kuntze," Ann. Botany, 45: 1-15, 1931. 
Edgerton, C. W., S. J. P. Chilton, and G. B. Lucas, "Genetics of Glome - 
rella. II. Fertilization between strains," Am. J. Botany, 52:115-118, 
1945. 
Fischer, G. W., "Two cases of haplo-lethal deficiency in Ustilago bullata 

operative against saprophytism," My col., 32: 275-289, 1940. 
Flor, H. H., "Heterothallism and hvbridization in Tilletia tritici and T. 

levis," J. Agr. Research, 44:49-5$, 1932. 
Hanna, W. F., "The problem of sex in Coprinus lagopus," Ann. Botany, 
39:431-457, 1925. 
"Studies in the phvsiologv and cytology of Ustilago zeae and Sorosporhnn 
reilianum" Phytopathology, 72:415-442, 1929. 
Johnson, T., and M. Newton, "Mendelian inheritance of certain pathogenic 
characters of Puccinia graminis tritici" Can. J. Research, 18: 599-611, 
1940. 
Kernkamp, AI. F., "Genetic and environmental factors affecting growth tvpes 
of Ustilago zeae," Phytopathology, 29:473-484, 1939. 
"The relative effect of environmental and genetic factors on growth 
types of Ustilago zeae," Phytopathology, 32: 554-567, 1942. 
Kniep, H., "Uber morphologische und phvsiologische Geschlechtsdifferen- 
zierung," Verhandl. physik.-med. Ges. Wurzburg, 46: 1-18, 1919. 
"Uber Schlechtsbestimmung und Reduktionsteilung," Verhandl. physik.- 
med. Ges. Wurzburg, 47: 1-29, 1922. 
"Verehrbungserscheinungen bei Pilzen," Bibliogr. Genet., 5: 371-478, 1929. 
Lindegren, C. C, "The genetics of Neurospora. II. The segregation of sex 
factors in asci of N. crassa, N. sitophila, and N. tetrasperma," Bull. 
Torrey Botan. Club, 59: 119-138, 1929; III, 60: 133-154, 1933. 
"A six-point map of the sex chromosome of Neurospora crassa," J. 
Genetics, 32: 234-256, 1936. 



338 GENETICS OF FUNGI 

Lindegren, C. C, "Non-random crossing-over in the second chromosome 

of Neurospora crassa," Genetics, 24: 1-7, 1939. 
"Yeast o-enetics: life cycles, hybridization, vitamin synthesis, and adaptive 

enzymes," Bact. Rev., 9: 111-170, 1945. 
Macrae, Ruth, "Interfertility studies and inheritance of luminosity in 

Panus stipticm," Can. J. Research, 20:411-434, 1942. 
Newton, D. E., "Bisexual ity of individual strains of Coprimts rostrupianus" 

Ann. Botany, 40: 105-128, 1926. 
"The distribution of spores of diverse sex on the hymenium of Coprimts 

lagopus," Ann. Botany, 40:891-917, 1926a. 
Newton, D. E., and T. Johnson, "Variation and hybridization in Puccinia 

grawinis," Proc. 3rd Intern. Congr. Microbiol., p. 544, 1940. 
Sass, J. E., "The cytological basis for homothallism and heterothallism in the 

Agaricaceae," Am. J. Botany, 76:663-701, 1929. 
Schmitt, C. G., "Cultural and genetic studies on Ustilago zeae," Phyto- 
pathology, 30:381-398, 1940." 
Shear, C. L., and B. O. Dodge, "Red bread-mold fungi of the Monilia sito- 

phila group, life histories and heterothallism," /. Agr. Research, 

34: 1019-1042, 1927. 
Stakman, E. C, and J. J. Christiansen, "Heterothallism in Ustilago zeae," 

Phytopathology, 77:827-834, 1927. 
Stakman, E. C, M. F. Kernkamp, T. H. King, and W. J. Martin, "Genetic 

factors for mutability and mutant characters in Ustilago zeae," Am. J. 

Botany, 30: 37-48, 1943. 
Tatum, E. L., "Nutrition, genetics, and 'Neurospora,' " Stanford Med. Bull., 

2: 1-4, 1944. 
"Biochemistry of fungi," Ann. Review Biochem., 73:667-704, 1944a. 
Wilcox, M. S., "The sexuality and arrangement of the spores in the ascus 

of Neurospora sitophila," Mycol., 20: 3-16, 1928. 
Wolf, F. A., "Spore formation in Podospora anserina (Rabh.) Winter," 

^7272. Mycol., 70:60-64, 1912. 



Chapter 15 

POISONOUS AND EDIBLE FUNGI 

When mention is made of poisonous fungi, most persons im- 
mediately think of toadstools, regarding them as comprising all 
the poisonous forms. These persons separate toadstools (Todes 
Stuhl) from mushrooms, placing all poisonous species in the toad- 
stool group and all edible ones in the mushroom group. Such a 
distinction is unwarranted and mycologically meaningless. In 
the present account, which is by no means comprehensive, con- 
sideration will be given to certain fleshy fungi and also to other 
well-known species that are poisonous, especially to humans. 

POISONOUS FLESHY FUNGI 

Fleshy fungi have long been employed for food, and it has as 
long been known that some species are extremely poisonous. . 
Thousands of species are edible, however, whereas relatively few 
are toxic to man. Sickness and fatalities from eating mushrooms 
can be attributed only to lack of knowledge. Anyone can learn 
to recognize the poisonous species, and it cannot be too strongly 
emphasized that such knowledge constitutes the only safe guide 
to determining which species are to be avoided. A beginner can 
soon learn to recognize a few of the choicest and most common 
edible species and can confine his collections for the table to these 
species, which include the common mushroom, Fsalliota campes- 
tris, the shaggy mane, Coprinas comatiis, the common ink cap, 
C. atr anient arms, the glistening ink cap, C. micaceus, the oyster 
mushroom, Pleurotus ostreatus, the parasol mushroom, Lepiota 
procera, the honey agaric, Armillaria mellea, the velvet-stemmed 
mushroom, Colly bia vehitipes, the morel, Morchella esculent a, all 
coral fungi, and all puffballs that are pure white in section. While 
gathering these, the student will gradually become acquainted 

with the poisonous ones. 

339 



340 



POISONOUS AND EDIBLE FUNGI 




Fig. 58. Sonic common poisonous fungi. A. Amanita phalloides. B. Clito- 
cybe Mud ens. C. Pavaeoiits retirugis. D. Amanita caesarea. E. Morchella 

escitlenta. 



POISONOUS FLESHY FUNGI 341 

A compendium bv Dujarrac de la Riviere and Heim (1938) 
constitutes an invaluable source of information about poisonous 
fun^i. The earlier series of researches on poisonous mushrooms 
and their toxic properties by Ford and his associates (1906, 1906a, 
1907, 1907a, 1911, 1913, 1914, and 1926) should also be read by 
all mycologists and laymen who collect fungi for food. 

It appears that in ancient times the Babylonians, Romans, and 
Greeks, both those of hig-h estate and of the lower classes, em- 
ployed mushrooms in season as delicacies and as daily food. The 
fact that deaths from poisoning occurred among their notables 
may be regarded as evidence that the ancients were not able to 
distinguish between noxious and harmless species. History re- 
cords that such outstanding civic and political leaders as Pope 
Clement VII, Emperor Jovian, Emperor Charles VI, Emperor 
Claudius (his wife Agrippina is said to have added poison to his 
dish of boleti), the widow of Czar Alexis, and the wife, two sons, 
and a daughter of the Greek poet Euripides were among the 
victims of poisonous mushrooms. Galen cautioned his patients 
against using mushrooms, stating, "Few of them are good to be 
eaten, and most of them do suffocate and strangle the eater," 
although his "Amanitae" almost certainly were Psalliota campes- 
tris. The renowned Greek physician Dioscorides states, "Fungi 
have a two-fold difference, for they are either good for food, or 
are poisonous; their poisonous nature depends on various causes, 
for such fungi grow amongst rusty nails, or rotten rags, or near 
serpents' holes, or on trees producing noxious fruits." 

Some appreciation of the number of fatalities from mushroom 
poisoning can be gained from Ford and Clark's (1914) report 
assembled from various sources. In the Les Vosges area of south- 
western France the annual death toll is about 100, and in Japan 
480 deaths occurred in 8 years. Sartory [Ford and Clark (1914), 
p. 169] lists 153 fatal cases in a 2-week period in 1912 in France. 
In 1911 there were 22 deaths in\he vicinity of New York City 
within a 10-dav period. Throughout the world a large number 
of fatalities undoubtedly are due to poisonous mushrooms every 
year. A vast majority is caused by the "death angel," Amanita 
phalloides. Rolfe (1928, p. 232) has expressed the opinion, "This 
inglorious trio [Amanita phalloides, A. virosa, and A. verna] is 
responsible for fully ninety per cent of the deaths from fungus 
poisoning- 



•>■> 



542 



POISONOUS AND EDIBLE FUNGI 



Classification of fleshy fungi according to toxic effect. 
Toxicologists have classified poisonous fleshy fungi according to 
the effects that thev produce upon the human system [Ford 
(1926)]. On this basis they may be divided into the following 

groups: 

1. Fungi whose toxicity is first manifest 6 to 15 hours after in- 
gestion and that cause degeneration of the nervous tissues and 
glandular parenchymatous tissues, especially the liver. The clini- 




Conic 



Infundibuliform 



Umbonate Campanulate 



Fig. 59. Types of pilei of agarics in diagram, indicating variation in form 

used in ireneric determinations. 

cal symptoms consist of sudden seizure by severe abdominal 
pains, accompanied by vomiting and diarrhoea. Abundant blood 
and mucus appears in the vomitus and stools. The victim loses 
weight rapidly, passes into a coma after 2 to 5 days, and succumbs. 
Recovery is very rare. Poisoning of this type is caused by inges- 
tion of Amanita phalloides, A. virosa, and A. verna, more rarely 
by Pholiota autumnalis and Hygrophoms conic us. 

2. Funoi whose poisonous effects appear soon after ingestion 
and that act chiefly by stimulating and then paralyzing the cen- 
tral nervous system. Poisoning is manifest by profuse perspira- 
tion and salivation, retching, and diarrhoea, accompanied by de- 
lirium, hallucinations, and convulsions. The patient may die from 
paralysis of respiration. This complex is caused mainly by Ama- 
nita muse aria. Similar clinical symptoms may be induced by A. 
pant her in a, Inocybe infelix, I. infida, and Clitocybe illudens. In 
Siberia decoctions of dried A. muse aria are sometimes used to 



POISONOUS FLESHY FUNGI 343 

induce orgies of intoxication somewhat similar to those from 
hashish. 

3. Fungi whose irritant principles act on the mucous mem- 
branes of the gastrointestinal tract soon after the fungi are eaten. 
The clinical symptoms, consisting of griping pains in the stomach, 
dizziness, nausea, vomiting, and diarrhoea, subside rather abruptly, 
and recovery proceeds rapidly. This type of poisoning is induced 
by Riissula emetica, Lactariits torminosus, Lepiota morgani, Ento- 
lovia lividum, Boletus sat anus, B. mineato-olivaceus, and some of 
the species of Amanita. 

4. Fungi that contain hemolytic principles. The symptoms are 
abdominal distress, dizziness, and vomiting. The vomitus contains 
blood. The victim may have convulsions and may pass into pro- 
found sleep. During convalescence mild jaundice develops. The 
ingestion of Helvetia esculenta commonly causes this type of 
poisoning, which may also be induced by other species of Helvella 
and by Amanita rubescens. 

5. Fungi that stimulate the central nervous system in a manner 
somewhat like alcoholic intoxicants. The victim feels greatly ex- 
hilarated and laughs immoderately. His gait is staggering, and he 
has the feeling that he is walking on air. This type of intoxica- 
tion lasts for 24 to 48 hours after the ingestion of Fanaeolus papi- 
lionaceus or P. campanulatus. 

Identification of poisonous mushrooms. As has previously 
been stated, a person can learn to recognize the poisonous fleshy 
mushrooms if he applies himself to the task. In order to do so, 
he must become familiar with the salient structural features that 
are employed by the mycologist in identifying mushrooms. Rela- 
tively few species will be found to be poisonous. Each of the 
more common poisonous species will be briefly characterized in 
the following account. 

Amanita phalloides. The fructifications of this species vary in 
color, being white, green, olive, amber, and rarely yellowish. 
They grow singly and are 5 to 7 in. tall. The pileus or cap is 
white, viscid, and convex, with or without scales at the surface. 
The stipe or stem is smooth and of the same color as the pileus. 
The gills are white and free from the stipe. The annulus or veil 
breaks at the margin of the cap and clings skirt-like near the top 
of the stipe. The volva or cup is variable because of the manner 



344 



POISONOUS AND EDIBLE FUNGI 



in which it ruptures. It may be cup-like or appear as a bulbous 
expansion at the base of the stipe. Since the volva may be deeply 
buried in the leaf mold, care must be exercised while collecting 
to remove the entire fructification. This is of special importance, 
because A. phalloides mav be mistaken for Lepiota naucina, a 
common edible species. 





Free 



Adnate 




Adnexed 

Fig. 60. Diagrams illustrating types of attachment of gills to the stipe, a 
character used in determination of genera among agarics. 

Amanita mm c aria. This fungus, called the fly agaric because 
it has been used as a fly poison, grows in fields or open woods. 
The striking yellow to orange and even red color of the fructifi- 
cations characterizes this species, which is 4 to 6 in. tall. The 
pileus is 3 to 5 in. broad. Prominent warty scales cover the 
pileus. The gills are white. The veil remains around the stem as 
a large, membranous, pendent collar. The base of the stipe is 

bulbous. 

Other white species of Amanita. Several other typically white 
species of Amanita, including A. verna, A. virosa, and A. spreta, 
are as deadly poisonous as is A phalloides. All possess a volva, 
an annulus, and white gills and have scales on the expanded pileus. 
The possession of these characters positively identifies the fungus 
as a species of Amanita. Since nearly all species of Amanita, 



POISONOUS FLESHY FUNGI 



345 



whether white or some color, are known to be poisonous, it is pru- 
dent sedulously to avoid all of them. 

Lepiota morgani. The genus Lepiota lacks the volva but in 
other features looks like Amanita. Lepiota morgani grows in 
fields and open woods, especially in the Ohio Valley, and is not 
uncommon in the vicinity of Durham, North Carolina. The fruc- 




Fig. 61. Shapes of spores in outline. A. Amanita phalloides. B. Amanita 
verna. C. Lepiota morgani. D. Amanita muscaria. E. Lepiota procera. 
F. Clitocybe illudens. G. Rnssida emetica. H. Psalliota campestris. I. 
Panaeolus retimgis. J. Amanitopsis strangulata. K. Entoloma sp. L. Bole- 

tinus sp. M. Hebeloma crnstidtf orme . 

tifications are 4 to 8 in. tall, and the convex-to-flat cap may be 
equally broad. The stipe has a club-shaped base. The color 
varies from grayish white to buff or pale amber. Irregular scales 
or patches occur on the cap. The annulus is large, thick, and 
movable. The gills are free, rather broad, ventricose, and white 
at first, changing to bright green and then to dull green. The 
color is so striking as to prevent this species from being mistaken 
for any edible one. 

Clitocybe illudens. The fructifications of this fungus occur in 
dense clusters, each being 3 to 7 in. tall and 2 to 5 in. broad. They 
are luminescent, hence the common name jack-o'-lantern. The 



346 POISONOUS AND EDIBLE FUNGI 

color ranges from saffron yellow to orange. The caps are plane 
to centrally depressed. The gills are decurrent, of the same color 
as the cap, and narrowed at each end. The stipes are firm, smooth, 
and solid, tend to be excentric, and are darker near the base. 

Clitocybe dealbata yar. sudorifica. Clusters of fructifications of 
this fungus, also called C. sudorifica, occur on lawns or on other 
grassy sites. They are % to 1 Y s in. tall, and the caps are % to 1 % 
in. broad. The color throughout is grayish white. The caps are 
plane, depressed, or umbilicate, and the margin splits irregularly. 
The stipes have a spongy center. The gills are thin, narrow, and 
adnate or slightly decurrent. A few cases of poisoning have also 
been attributed to C. vwrbrfera and C. nebulosus. 

Lactarius torrmnosus. When the fructifications of Lactarius 
are broken, a milky or colored juice exudes. This characteristic 
serves to distinguish Lactarius from all other ^ill-bearingr fungi. 
The flesh is always very brittle. Lactarius torminosus occurs on 
the ground in woods in late summer. The fructifications occur 
singly. They are 2 to 4 in. tall with a pileus of approximately the 
same breadth. They are convex and depressed in the center. The 
gills are crowded, thin, and whitish. The stipe is cylindrical, even, 
and hollow. The milk is white, unchangeable, and acrid. The 
pilei have an uneven mixture of pink and ochre colors and are 
very hairy at the margins. 

Russula emetica. Species of Russula are at once separated from 
Lactarius by the absence of milky juice, although they resemble 
Lactarius in all other respects. Russula emetica fruits during sum- 
mer and autumn. It is a very beautiful and very fragile species. 
The fructifications are 2 to 4 in. tall, and the cap is equally broad. 
They are pink when young and darker red when older. The 
stipes are stout and spongy within. The caps are plane to de- 
pressed and are furrowed near the margin. The gills are free, 
broad, not crowded, and white. 

Pholiota autuiunalis. Species of Pholiota are ochre-spored. 
They possess an annulus, and the gills are adnate. Pholiota auturn- 
nalis fruits on decaying wood. The fructifications are clustered 
and are 1 to 2 in. tall. The caps are convex, cinnamon-rufous to 
dingy yellow, and striate on the margin. The stipes are slender, 
fibrillose, hollow, and somewhat paler than the cap. 

hwcybe bifida. Ochre-spored species with a fibrillose uni- 
versal veil are included in Inocybe. hwcybe infida occurs on 



POISONOUS FLESHY FUNGI 



341 



mossy ground during autumn. The fructifications are 1 to 2 in. 
tall. The caps are % to 1 in. broad, campanulate, and whitish 
with a brownish umbo. The gills are annexed, close, narrow, and 
cinnamon-colored. The stipes are slender, hollow, and white. 
Inocybe injelix. This species occurs throughout summer among 
mosses or on bare soil. Its size agrees with that of /. bifida. The 
campanulate pilei are floccosely squamulose and grayish brown or 




Fig. 62. Relationship of structural features in Amanita. A. Undifferentiated 
"egg" B. Button stage, in which the gills are differentiating in the upper 
portion. C. Opening of the pileus, showing at one side the ruptured uni- 
versal veil and annulus. D. Expanded pileus, in section, with scales on the 
pileus and volva surrounding the base of the stipe. Both structures are re- 
mains of the universal veil. 

amber. The gills are adnexed, close, broad, ventricose, and rusty. 
The stipes are equal, solid, silky fibrillose, whitish above and 
brownish below. 

Hebeloma criistidijorme. In Hebeloma are placed ochre-spored 
species with adnate gills and with a delicate fibrillose veil that is 
present only on the young caps. All species are unwholesome. 
Hebloma cmstulij orme appears on lawns in autumn. It is 2 to 3 
in. tall, and the caps are equally broad. The caps are convex to 
umbonate, tan-colored, darker over the center, and viscid. The 
gills are adnate but rounded near the stem, and their edge is 
white and irregular. The stipe is stuffed, enlarged below, and 
w T hitish. It is reported that H. fastibile causes the same type of 
symptoms as does Amanita miiscaria. 



348 POISONOUS AND EDIBLE FUNGI 

Entoloma lividum and E. sinuatum. Entoloma is characterized 
by being pink-spored with gills that are adnate to sinuate. All the 
species should be avoided. Entoloma lividum and E. sinuatum 
have been proved to be poisonous. Thev differ mainly in that 
the stipe of E. lividum is solid and of E. sinuatum hollow. They 
grow gregariously in the woods. The fructifications are 3 to 5 in. 
tall. The caps are 2 to 3 in. broad, convex, becoming centrally 
depressed, moist, even, and vellowish white, with a wavy margin 
and sulcate surface. 

Panaeolus papilionaceus and P. retirugis. In Panaeolus are in- 
cluded black-spored agarics that grow on dung or on grassy, 
manured ground. The pilei are thin, with even margins that ex- 
tend beyond the gills. The gills are spotted with brown and black; 
the stipes are long and slender. The pilei of both P. papilionaceus 
and P. retirugis are conic and grayish to smoky, with fragments of 
veil attached around the margin. The centers of the pilei are com- 
monly darker than the margins. The gills are adnate and, as the 
caps expand, tend to separate more and more from the stipe. 

Boletus sat anus, B. luridus, and B. mineato-ol'rcaceus. In Boletus 
are placed fleshv, central-stalked polypores. The caps are con- 
vex, and the pore layer is quite readily separable from the sub- 
stance of the cap. Many discolor immediately on being bruised. 
Some persons maintain that none of the species should be eaten. 
Many are bitter and possess disagreeable odors. Boletus mineato- 
olivaceus possesses caps 2 to 6 in. broad. They are red, becoming 
ochre-red with a«e. The flesh is yellow but instantly becomes 
blackish blue when bruised. 

The caps of B. luridus, about 8 in. across, are dirty olivaceous 
yellow; the flesh is vellowish, becoming blue. The tubes are yel- 
lowish, becoming green. The stipes are approximately 6 in. long 
and yellowish above and blackish at the base. 

Hygrophorus conicus. Hvgrophorus contains the white-spored 
species in which the tissue of the cap is continuous with that of the 
stem. The <nlls are distant, the edge being acute at the margin, 
are gradually thickened toward the stipe, and are characteristically 
waxy, appearing to be sodden. Hygrophorus conicus grows in 
woods in mossy or grassv situations. It has conic pilei, about 2 in. 
broad, fragile, slightly viscid, and red, orange, or yellow, blacken- 
ing when bruised. Gills are close, ventricose, almost free, and 
yellowish. Stipes are yellow, fibrous, equal, and striate. 



POISONOUS FLESHY FUNGI 



349 



Morchella esculenta. The morels are disk fungi that appear in 
late spring in damp situations. Morel fruits possess a distinct cap 
and stalk. The cap varies in shape with the species, being spheri- 
cal, ovate, cylindrical, or conic. A network of ridges and pits 
covers the outer surface. The stipe is stout and irregularly 
wrinkled. All species are buff to light ochre. The fructifications 
of M. esculenta may be as high as 6 in. tall. The cap is oval in 
outline, and the pits are irregularly arranged. , 




Fig. 63. Structural diagrams of Boletus. A. Young unopened pileus in which 
the entire fructification is still enclosed within the universal veil. B. Ex- 
panded pileus, showing the annulus and remnants attached to the rim 
of the pileus. C. Opened, mature pileus with stipe, cap, and pore surface. 

Persons who gather mushrooms should learn to recognize the 
foregoing poisonous species and should sedulously avoid eating 
them. Although the judicious use of such knowledge constitutes 
the best and only safeguard, many other so-called tests to deter- 
mine whether a given form is poisonous may be mentioned. Silver 
spoons or coins are said to turn black when dipped into a dish of 
cooked poisonous mushrooms. Poisonous species are said to peel 
with readiness. Species that are bright colored, that have unde- 
sirable odors, or that have a bitter taste when freshly gathered are 
claimed to be toxic. The reliability of these and similar tests is 
vouched for bv the world-famous authority, "They say.' 1 All 
such tests are without foundation and must be regarded as sheer 
nonsense. 



350 POISONOUS AND EDIBLE FUNGI 

Toxicology. The vast amount of experimentation that has been 
conducted to determine the nature of the toxic principles in 
poisonous fleshy fungi can be appreciated from Ford and Clark's 
report (1914). They indicate that in 1826 Letellier extracted 
from Amanita phalloides a heat-stable substance that he called 
amanitin. Later he found, in addition to this thermostable sub- 
stance that he thought to be a glucosidal alkaloid, a substance 
capable of attacking mucous membranes. In 1877 Ore [Ford and 
Clark (1914), p. f 71 ] ascribed poisoning by A. phalloides to a hy- 
pothetical alkaloid that he named phalloidin. In 1891 Robert 
[Ford and Clark (1914), p. 177] extracted from A. phalloides a 
hemolytic substance, readily destroyed by heat, which he named 
phallin. At first he believed it to be the essential poison, but he 
later extracted an alcohol-soluble alkaloid that was extremely 
poisonous to his experimental animals. 

The analyses by Ford (1906) showed that A. phalloides con- 
tains, besides phallin, the hemolytic principle of Robert, another 
substance of toxic nature. Ford verified the thermolabile nature 
of phallin and found that the other substance was heat-stable and 
resistant to digestion by pepsin and pancreatin. He also prepared 
antiserum that was effective against the stable substance but had no 
neutralizing effect on phallin. To this stable extractive Ford 
gave the name amanita-toxin. 

Schlesinger and Ford (1907) purified amanita-toxin to the ex- 
tent that it did not give the reactions of proteins, glucosides, or 
alkaloids, and concluded that it ". . . appears to be an aromatic 
phenol so combined with an amine group that it readily forms an 
indol or pyrrol ring." 

Since Amanita virosa, A. verna, Pholiota autumnalis, and Hygro- 
phorns conicns induce similar clinical symptoms, they may be as- 
sumed to contain the same amanita-toxin as does A. phalloides. 
Other species which contain amanita-toxin are A. porphyria, A. 
strobilijorvris, A. radicata, A. chlorinosoma, A. viappa, A. vior- 
risii, A. citrina, A. cremilata, and Avianitopsis volvata. 

The toxic principle in Amanita vniscaria was isolated by 
Schmiedeberg and Roppe [Ford and Clark (1914), p. 1771 in 
1869 and given the name muscarine. There was also isolated from 
this same species the alkaloid choline, which, on uniting with 
oxygen, as it does when the flv agaric decays, becomes muscarine. 
Robert [Ford and Clark (1914), p. 177 J maintained that this 



FOOD VALUE OF FLESHY FUNGI S51 

fungus contains a third alkaloid, which he called "pilz-atropin." 
Muscarine depresses the same nerves that are stimulated by "pilz- 
atropin" and atropin, both of which therefore are physiological 
antitodes for muscarine. Persons who have been poisoned by 
A. miiscaria and whose heart has nearly ceased pulsating may be 
given atropin, with the result that heart action will again become 

strong. 

Studies on muscarine indicate that it is not a chemical entity 
but a group of at least five substances, having the empirical form- 
ula C5H15NO2. Muscarine occurs in Amanita pantherina, Bole- 
tus satanus, B. luridus, and Russula emetica. Clitocybe illudens, 
Lactarius torminosns, lnocybe bifida, and /. decipiens contain a 
muscarine-like principle that may be similar to that in Amanita 

miiscaria. 

There occurs in Helvetia esculenta a water-soluble, heat-labile, 
hemolytic principle that has been identified as helvellic acid, 
C12H20O7. It is generally agreed that fresh specimens are free 
from this poison but that it occurs in old or decaying morels. 

FOOD VALUE OF FLESHY FUNGI 

The food value of mushrooms is indicated by analyses made 
years ago by Mendel (1898). Certain of his data are presented 
in Table 27. Mendel pointed out that these percentages do not 
represented the digestible fraction. For example, he found that 
only about one-seventh of the total nitrogen in Coprinus comatus 
is actually digestible. No determinations were made of the nu- 
tritive value of the ether extract, that is, the fatty substances, but 
Mendel assumed that the digestible portion of this fraction must 
be similar to that of the total-nitrogen fraction. 

TABLE 27 
Composition of Certain Edible Fungi 

Constituents 



Dry 



{percentage on dry-weight basis) 



Matter Total Protein Ether 

Species (percentage) N N extract Ash 

Coprinus comatus 7.81 5.79 1.92 3.3 12.5 

Morchella esculenta 10.46 4.66 3.49 29.3 10.4 

Pleurotus ostreatus 26 . 30 2 . 40 1.13 31.5 6.1 

Psalliota campestris 8 . 20 4.75 3.57 11.6 



352 POISONOUS AND EDIBLE FUNGI 

Mendel concluded that mushrooms have a low caloric value. 
Nevertheless he properly regarded them as being among the most 
appetizing of culinary delicacies and as adding greatly to the 
palatabilitv of many foods when cooked as savories with them. 

Later workers are inclined to regard mushrooms as having 
amounts of nitrogenous substances, carbohydrates, and fats that 
would rank them, in regard to nutritive value, along with fresh 
vegetables. Data of Sabalitschka [IvanofT and Zwetkoff (1932)] 
showed that Psalliota campestris and Boletus edulis have a high 
protein content. This finding was confirmed by Saburow and 
YVasiliew [IvanofT and Zwetkoff ( 1932)], who recorded the pro- 
tein content of these two species as 32.06% and 31.25%, respec- 
tively, these figures being based on the weight of dry substance. 
On the other hand, other edible species may be low in proteins, 
since Saburow and Wasiliew found in Colly bia velutipes 8.87% 
and in Tricholoma portentosum 10.50%. They also reported 
lar^e variation in fat content between species, Boletus edulis hav- 
ing 1.6% and B. scaber 9.69%. Since the proportions of proteins, 
carbohydrates, and fats that are digestible by man remain un- 
known, the true nutritive value of mushrooms likewise remains 
a mystery. 

Fleshy species most used as food. The fleshy fruit-bodies of 
fungi that are used as food in most parts of the world are not 
cultivated but occur in forests, mainly on the forest floor. The 
choicest species include Psalliota (Agaricus) campestris, P. arven- 
sis, Boletus edulis, Lepiota procera, Lactarius deliciosus, Coprinus 
comatus, Cantharellus cibarius, Pleurotus ostreatus, and Fistulina 
hepatica. Among other highly prized species are the morels, in- 
cluding Morchella esculenta, M. conic a, Gyroviitra gigas, and G. 
esculenta, the truffles, especially Tuber aestivum and T. vielano- 
spervnmu and certain puffballs. In parts of Australia and Tas- 
mania use is made of the large sclerotia of Polyporus viylittae, 
which are called "native bread" and "black-fellow's bread." The 
natives in Tierra del Fuego eat large quantities of Cyttaria, espe- 
cially C. gunnii, C. hookeri, C. darwinii, and C. harioti, which 
grow parasiticallv on the branches of Nothofagus. 

Artificial cultivation of fleshy fungi. In light of the fact 
that the excellence of certain species has long been appreciated, 
it is not surprising to find that attempts were made by the ancients 
to cultivate them. At present, however, few species are culti- 



FOOD VALUE OF FLESHY FUNGI 353 

vated in any country. Psalliota campestris, the common mush- 
room, is apparently the species most widely grown under arti- 
ficial conditions. Precise directions for the commercial growing 
of this mushroom are available but are not relevant to this account. 
A few of the general features involved in its culture, however, 
seem pertinent. Caves, cellars, abandoned mines, and special types 
of glasshouses are suitable for growing mushrooms, provided that 
temperature, moisture, and ventilation are properly controlled. 
Of these factors, temperature is perhaps the most vital; it should 
be kept within the range 53° to 63° F. High relative humidity 
is required, but the site should not be wet. 

Mushroom growers attach great emphasis to proper prepara- 
tion of the manure. Stable manure, including the litter used for 
bedding, is piled deeply, mixed with loam, and turned and re- 
piled until a suitable compost is formed. The compost is then 
placed in beds and is implanted with spawn, that is, with blocks 
of humus permeated with the mycelium of the mushroom. After 
several weeks the beds are cased. This process consists in cover- 
ing the beds to a depth of 1 or 2 in. with a layer of loam. The 
beds then require occasional sprinkling to keep them moist. The 
mushrooms should soon begin to appear. In France morels are 
grown in much the same way, except that bits of fruit bodies are 
used as spawn. 

In parts of China Hirneola polytricha, under the Chinese name 
Mil Erh, is grown under semiartificial conditions. Sapling oaks 
{Quercus variabilis) are cut into poles, allowed to lie on the 
ground for several months, and then stacked in small piles in moist 
places. The gelatinous fruit bodies are developed the following 
year. The Chinese similarly grow the large sclerotia of Poria 
cocos on partly buried pine poles. 

The fruit bodies of Armillaria shii-take are produced artificially 
on a large scale in Japan and are marketed under the name shii- 
take. The name shii applies to an evergreen oak, Quercus cuspi- 
data. Recently cut logs of this oak are soaked in water, and the 
bark is loosened by pounding; holes are then made in the logs, 
and pieces of wood decayed by the fungus are placed therein. 
After about 2 years the mushrooms appear. By proper manage- 
ment of cutting, the coppice growth from the stumps attain 
cutting size in about 20 years. Tracts are thus reforested to con- 
tinue the production of crops of shii-take. 



354 POISONOUS AND EDIBLE FUNGI 

ERGOT AND ERGOTISM 

The name ergot, which is properly applied to the sclerotial stage 
of Claviceps purpurea, is derived from the old French argot and 
refers to the resemblance of the sclerotium to a cock's spur. 
Er^ot, when ingested bv man and various animals, has long been 
known to be poisonous, causing a disease known as ergotism. 
Both the disease and its cause have come to be well known and 
have attracted the attention of a large number of investigators. 
Two monographic treatises on this subject, one by AtanasofT 
(1920) and the other by Barger (1931), are especially note- 
worthy. That bv AtanasofT is concerned primarily with matters 
of plant-pathological and mycological interest, whereas that by 
Barger deals primarily with ergotism. Barger's interests were 
centered on this problem for more than 20 years, and his compre- 
hensive report, although intended primarily for the student and 
the practitioner of medicine, is also of wide general usefulness. 

Historical account. It becomes apparent from the account 
by Barker (1931) that the antiquity of ergotism cannot be estab- 
lished with certainty. There is little likelihood that the ancient 
Greeks and Romans knew this disease, as is maintained by Robert 
[Barger (1931), pp. 40-42]. Certain diseases mentioned by Hip- 
pocrates and Galen and interpreted by Robert and others to be 
ergotism seem to have been some other disorder. It seems highly 
probable that an outbreak of ergotism was first chronicled by 
some unknown writer in the Annates Xanthensis in a.d. 857. 
Translated, his statement is: "A great plague of swollen blisters 
consumed the people by a loathesome rot, so that their limbs were 
loosened and fell off before death." Confusion also exists regard- 
ing the cause of the epidemics called "holy fire" {ignis sacer) that 
occurred throughout the succeeding period of about 800 years. 
The gangrenous condition of limbs, resulting in death or the loss 
of hands and feet, undoubtedly was ergotism, although anthrax, 
erysipelas, scurvy, and plague may have accounted for a portion 
of the mortality. 

The modern history of ergotism begins with an account by 
Dodart [Barger (1931), pp. 59-60] of an epidemic in the Sologne 
district of France in 1676. In 1777, in this same district, about 
8000 persons are said to have succumbed from ergotism. In 1770 



ERGOT AND ERGOTISM 



355 




Fig. 64. Poisonous Ascomycetes on grasses. A. Gibber -ell a saubinettii (G. 
zeae) in small clusters on barley glumes. B. Cluster of perithecia of G. 
saubinettii. D. Ascospores. E. Conidia of the Fusarium stage. F. Branch 
of panicle of Paspalum laeve, certain of the ovaries having been replaced 
by sclerotia of Claviceps paspali. G. Sclerotium of C. paspali that has hiber- 
nated, after which three perithecial stromata developed. H. The conidia of 
C. paspali belong to the form Genus Sphacelia and occur on the surface of 
the stromata that later become sclerotia. 



356 POISONOUS AND EDIBLE FUNGI 

an outbreak involved the inhabitants of several European coun- 
tries, and subsequentlv there have been manv epidemics through- 
out the whole of Europe, some of them widespread and all of them 
producing horrible suffering and disfigurement. 

The date of the first use of ergot as a drug cannot be fixed, but 
the first published mention of its use to induce uterine contrac- 
tions occurs in Adam Lonicer's Kreuter bitch in 1582. The ergot 
grains are therein described as "long, black, hard, narrow corn 
pegs, internallv white, protruding like long nails from between 
the grains in the ear," and three sclerotia are designated as consti- 
tuting a dose. Subsequentlv for a period extending throughout 
the eighteenth century midwives in various European countries 
used ergot to expedite lingering parturition. Its use did not enter 
into pharmaceutical practice, however, nor was it employed by 
the medical practitioner. In the United States ergot was medically 
introduced under the name of puhis parturiens early in the nine- 
teenth century. 

Early writers were not in accord on the true nature of ergot. 
Caspar Bauhin refers to it as Secale luxurious in his Fhytopinax, 
published in 1596 [Barger (1931), p. 10]. Until the middle of the 
nineteenth century many writers regarded eroot grains as degen- 
erated kernels. Among the causes assigned for this degeneration 
were improper nutrition, failure of the flowers to become fertil- 
ized, injury from insects, and excessive rainfall. Fries (1822) 
considered the ergot grain as a fungus structure; he gave it the 
name Spervwedia davits but later (1849) changed this name to 
Claviceps purpurea. Leveille (1827) observed that the sugary 
secretions on young sclerotia contained conidia. Thinking that 
the conidia were reproductive structures belonging to a fungus 
parasitizing the sclerotia, he named this supposed parasite Sphacelia 
segetum. He' (1842) maintained that the ergot itself was a de- 
generated kernel. Mcvcn's observations (1841) on ergot led him 
to conclude that the sclerotium is an early stage of the Sphacelia 
segetum that Leveille had described nearly 15 years before. The 
chapter on the nature of ergot was finally concluded by Tulasne 
(1853), who established that the conidia, sclerotia, and perithecial 
stromata constitute developmental stages of one and the same 
fungus, which he called Claviceps purpurea (Fr.). 

The structure and development of Claviceps purpurea have been 
recounted in some detail [Falck (1911), Stager (1903), Zimmer- 
man (1906), Kirchhoff (1929), Killian (1919)]. This fungus at- 



ERGOT AND ERGOTISM 



351 



tacks various cereal and forage grasses. It is most commonly 
known in the sclerotial or ergot stage as it occurs on rye. The 
rye grain is replaced by the ergot "grain" or sclerotium. The 
fungus may first be noted at the time of the flowering of the rye, 




Fig. 65. Stages in the development of ergot, Claviceps purpurea, on rye. A. 
Ergot grains (sclerotia) appearing as dark spurs and replacing the rye grains. 
B. Young infected rve ovarv, at whose surface conidia are formed. C. De- 
tail of surface of young infected ovarv. D. Mature ascus of C. purpurea, 
bearing eight thread-like ascospores. E. Diagram of apex of perithecial 
stroma, showing perithecia arranged near the periphery. F. Sclerotium 
in spring, bearing several club-shaped perithecial stromata. 

when the young rye ovaries are covered with masses of conidia 
that collect in droplets. These droplets ("honey dew") are dis- 
persed by insects. By the time the normal grain is ripe, the ergot 
grains are also mature. When the grain is threshed, the ergot 
grains are admixed with the rye. 

Ergotism in livestock. There is reason to believe that sclerotia 
from all species of Claviceps are poisonous. From various parts of 
the world have come reports of the poisonous effect of ergotized 



358 POISONOUS AND EDIBLE FUNGI 

pasture grasses and of ergotized hav when consumed by domestic 
animals. The fungi involved are mainly Claviceps purpurea and 
C. paspali. Poisoning from C. purpurea occurs when animals are 
fed rye as grain or are pastured on ergot-infected Loliwn perenne, 
Poa pratensis, or Agrostis alba. Several serious outbreaks of 
ergotism among cattle and horses, caused by C. purpurea on 
Ely mils canadensis, fed as hay, have been reported from the prairie 
regions of the central United States. Claviceps paspali, occurring 
on several species of Paspalum used as forage grasses, is known 
to cause poisoning of cattle in the Argentine, Natal, and the south- 
ern United States. 

Poisoning bv C. purpurea causes lameness and swelling of one 
or more limbs; in severe cases, as the result of impairment of cir- 
culation, the extremities may become oangrrenous. Ears, horns, 
hoofs, toes, feet, and tails may become necrotic and may slough 
off. The loss to stockmen from abortion by cows and mares is 
heavy when ergot is abundant. Gastric disturbances and varying 
degrees of paralysis are other symptoms of ergot poisoning in 
horses and cattle. 

An epidemic of poisoning by C. paspali in Mississippi was 
studied by Brown and Ranck (1915) and Brown (1916). Their 
feeding trials involved guinea pigs, young calves, and more mature 
young cattle. Thev noted that affected animals are highly nerv- 
ous and are unable to coordinate their movements. Paralysis may 
ensue, and in consequence affected cattle are unable to reach water 
to drink. If ergotized grass was kept from the sick animal, and 
water and feed were given after the administration of a purgative, 
recovery followed. 

Toxicology. Many painstaking chemical analyses have been 
made to isolate and identify the active principle in ergot, as is 
apparent from Barger's (1931) monograph. In 1875 Tanret 
[Barger (1931)] isolated what he regarded as a pure alkaloidal 
substance and as the active principle, and called it ergotinine. In 
1884Kobert [Barger (1931)] identified three substances, ergotinic 
acid, sphacelic acid, and cornutine. Ergotinic acid is a nitrogen- 
ous elucoside that causes inflammation of mucous membranes and 
hemolysis. Robert first thought sphacelic acid caused uterine 
contractions but later attributed this action to cornutine. At 
first cornutine was thought to cause convulsions and paralysis. 
In 1897 Jacoby isolated a phenol-like substance that he called 



TOXICITY OF GIBBERELLA SAUBINETTII 359 

sphacelotoxin. When acted upon by an alkaloidal base, sphacelo- 
toxin became secalintoxin, and when purified, it became the 
ergotinine of Tanret. Analysis by Kraft in 1901 yielded two alka- 
loids, Tanret's crystalline ergotinine and an amorphous hydro- 
ergotinine. In 1906 Barger and Dale [Barger (1931)] maintained 
that Kraft's hydroergotinine is ergotoxin, CssH^OeNg, an alkaloid 
capable of increasing blood pressure, of causing gangrene on the 
combs of hens, and of inducing uterine contractions. Tanret, who 
first gave the formula of his ergotinine as C35H40O6N4, later 
changed it to C35H40O5N.-,. After years of study Barker con- 
eluded that the correct formula of ergotinine is C33H35O5N5. 

Reports of studies on the nature of the toxic principle in ergot 
appeared almost simultaneously from several laboratories. Stoll 
and Burkhart (1935) called their purified alkaloid ergobasine; 
Thompson (1935) called his ergostetrine. Dudley and Moir 

(1935) named their substance ergometrine. Kharasch and Le- 
gault (1935) called their product ergotocin. They got 0.1 to 0.3 
mg of ergotocin from 3 to 4 grams of ergot grains, an amount 
held to constitute a dose. Kharasch, King, Stoll, and Thompson 

(1936) compared the melting points of the four alkaloids, ergoba- 
sine, ergostetrine, ergometrine, and ergotocin, and those of certain 
of their salts, and also the optical properties of each in different 
solvents, and came to the conclusion that the four names are 
synomymous. 

The alkaloidal content of ergot varies with the year and with the 
locality. Spanish and Portuguese ergot assays 0.05 to 0.30%, 
whereas ergot from Russia and Poland varies from 0.02 to 0.10%. 
The superiority of the Spanish and Portuguese ergots may be 
causally related to moisture. When stored at high humidities, 
ergot deteriorates, deterioration being correlated with increased 
histamine content. When stored dry, it keeps for long periods, al- 
though pharmaceutical supply manufacturers avoid buying ergot 
that is more than a year old. 

TOXICITY OF GIBBERELLA SAUBINETTII (G. ZEAE) 

AND FUSARIUM SPP. 

The heads or inflorescences of various grasses may be para- 
sitized by a polymorphic ascomycetous fungus, Gibberella scaibi- 
nettii, that is especially destructive to barley, oats, rye, wheat, and 



360 POISONOUS AND EDIBLE FUNGI 

corn. On small qrains the disease is known by the common name 
scab. The causal fungus is most frequently encountered in its 
conidial stage, which is of the Fusarium type. As the cereal crop 
approaches maturity, conidia of the Fusarium stage are present in 
profusion at the surface of the grains and glumes. This fungus, 
especially as it occurs on barley, has long been known to be 
poisonous. The status of present-day knowledge of its toxicity 
in connection with scabby barley is summarized in a report by 
Christiansen and Kernkamp (1936). 

Long ago peasants in Russia found that scabby barley, when 
used in bread-making or when fed to livestock, is toxic. In 
northern Russia this toxicity came to be attributed to Fusarium 
avenaceum (Fr.) Sacc, and in southern Russia, to Fusarium 
gramineum Schwabe. In the United States about a dozen species 
of Fusarium are known to be associated with barley scab. 

Barley scab was unusually abundant in 1928 in the Upper Mis- 
sissippi Valley. Much of this diseased barley was used to feed 
swine, and in consequence of complaints of sickness in the herds, 
special efforts were made to learn more about the poisonous prop- 
erties of Fusarium-affected barley. Some of the diseased crop was 
exported to Europe, where similar complaints arose from its use 
as feed for swine. The results of experimentation that was initi- 
ated in the United States and in Europe leave no doubt that the 
feeding of scabby barley is responsible for sickness among do- 
mestic animals. In their entirety these experiments showed that 
such barley is poisonous to horses, cattle, sheep, pigs, chickens, 
and dogs, ruminants being able to tolerate greater proportions of 
affected grains. 

Christiansen and Kernkamp (1936) observed that pigs refuse to 
eat scabby barley unless they can get nothing else. If the propor- 
tion of affected kernels is as much as 16° o, it is extremely toxic, 
and if as much as 32%, the pigs refuse to eat it. Poisoning is 
manifested by loss of appetite, listlessness, and weakness and 
nausea; death may ensue. These investigators found that the 
poisonous principle is v\ater-soluble and heat-stable. An aqueous 
extract from 15 grams of scabby barley, when administered orally 
through a stomach tube to a pig weighing 100 lb, caused vomiting. 
An overdose of extract from Fusarium-infected corn caused 
death. 



IMPLICATIONS 361 

Christiansen and Kernkamp isolated several species of Fusarium 
from affected barley kernels, F. gramineimi being most common. 
Extracts from pure cultures of these species did not prove toxic 
to pigs, although they refused to eat barley that had been used 
as a culture medium unless it was masked by mixture with a suffi- 
cient quantity of other feed. When Christiansen and Kernkamp 
inoculated wheat, barley, and corn with F. gram'meum at a time 
when the grain was developing, the ripened kernels were found 
to contain the toxic principle. Moreover, affected grain retained 
its toxicity for long periods, at least 3 years. Affected kernels 
tend to float at the surface of water; this fact can be utilized in 
separating normal and scabby grains. 

Concerning the chemical nature of the toxic principle little has 
been established to date, except that it is water-soluble and ther- 
mostable. Schroeter and Strassberger (1931) found large quanti- 
ties of choline and fatty acid esters of choline in Fusarium- 
affected grain and expressed the opinion that these substances 
constitute the toxic principle. 

It may be recalled that the proximate cause of wilting in vascu- 
lar diseases of crop plants associated with species of Fusarium is 
commonly regarded as a toxin. The experiments with Fusarium- 
affected grain indicate, as a line of departure, the employment of 
animals in studies involving the nature of such toxins in Fusaria 
causing vascular-wilt diseases. 

IMPLICATIONS 

In the past, studies of poisonous fungi have been concerned 
mainly with the identity of the poisonous fungus, with the na- 
ture of its toxic principle, and with the effects of this principle 
upon animals and man. Too little is yet known about fungi 
poisonous to seed plants. It is indicated that future studies should 
stress plant toxemias to a greater extent than have those of the 
past, in order to account for the disease syndrome. By the use 
of plant toxins as antigens, it should be possible to produce specific 
antitoxins. Furthermore the ultracentrifuge and electron micro- 
scope should enable the worker to purify fungus toxins and anti- 
toxins and thus to learn something more of their physical prop- 
erties and eventually of their chemical constitution. 



362 POISONOUS AND EDIBLE FUNGI 



LITERATURE CITED 

Atanasoff, D., "Ergot of grains and grasses" (stenciled copy, 107 pp.). U. S. 
Dept. Agr., Bur. Plant Industry. 1920. 

Barger, G., "Ergot and ergotism," a monograph based on the Dohme lectures 
delivered in Johns Hopkins University. 279 pp. Gurney and Jackson, 
London. 1931. 

Brown, H. B., "Life history and poisonous properties of Claviceps paspali," 
J. Agr. Research, 7:401-406, 1916. 

Brown, H. B., and E. AI. Ranck, "Forage poisoning due to Claviceps paspali 
on Paspalum," Miss. Agr. Expt. Sta. Tech. Bull., 6:3-35, 1915. 

Christiansen, J. J., and H. C. H. Kernkamp, "Studies on the toxicity of 
blighted barley to swine," Minn. Agr. Expt. Sta. Tech. Bull., 113. 38 
p. 1936. 

Dudley, H. W., and J. C. .Moir, "The new active principle of ergot," 
Science, 81:559-560, 1935. 

Dujarrac de la Riviere, D., and Roger Helm, Les champignons toxiques. 
Paris. 1938. 

Falck, R., "Uber die Luftinfektion des Mutterkorns {Claviceps purpurea 
Tul.) und die Verbreitung prlanzlicher Infektionskrankheiten durch 
Temperaturstromungen," Z. Forst- und Jagdwese?i, 43:202-221, 1911. 

Ford, W. W., "The toxicological constitution of Amanita phalloides" /. 
Expt. Med., 5*: 437-450, 1906. 
"The toxins and antitoxins of poisonous mushrooms," /. Injections Dis- 
eases, 3: 191-224, 1906a. 
"On the presence of hemolytic substances in edible fungi," /. Injections 

Diseases, 4: 434-439, 1907. ' 
"A clinical study of mushroom intoxication," Johns Hopkins Hosp. Bull., 

18:1-21, 1907a. 
"The distribution of haemolysins, agglutinins, and poisons in fungi, espe- 
cially the Amanitas, the Entolomas, the Lactarius, and the Inocybes," 
/. Pharmacol., 2:285-318, 1911. 
"A new classification of mycetismus (mushroom poisoning)," /. Pharma- 
col., 29: 305-309, 1926. 

Ford, W. W., and E. D. Clark, "A consideration of the properties of 
poisonous fungi," Mycol., 6: 167-191, 1914. 

Ford, W. W., and J. L. Sherrick, "On the properties of several species of 
the Polvporaceae and of a new variety of Clitocvbe, Clitocybe dealbata 
sudorifica Peck," /. Pharmacol, 2: 549-558, 1911. 
"Further observations of fungi, particularly- Clitocybe sudorifica Peck, 
Pholiota autumnalis Peck, and Inocybe decipiens Bresadola," /. Pharma- 
col., 4: 321-332, 1913. 

Fries, Elias M., Systema Mycologicum, 2: p. 268. 1822. 
Swmna vegetab'ilium Scandinaviae, p. 381. 1849. 

Iwanoff, X. X., and E. S. Zwetkoff, "The biochemistry of fungi," Ann. 
Rev. Biochem., 2:521-540, 1932. 



LITERATURE CITED 363 

Kharasch, M. S., H. King, A. Stoll, and M. R. Thompson, "The new ergot 

alkaloid," Science, 83: 206-207, 1936. 
Kharasch, M. S., and R. R. Legault, "Ergotocin," Science, ^7; 388, 1935. 
Killian, Charles, "Sur la sexualite de l'ergot de Seigle, le Claviceps purpurea 

Tulasne," Bull. soc. my col. Fra?ice, 25: 182-197, 1919. 
Kirchhoff, H., "Beitrage zur Biologie und Physiologie des Mutterkorn- 

pilzes," Zentr. Bakt., Parasitenk., 11 Abt., 77:310-369, 1929. 
Leveille, J. H., "Memoire sur le genre Sclerotium," Comp. rend., 14:446- 

448, 1842. 
"Memoire sur l'ergot, an nouvelles recherches sur la cause et les effets de 

l'ergot, considere sous le triple rapport botanique, agricole et medical," 

Mem. Soc. Limn. Paris, 5: 565-569, 1927. 
Mendel, L. B., "The chemical composition and nutritive value of some edible 

American fungi," Am. J. Physiol., 1: 225-238, 1898. 
Meyen, F. J. B., Pflaiizenpathologie. p. 192. 1841. 
Rolfe, R. T., and F. W. Rolfe, The romance of the fungus world. 309 pp. 

J. B. Lippincott Co., Philadelphia. 1928. 
Schlesinger, H., and W. W. Ford, "On the chemical properties of Amanita 

toxin," /. Biol. Chem., 5:279-383, 1907. 
Schroeter, G., and L. Strassberger, "Cholin als Schadstoff in kranker 

Gerste," Biochem. Z., 232:452^58, 1931. 
Stager, R., "Infektionsversuche mit Gramineen bewohnenden Claviceps- 

Arten," Botan. Z., 61: 111-158, 1903. 
Stoll, A., and E. Burkhardt, "L'ergobasine, nouvel alcaloide de l'ergot de 

seigle, soluble dans l'eau," Compt. rend., 200: 1680-1682, 1935. 
Thompson, M. R., "The new active principle of ergot," Science, 81:636- 

638, 1935. 
Tulasne, L. R., "Memoire sur l'ergot des Glumacees," Ann. soc. nat. botan., 

3 ser., 20:5-56, 1853. 
Zimmerman, A., "Erganzende Versuche zur Feststellung der Keimfahigkeit 

altere Sklerotien von Claviceps purpurea," Z. Pflanzenkr., 16:129-131, 

1906. 



Chapter 16 

MEDICAL MYCOLOGY 

The fungi which are pathogenic to man occupy a position 
which mav well be designated as a "no man's land" for both the 
mycologist and the medical practitioner. Even well-trained my- 
cologists have no first-hand knowledge of humanly pathogenic 
fungi, and these organisms remain quite unknown to the physician, 
since they are given little, if any, attention in the curricula of our 
best medical schools. Lack of proper appreciation of these fungi 
may also be attributed in part to the fact that the mycologist is 
quite unacquainted with the clinical aspects or clinical variations 
and pathological anatomy of the diseases which they produce and 
that the physician is lost in the maze of controversial taxonomic 
and cultural difficulties which both he and the mycologist have 
fostered. Some of these problems have arisen because the patho- 
genic fungi exhibit so much variation in appearance when in 
lesions and when grown on various culture media. In addition, 
some confusion may be attributed to difficulties in interpreting 
many of the studies and descriptions of the pathogens. Experi- 
enced, well-trained mycologists with the organisms available for 
critical study find these taxonomic problems very puzzling and 
time-consuming. As a consequence a confusion has developed 
which will depend for clarification upon collaborative studies 
among clinicians, pathologists, taxonomists, serologists, biochem- 
ists, and epidemiologists. No single investigator, working inde- 
pendently, can hope to establish order in a field so chaotic. 
Thousands of papers on medical mycology, many of them case 
reports, have been published since 1900. An appreciation of the 
status and scope of this subject can be gained from Dodge's (1935) 
Medical Mycology and from the excellent recent summaries by 
Tate (1929), Ramsbottom (1931), Gregory (1935) and Emmons 
(1940). The medical practitioner will find the volume by Lewis 

364 



MEDICAL MYCOLOGY 365 

and Hopper (1939) especially helpful in diagnosis and in identi- 
fication. 

Similarly the physician will find the Manual of Clinical Mycol- 
ogy by Conant, Martin, Smith, Baker, and Callaway (1944) indis- 
pensable in dealing with mycotic diseases. It discusses systemati- 
cally and briefly symptoms, differential diagnosis, prognosis, im- 
munology, etiology, identification, isolation and cultivation of the 
fungus, and range of the disease. 

It would seem that medical mycology is not surpassed bv any 
other field of mycological study in potential importance and in 
appeal to the scientific imagination of the young investigator 
seeking new and difficult problems whose solution means much 
to the welfare of the human race. 

On the basis of present-day knowledge certain general state- 
ments regarding fungi pathogenic to man appear to be war- 
ranted. These statements are therefore categorically presented 
in the following introductory paragraphs. In the first place the 
number of species known to be pathogenic to man is limited. 
These are mostly imperfect fungi; a few are Ascomycetes, closely 
related to the yeasts, and a few are Actinomycetes, whose syste- 
matic position is still a matter of dispute. 

Little is definitely known about their source in nature except 
that circumstantial evidence indicates that some of them originate 
on plants. Some species, especially among the Trichophytoneae, 
occur also on wild and domestic animals and are transmitted to 
man only by being implanted. 

Entrance to the body is gained (a) through hair follicles, but 
never through sweat glands, (b) through the nasal passages and 
thence into the lungs, and (c) through abrasions or injuries, as 
through scratches or wounds made by thorns or splinters. In a 
few species entrance appears to be gained through the enteron. 

The types of tissue reactions induced in man by fungi are ex- 
tremely variable. Some species remain quite superficial in their 
effect, whereas others produce deep lesions or involve such internal 
organs as the lungs, spleen, and liver. Some are local, and some 
systemic. Among the common tissue changes are congestion, 
edema, exudation, hyperplasia, necrosis, scar-tissue formation, and 
suppuration with accompanying migration of polymorphonuclear 
cells. 



366 MEDICAL MYCOLOGY 

In artificial culture many of these pathogens have a very differ- 
ent appearance from the way they look in tissues. Some of them 
are filamentous when grown at room temperature but under 
otherwise similar conditions are yeast-like in appearance when 
cultivated at incubator temperature, 37.5° C. 

HISTORICAL MATERIAL 

Medical mycology may be said to begin with Schoenlein, who 
in 1839 associated a fungus with favus, a form of ringworm char- 
acterized by lesions and having bright yellow crusts composed of 
small cup-like scales. The causal organism was given the name 
Achorion schoenleini by Remak 6 years later. In the same year 
Malmsten employed the generic name Trichophyton for the ring- 
worm pathogen. The deeply seated, suppurative form of ring- 
worm known as kerion was shown in 1856 to be induced by a 
Trichophyton originating from animals. Further proof of trans- 
mission from animal to animal followed, as well as demonstration 
by various workers that ringworm can be transmitted to man 
from horse, cow , dog, or cat. All in all, however, little important 
work in this field was accomplished until Sabouraud began his 
studies in the early 1890's. The publication of his monumental 
Les Teignes (1910) constitutes the beginning of the modern era 
of investigation and is the foundation upon which all present-day 
studies in medical mycology are based. 

The medical worker has found it convenient to designate by 
the term "mycoses" (literally "filled with, or full of, fungi") the 
diseases of man and animals caused by fungi. This terminology 
has a definite significance for the mycologist, since the generic 
name of the pathogen and the suffix "osis" are combined, as in 
Actinomycosis, Torulosis, Histoplasmosis, and Blastomycosis, and 
it will be employed in the discussion that follows. Confusion 
arises, however, when "osis" and "mycosis" are applied to clin- 
ically distinct mycoses, such as may be produced by one and the 
same fungus involving different organs and tissues, for example, 
"onchomycosis" when the nails are involved, "sychosis" when 
the beard is involved, and "dermatomycosis" when the glabrous 
skin is involved. Similarly, the wisdom of retaining the name "der- 
matophytes" or "dermatomycetes" for those fungi that invade 
the keratinized layers of the epidermis and such appendages or 



COCCIDIOIDES IMMITIS 



361 



modifications as the hair, nails, hooves, feathers, and horns may 
be questioned. One might with equal reason indicate by the term 
"caulophytes" those fungi involving plant stems, "fructophytes," 
those involving fruits, and "phyllophytes," those attacking foliage! 

The account that follows is 
intended as an introduction to 
the mycologic features of some 
of the better-known human 
pathogens. The scope of the 
field can be appreciated only 
by consultation of certain vo- 
luminous monographic studies, 
such as those of Sabouraud 
(1910), Brumpt (1935), and 
Dodge (1935). It is also quite 
apparent that all too little is as 
yet known of the mycotic flora 
of the surface of the normal 
body and of the protective 
mechanisms which the skin af- 
fords to invasion by fungus 
pathogens. 

COCCIDIOIDES IMMITIS 




Fig. 66. Coccidioides immitis. A. 
Hypha from nutrient agar, tending 
to be racket-shaped. B. Arthro- 
spores from culture. C. Spores, one 
of them germinating from globular 
sporangium-like cell, which con- 
tains numerous spores. (After 
Moore.) 



This organism, w T hich causes 
a highly fatal disease, was first 
reported in Argentina but is 
best known in California, Ari- 
zona, and Texas. The disease 
is commonly known in its acute 
form as valley fever; the medi- 
cal profession calls it coccidioidal granuloma. It has been re- 
ported to occur among cattle, sheep, and dogs, but as yet there is 
little evidence of transmission from animals to man or man to ani- 
mals. Emmons (1942) reported that the pathogen occurs in ro- 
dents, including deer mice, pocket mice, kangaroo rats, and ground 
squirrels, in Arizona and also that he was able to isolate it from soil. 

The early history of coccidioidomycosis is summarized in an 
account by Rixford, Dickson, and Beck (1931). The disease 



568 MEDICAL MYCOLOGY 

may be manifest as a mild, pleurisv-like, respiratory infection, 
with chills, night sweats, and headache. After 2 or 3 weeks papil- 
lomatous eruptions appear on the arms, thighs, and scalp, and 
occasionally the knee and ankle joints are arthritic. Examination 
by X-ray may reveal pulmonary nodules resembling primary 
tuberculosis. The sputum is mucopurulent and may contain 
blood. 

Ulcerative lesions on the face and neck may characterize an- 
other form of the disease. Such lesions slowly become subcu- 
taneous and may spread to the meninges and spinal cord. If the 
miliary type of involvement develops, the fever is high, prostra- 
tion is marked, and death occurs after a few weeks. 

When present in the tissues, Coccidioides 'nmriith, described by 
Stiles in a report by Rixford and Gilchrist (1896), consists of 
large, thick-walled, spherical cells that may reach a diameter of 
50 to 70 /*. At maturity these cells function as sporangia, although 
they have been misinterpreted by some to be asci. By cleavage 
their content gives rise to a large number of spores, which escape 
by rupture of the sporangial wall. From a comparative study of 
15 strains by Baker, Alrak, and Smith (1943) it has been con- 
cluded that the organism is a Phycomycete. 

This fungus in cultures on semisolid media forms creamy white, 
cottony mycelium. By fragmentation chlamydospore-like oidia 
are produced. Sporangia and sporangiospores are developed, how- 
ever, if cultured under reduced oxygen tension in the presence of 
tcrcf albumen or serum. 

The acute type of the disease probably enters through the pul- 
monary route. The pathogen has been isolated from the soil, but 
soil may not constitute its natural habitat. In patients who re- 
cover spontaneously, and among residents of the San Joaquin 
Valley generally, intradermal injection of killed cultures of the 
fungus results in rather severe skin reactions. 

CRYPTOCOCCUS HISTOLYT1CUS 

Approximately 30 species of Cryptococcus are reported to be 
pathogenic to man, Cryptococcus histolyticus, a cause of blasto- 
mycosis, being perhaps the best known. Reports of blastomy- 
cosis include a disease which in the United States manifests itself 
by a disturbance of the central nervous system, clinically like 



HISTOPLASA1A CAPSULATUM 369 

chronic meningitis, whereas in Europe ulcerative lesions of the 
skin and underlying tissues are a more common manifestation. 
The evidence by Benham (1934) indicates that European blasto- 
mycosis and American torulosis are identical. Freeman's (1931) 
account of clinical appearance and pathology shows that there 
may be chronic respiratory involvement which leads to a diagnosis 
of tuberculous meningitis. The pathogen is presumed to enter 
through the respiratory tract. 

The etiology of this disease remains confused. Freeman (1931) 
indicates that several organisms may produce the same disease 
complex. 

The pathogen is usually known as Torida histolytica or Crypto- 
coccus hominis. It is one of the Saccharomycetaceae, having ovoid 
to elliptical cells occurring singly or in groups and invested by a 
thick gelatinous capsule. It forms white to yellowish white, 
pasty, opaque colonies on agar. Its only known method of repro- 
duction is by buds, unless the researches of Todd and Hermann 
(1936) are confirmed. Their study of the developmental cycle 
shows endospore formation of the type found in Debaryomyces, 
in consequence of which they referred the pathogen to D. homi- 
nis. It has been suggested, on the basis of priority, that the proper 
binomial is D. neoformans. 

A generalized blastomycosis, manifest as cutaneous abscesses, is 
caused by the closely related Blastomy ces dermatitidis, also known 
as Gilchristia dermatitidis or Xymonema dermatitidis. 

HISTOPLASMA CAPSULATUM 

Approximately 30 years ago Darling reported the occurrence 
among the natives of Panama of a disease characterized clinically 
by fever, emaciation, anemia, splenomegaly, leucopenia, and ulcer- 
ation of the nose, throat, and intestines. He was not able to iso- 
late the etiologic agent but believed it was a protozoan, to which 
he gave the name Histoplasma capsidatwn. Subsequently other 
cases of histoplasmosis were recorded in widely separately places, 
and in 1932 de Monbreun (1934) isolated and described the causal 
fungus. In the mononuclear blood cells and lymph vessels it exists 
as yeast-like cells with thick capsules. When the organism is kept 
at body temperature on blood or serum media, this form of the 
pathogen persists. When grown on other agar media, however, it 



310 



MEDICAL MYCOLOGY 



is filamentous and produces peculiar spherical conidia or chlamy- 
dospores, covered with finger-like outgrowths, 10 to 25 /x in 
diameter. 

Its relationship to other fungi is not clearly established. It has 
been interpreted to be related to Coccidioides and placed in the 
Genus Posadasia anions the Endomvcetaceae. Studies by Howell 
( 1939), however, in which Histoplasma was compared with Sepe- 




Fig. 67. Histoplasma capsulatimi. A. Mycelium from culture. B. Aleuro- 
spores that form on aerial mycelium, showing characteristic protrusions. 
C. Aleurospore in optical section. D. Aleurospores that form submerged. 

Their walls are smooth. 

donium and other fungi related to Sepedonium show that these 
two genera are closely related Fungi Imperfecti. Sepedonium is 
never yeast-like, however, and it may produce phialospores, which 
are not known to be developed among species of Histoplasma. 
Conant (1941) reported that in its parasitic form within tissues 
Histoplasma is always yeast-like with thick capsules. On blood 
agar incubated at 37° C it buds, yeast-like, but at room tempera- 
ture it is myceloid and forms tuberculate chlamydospores. 
Conant too regards it as closely related to Sepedonium. 



PHIALOPHORA VERRUCOSA 

This is amono; the organisms involved in a chronic infection of 
the skin and subcutaneous tissues, characterized by the presence 



PHIALOPHORA VERRUCOSA 



311 



of warty or cauliflower-like excrescences. Usually the hands are 
involved; the feet are especially susceptible to infection. Other 
parts also are known to bear the nodular ulcers. The disease has 
a wide geographical distribution [Emmons (1940)] in the tropics 
of both hemispheres, especially among laborers who work bare- 
footed. There is no evidence of spread from person to person. 
The pathogen appears to enter through injuries, such as those 
from thorns or splinters. 




Fig. 68. A. Phialophora verrucosa, the spores borne in a phial and adhering 

in a mass at the opening of the phial. B. Hormodendroii pedrosoi, conidio- 

phores and chains of conidia that arise as buds. 

Two names, chromoblastomy cosis and dermatitis verrucosa, 
both of which have been criticized, have been applied to the dis- 
ease. The name chromoblastomycosis is criticized on the grounds 
that the fungus cells within the tissues, although pigmented, do not 
bud in yeast-like fashion but divide by septation. The roughening 
of the skin indicated by the name dermatitis verrucosa does not 
give an adequate clinical picture, since other tissues and related 
conditions are included in the disease complex. 

The causal agency was first described by Medlar (1915) as 
Phialophora verrucosa, one of the Dematiaceae, although the dis- 
ease was first observed by Pedroso in Brazil 4 years earlier. Phialo- 
phora, when seen in scrapings or in biopsied dermal papillae, con- 



312 MEDICAL MYCOLOGY 

sists of thick-walled, brown, spherical cells or two or three closely 
associated cells. In its saprophytic phase on agar it is mvceloid 
and grayish black. Sporulation occurs from lateral conidiophores, 
which are phial-like or cup-like with funnel-shaped mouths. The 
small spores are formed in the base of the cup and are extruded 
but adhere in a spherical mass at the mouth of the cup. 

The natural habitat of P. verrucosa is revealed bv the work of 
Conant (1937). He found that Cadophora americana, one of sev- 
eral species that cause a blueing of wood pulp, is morphologically 
and culturally identical with P. verrucosa. Further evidence of 
their identity comes from their antigenic similarity, established by 
Martin (1938). 

iVnother closely related species, Hormodendrwn pedrosoi, de- 
scribed by Brumpt in 1922 [Brumpt (1935)], causes an involve- 
ment whose clinical aspects cannot be distinguished from those 
induced by P. verrucosa. This fact has been established by several 
investigators, among whom are Emmons (1936) and Martin, 
Baker, and Conant (1936). Further evidence adduced by Em- 
mons and Carrion (1937) showed that some strains of H. pedrosoi 
may form phialospores in culture. Not only have morphologic 
relationships been established between these two fungi that pro- 
duce chromoblastomycosis, but also serologic evidence of Martin, 
Baker and Conant (1936) and Conant and Martin (1937) shows a 
very close relationship. These workers found that H. pedrosoi 
causes specific complement-fixing antibodies to form in the pa- 
tients' serum and that there is a cross-antigenic relationship be- 
tween strains of Hormodendrum and Phialophora. The taxono- 
mic difficulties that have arisen in this complex are indicated by 
combinations which have placed the pathogen in such genera as 
Gomphinaria, Fonsecaea, Carrionia, Acrotheca, and Trichospor- 
ium. Presumably one variable species only is involved in the pro- 
duction of chromoblastomycosis, as is indicated in the brief but 
comprehensive account by Carrion (1942). 

MALASSEZ1A OVALIS 

Approximately 75 years ago Malassez reported the occurrence 
of an organism, Pityrosporimi ovale, in the squamae, follicles, and 
sebaceous glands of the scalp. Since then many papers have been 
published, interest in this organism being centered on its possible 



ACTINOMYCES BOVIS 313 

relationship to baldness. Some workers have maintained that this 
organism is the cause of dandruff and seborrheic dermatitis; 
others, that it is a harmless saprophyte. Unna, one of the foremost 
students of this problem, is among those who believe that P. ovale, 
which he called the "bottle bacillus" because of the shape of the 
cells, is the etiologic agent in this scaly condition of the scalp; 
he designated the disease "pityriasis capitis." 

Among the recent workers who regard this organism as patho- 
genic is xMoore (1935), Ota and Huang (1933), on the other hand, 
concluded that their yeast-like isolates from seborrheic dermatitis, 
belonging to Pityrosporum, were saprophytes. The most critical 
study of this whole problem is that of MacKee and his associates 
(1938). They made direct examination of the scrapings of 
normal and diseased scalps and in one series found P. ovale in 86 
of the 100 cases examined, prevalence being little different on the 
normal and on the diseased scalps. From these scrapings they also 
cultured species of molds belonging to Aspergillus, Rhizopus, Al- 
ternaria, Chaetomium, Torula, Dematium, and Mycoderma, and 
in addition several species of Staphylococcus. MacKee and his 
associates conclude: 'The occurrence at times of the organism 
[P. ovale] on all types of scalps and the fact that it may occa- 
sionally be found in as large numbers on the normal scalp as on one 
with severe dandruff leads one to consider the possibility that this 
yeast is a saprophyte, and grows well in the presence of scaling 
or in sebaceous material but is not responsible for the presence 
of these findings." 

D 

ACTINOMYCES BOVIS 

Bacteriologists and mycologists are not in accord on the sys- 
tematic position of Actinomyces, bacteriologists regarding it as 
among the Schizomvcetes, and mycologists including it among 
the Hvphomycetes. Actinomyces is a large genus and includes 
not only many species that are pathogenic to man and other ani- 
mals, but also a few plant pathogens and many species that are 
normal inhabitants of the soil. 

The mycelium of Actinomyces consists of very slender, 
branched hyphae, commonly about 1 [x in diameter. More or less 
specialized branches become sporogenous and by segmentation 



314 



MEDICAL MYCOLOGY 



form chains of spores. These sporogenous hyphae are coiled, the 
rotation of the helix and the type of coiling being characteristic 
of the species. 

The best known of the species pathogenic to man is Actino- 
myces bovis, described by Harz (1879) in 1879. It causes a 
chronic disease known to the medical profession as actinomycosis 
and characterized by the formation of suppurative tumors. 




Fig. 69. Actinomyces bovis. A. Filamentous appearance of colony in cul- 
ture. B. The hyphae from culture when smeared on microscopic slide 
fragment to become bacteria-like. C. "Sulphur granule" taken from abscess 
and stained to show peripheral clubs. D. View of clubs with magnification 

slightly increased over that in C. 

Farmers and cattlemen are more commonly afflicted than are 
persons in other occupations. The disease involves not only man 
but also such other animals as horses, cows, sheep, and pigs, as 
well as many species of wild animals. In cattle the disease is called 
"lumpy jaw," "wooden tongue," or "sarcoma of the jaw." In 
man A. bovis may involve any part of the body but is most com- 
mon on the head and neck. About 60° of all cases are cervico- 
facial, 14 are thoracic, and 8 to 18°o involve the abdominal 
organs. 

Many cervico-facial cases arise from dental defects or after 
the extraction of teeth. Studies by Emmons (1935) show that in 
a high percentage of instances the causal fungus can be isolated 
from the normal mouth, carious teeth, tonsillar crvpts, or drain- 



SPOROTRICHUM SCHENCKII 315 

ing sinuses. He found it in 47% of extirpated tonsils in Puerto 
Rico. 

Meningitis and endocarditis are amon<? the occasional manifes- 
tations of actinomycosis. In generalized actinomycosis evidence 
indicates that the pathogen is spread through the blood stream. 

Emmons (1935) has shown that some of the confusion regard- 
ing the causal fungus has arisen because it is a microaerophilic 
species and must not be confused with aerobic contaminants. It 
appears to be widespread on vegetation, so that it is inadvisable 
to chew straws, sticks, weeds, or plant stems. Slight wounds ap- 
pear to serve as portals of entry for the fungus into the tissues. 
Further confusion in etiology arises because the aerobic species, 
Actinomyces hominis, is primary in approximately 10% of Actino- 
myces cases. 

Such generic names as Nocardia, Streptothrix, Oospora, and 
Discomyces have been applied to this fungus. Some workers pre- 
fer to use for it the name Actinomyces Israeli. An extensive bibli- 
ography on actinomycosis exists. In a publication by Musgrave 
and his associates (1908) that appeared in 1908 more than 1500 
titles of papers on this disease are assembled. 

In the diagnosis of actinomycosis the presence of granulation 
tissue and of pus-containing "sulphur granules" should be sought. 
These granules are composed of radially arranged hyphae, which 
are terminated peripherally by eosin-staining clubs, the clubs be- 
ing sheathed hyphal tips. Emmons (1935) states that these clubs 
are not formed within tonsillar tissues. Observations by Lentze 
(1938), involving 55 cases of true actinomycosis, showed that 
granules can be demonstrated in 80% of the cases. He placed 
the pus in a drop of methylene blue and noted that the leucocytes 
which take the methylene blue invest a cauliflower-shaped mesh- 
work of threads whose periphery consists of bluish-green clubs. 

SPOROTRICHUM SCHENCKII 

A comprehensive account of the Genus Sporotrichum and the 
clinical aspects of diseases it induces are contained in the mono- 
graphic treatise by Beurmann and Gougerot (1912). One species, 
.S. schenckii, first described by Schenck in the United States in 
1898 [Emmons (1940)] is definitely pathogenic to man. Certain 
other species, including 5. beiirmanni and 5. equi, are believed to 



316 



MEDICAL MYCOLOGY 



be specifically identical with 5. schenckii. This organism com- 
monly enters through some minor injury, such as a barberry, rose, 
or bramble puncture. Evidence also points to inoculation from 
splinters or into abrasions incurred in the work of clearing land. 
An ulcerative lesion that fails to heal develops. Gradually subcu- 
taneous abscesses form that spread along the lymphatics, dissemi- 
nation apparently being hematogenous. Once the disease becomes 




Fig. 70. Sporotrichum schenckii. (Adapted from Moore.) A. Germinat- 
ing spores. B. Yeast-like spores. C. Aleurospores laterally formed in cul- 
ture. D. Terminally formed spores that may become chlamydospore-like. 
E. Both intercalary and terminal chlamydospores may form in culture. 

F. Conidia formed abundantly. 

systemic, various organs, muscles, bones, lungs, joints, and other 
tissues, including the brain and viscera, become involved. 

Sporotrichosis may occur spontaneously in horses, dogs, cats 
and rabbits. Attention was called by du Toit (1942) to the occur- 
rence of 5. beitrmanni on wood, mud, and other materials in a 
mine in the Transvaal, where an outbreak of sporotrichosis in- 
volved 650 among 2500 native miners. The organism was intro- 
duced by a worker. Sterilized wood and mud constituted good 
substrata for the cultivation of S. beurmanni. 

On agar the fungus forms white colonies that may become 
brownish with age. Temperatures within the range 30° to 38° C 
are optimum. The hyphae are much branched, and chlamydo- 
spores appear on media poor in nutrients. Conidiophores are not 



MON1LIA (CANDIDA) SPP. 311 

differentiated, but profuse clusters of ovoid hyaline conidia arise 
laterally or terminally on short branches. 

Beurmann and Gougerot isolated from various plants species 
of Sporotrichum that they believed were pathogenic. Further 
evidence in support of its occurrence on vegetation is supplied 
by Benham and Kesten (1932). They inoculated carnation buds 
with S. schenckii, and a bud rot resembling the well-known bud- 
rot disease caused by S. poae developed. When reisolated, it was 
still virulent for man. These experiments are especially note- 
worthy, because they constitute the first successful transmission 
of a human disease to a plant. 

This fungus is among the few that produce specific agglutinins 
when spores are used as antigens. Spores may be agglutinated in 
high dilutions, indicating strong antigenic properties. 

MONILIA (CANDIDA) SPP. 

Within the Genus Monilia, as used in a medical sense, are in- 
cluded those fungi having sparse mycelial development and repro- 
ducing by budding to form white, smooth colonies on as^ar. The 
numerous species in this genus rather readily dissociate into rou^h 
and smooth colonies on artificial media; they vary in virulence. 
Some of them are filamentous at room temperature but veast- 
like at body temperature, and others are filamentous on ordinary 
agars and yeast-like on blood agar. They are entirely distinct 
from Monilia as used by the plant pathologist to designate conidial 
stages of Sclerotinia. Dodge (1935) places them in the Eremas- 
caceae Imperfectae. 

.Manifestly a number of generic types are represented anions; 
the medical Monilias, and accord has not been reached on their 
proper binomials. These taxonomic problems are set forth clearly 
in a recent report by Conant (1940); it is apparent that for final 
agreement action by the International Botanical Congress will be 
necessary. Amon^ those who have studied the classification of 
this group are Benham (1931), Langeron and Talice (1932), 
Shrewsbury (1934), Lamb and Lamb (1935), Dodge (1935), 
Martin and his associates (1937), and Langeron and Guerra 
(1938). The generic name Candida seems to be the preferred 
one for medical species of Monilia. 



318 



MEDICAL MYCOLOGY 



Morphologic bases for separating species seem to be inadequate, 
since clinically similar cases in the hands of a single investigator 
have vielded organisms that have been placed in as many as a 
half-dozen different species. This fact induced Benham (1931) 
to supplement morphologic differences with variations in fermen- 
tative ability and serologic characteristics. Lamb and Lamb 
(1935) used fermentation and precipitation reactions in specific 





Fig. 71. A. Hvphal elements and budding spores of Candida albicans. B. 

Malassezia oralis, showing thin-walled and thick-walled spores. Growth 

usually results in buds, but short hyphae may be formed. 

separation. Martin and his associates (1937) employed differ- 
ences in growth habits on blood agar, corn-meal agar, and Sabou- 
raud's agar, together with fermentation reactions, in their identifi- 
cation of species. All students of the group clearly recognize 
the inherent variability and dissociative potentialities of Monilia. 

Two species of Monilia are singled out from this aggregate as 
being of most interest. These include M. albicans (Candida tropi- 
calis) and M. psilosis (Syringospora albicans and S. psilosis, re- 
spectively, according to Dodge). 

Monilia albicans is best known in connection with thrush, a dis- 
ease of the throat and mouth of children; rarely it occurs also in 
old or debilitated persons. In addition it is of importance as an 
etiolouic accent in pulmonary moniliasis and may also attack the 
nails, producing chronic paronychia, may involve the mucous 
membranes of the genitalia, or may cause skin lesions on the palmar 



THE TRICHOPHYTON EAE 319 

and interdigital surfaces. It may establish secondary infection in 
pulmonary tuberculosis and has been recorded to be present in 
the mouths of normal, healthy persons in the proportion of 3 to 
24%. 

Monilia psilosis is associated with sprue, a disease primarily of 
the tropics, which involves the intestinal tract. Prolonged diar- 
rhoea and anemia are the most outstanding symptoms. Change of 
climate and vitamin deficiency have also been assigned etiologic 
roles in sprue, and M. psilosis is now generally believed to be a 
secondary cause of the disease. 

ASPERGILLUS FUM1GATUS 

Species of Aspergillus are predominantly saprophytes, but 
Dodge (1935) has assembled published reports showing that ap- 
proximately 30 species may at times be pathogenic. Aspergillus 
jumigatiis is among those that are regularly pathogenic. It attacks 
man, particularly in humid regions, most commonly producing 
symptoms that clinically resemble those of pulmonary tubercu- 
losis. If the sputum is examined, conidia will be found, but no 
trace of Mycobacterium tuberculosis. Neither are tubercles 
formed in the lungs, and upon treatment with potassium iodide the 
lung involvement usually clears promptly. 

Aspergillosis may be regarded as an occupational disease for the 
reason that it is most prevalent among those who work with abra- 
sives, force-feed fowls, or prepare furs or feathers for use as wear- 
ing apparel. The same species involves the lungs of birds, espe- 
cially quails and grouse, and may cause severe epizootics among 
them. 

Some of the other pathogenic species quite regularly involve the 
auditory passages or the nails or are associated with abscesses 
or asthma. 

THE TRICHOPHYTONEAE OR RINGWORM FUNGI 

The Trichophytoneae constitute a group of 100 to 200 species 
of Fungi Imperfecti that parasitize man and various animals by 
invading the keratinized layers of the skin and its modifications, 
such as hair, nails, feathers, hooves, and horns. The resulting 
dermatomycoses are commonly known as ringworm, tinea, dhobie 



380 MEDICAL MYCOLOGY 

itch, barber's itch, athlete's foot, herpes, favus, or kerion. Nearly- 
all these fungi grow readily on any common culture medium, but 
most laboratories employ the standard media of Sabouraud to cul- 
tivate them. On his "proof medium," containing- sugars, growth 
is especially luxuriant, and the various species exhibit their char- 
acteristic cultural aspects. On his "preserving medium," high in 
peptone and lacking sugars, growth is less rapid in most species, 
and pleomorphic changes are inhibited. 

There is nothing about their mycelium to enable the worker to 
differentiate the Trichophytoneae from many fungi commonly 
encountered in the laboratory. Various hvphal structures and 
various types of spores which develop in culture are employed, 
however, to identify and classify the numerous species. They 
may be briefly described, without reference to any particular 
genus or species, as follows: 

a. Racket-shaped hyphae. Sabouraud applied the term 
"raquette cells" to hyphae each of whose cells is of considerablv 
greater diameter at one end than at the other. When these hyphae 
occur in series, they have somewhat the appearance of tennis 
rackets placed end to end. 

b. Terminal clubs. When the apices of hyphae were vari- ' 
ously enlarged, they were called "terminal clubs" by Sabouraud. 

c. Pectinate hyphae. Hyphae bearing short, denticulate pro- 
jections along one side are called "pectinate hyphae." Usually this 
portion of the hypha is curved, and the projections form on the 
convex surface. If the projections appear as short hyphae, they 
are termed "nodular organs." 

d. Spiral hyphae. In certain species the terminal hyphae are 
coiled into a rather tight spiral, making up the so-called "spiral 
hyphae." These structures are regarded by some workers as com- 
parable with the hvphal ornamentations on the peridia of certain 
(A mnoascaceae and as an indication of relationship with this 
family. 

e. Arthrospores. In the parasitic stage the terminal hyphae 
become closely segmented, and the segments round up and be- 
come separate cells. These are the arthrospores, which constitute 
the sole means of reproduction in nature, if the possible existence 
of an ascal stage is disregarded. 

f. Chlamydospores. If chlamydospores are classified on the 
basis of their point of origin, there are three types, terminal, 



THE TRICHOPHYTONEAE 



381 




Fig. 72. Types of reproductive cells produced by Dermatophytes. A-F 
apply to Microsporum; G, to Epidermophyton; H-O, to Trichophyton; 
PS, to Achorion. A. Racket cells of Microsporum. B. Aleurospores. C. 
Pectinate hypha. D. Macroconidia^or fuseaux. E. Nodular organ. F. 
Spiral hypha. G. Epidermophyton macroconidia. H. Aleurospores budding 
from hvphae of Trichophyton in culture. /. Intercalary chlamydospores. 
/. Arthrospores. K. Pectinate hypha. L. Spiral hypha. M. Pedicellate 
chlamydospores. N. Racket cells in series. O. Fuseaux or macrospores. 
P. Aleurospores of Achorion. Q. Chlamydospores. R and S. Pectinate 

hypha. 



382 



MEDICAL MYCOLOGY 






lateral, and intercalary. When the chlamydospores are large and 
spindle-shaped, thev are known as fuseaux, according to the ter- 
minology of Sabouraud. Fuseaux may be borne terminally or 

laterally; they occur singly. 
Thev may be smooth, or the 
entire surface or a portion of it 
may be covered with projec- 
tions. They may consist of one 
cell or be septate. In some in- 
stances lateral chlamydospores 
are borne on a pedicel and 
hence are called "pedicellate 
chlamydospores." This type of 
chlamydospore may be sepa- 
rate from the parent hypha or 
may lack a septation. 

g. Aleuries or aleuro- 
spores. Spores that develop by 
migration of protoplasts from 
the hyphal cell into the devel- 
oping spores, leaving the hy- 
phal cell empty, are known as 
aleuries or aleurospores. They 
may not be abstricted and may 
remain attached, or a septum 
may be laid down. The hy- 
phae that bear aleuries may be 
simple or copiously branched. 
The aleuries of copiously 
branched hyphae may adhere 
in grape-like bunches. 

Classification. The clas- 
sification of the members of 
this group is a difficult task, and considerable disagreement exists 
concerning what characteristics constitute an adequate basis for 
distinguishing genera and species. Clinical aspects of the disease 
complex have been given precedence by some workers, purely 
mycological features by others, cultural characters by others, 
and host relations by others. This situation may be illustrated 
bv the delimitation of the Genus Achorion to include fungi which 







W ) 



/° #t °o 



>. 



•K 



Fig. 73. Diagrams illustrating the re- 
lationship of the fungi to hairs in: 
A. Microsporum. B. Endothrix Tri- 
chophyton. C. Ectothrix Tricho- 
phyton. Spores arise at or near the 
opening of the hair follicle. (After 
Henrici.) 



THE TR1CHOPHYTONEAE 383 

cause favus, whereas it is known that this disease may be caused 
by some species of Trichophytum (Trichophyton) and Micro- 
sporum (Microsporon) as well. Again Microsporum is under- 
stood to include those species which produce a sheath of closely 
aggregated tiny spores, never in chains, around the basal part of 
the hair. In Trichophyton, on the other hand, the infecting 
hyphae are intrapilar and become closely segmented, appearing 
as chains of spores. The spores, however, arise in chains from a 
circumpilar sheath. In some species the circumpilar portion dis- 
appears; these were regarded by Sabouraud as "endothrix." In 
others the circumpilar sheath is the most prominent feature; 
hence these species are termed "ectothrix." The transition group 
between these two constitute Sabouraud's "neoendothrix" species. 

Species of Epidermophyton and Endodermophyton are under- 
stood to be limited to the glabrous skin. The only kind of spores 
formed in culture by Epidermophyton are separate fuseaux; by 
Endodermophyton, arthrospores. 

Several systems of classification have been proposed, including 
those by Sabouraud (1910) and its modifications (1929), by Ota 
and Langeron (1923), by Langeron (1926), by Grigoraki (1925, 
1929), by Langeron and Milochevitch (1930), and by Dodge 
(1935). The system of Sabouraud is fundamental and is in general 
usage among students of this group, since it has the merit of being 
workable. A comparison of his larger groupings with those em- 
ployed by Dodge is shown in Table 28. 

Emmons (1934) found by a study of representative members of 
this group that botanical characteristics exist as means for classify- 
ing them and that such characteristics should replace clinical ones. 
He employed shape, size, and method of formation of conidia to 
separate Trichophyton, Microsporum, and Epidermophyton. 
Trichophyton possesses clavate conidia that are thin-walled and 
have few septations; Epidermophyton, conidia that are clavate to 
ovate and are thick- walled with few septations; and Microsporum, 
conidia that are spindle-shaped, thick-walled, and frequently sep- 
tate. Emmons regarded spirals, chlamydospores, and nodular 
organs as of little value in classification. 

In separating species Dodge (1935) based his key largely on cul- 
tural characteristics of giant colonies. This means of identifica- 
tion becomes increasingly useful as the student gains experience 



384 



MEDICAL MYCOLOGY 





TABLE 28 




Classification 


of Trichophyton eae, 


as Used by Sabouraud 


and by Dodge 


Sabouraud's 


Dodge's 




Type 


Classification 


Classification 


Synonyms 


Species 


Trichophyton 


Trichophyton 




T. tonsurans 


Endothrix 


Endothrix 








Malstenia 




T. tonsurans 




Sabouraudia 




T. sabouraudia 


Neoendothrix 


Neoendothrix 


Neotrichophyton Cast. 




Ectothrix 




Ectotrichophton Cast. 




Megaspores 


Megatrichophyton 


Enutotrichophyton 


M . roseum 


Faviformes 


Favotrichophyton 








Eufavotrichophyton 


Grubyella (pro parte) 


F. ochraceum 




Bodinia 


Bodinia Ota et Lang. 


F. violaceum 


Microides 


Ectotrichophyton 




E. mentagrophxtes 


gypseums 




Spiralia Grig. 


E. mentagrophytes 


niveums 




Microtrichphyton N. 


E. felinum 


Microsporum 


Microsporum 


Sabouraudites Ota et Lang. 




Neomicrosporum 


Neomicrosporum 






Eumicrosporum 


Eumicrosporum 






Achorion 


Achorion 


Grubyella Ota et Lang. 




Neoachorion 


Lophophyton 






Euachorion 


Euachorion 






Epidermophyton 


Epidermophyton 




E. floccosum 




Endodermophyton 




E. concentricum 



with cultures and becomes more and more familiar with them in 
the laboratory routine. 

Studies of the type conducted by Conant (1936, 1936a, and 
1937), in which the investigator has at his disposal a large number 
of species and strains, offers the best means of solving the confus- 
ing taxonomic problems of the Trichophytoneae. Conant made 
biometric studies but may not have had a sufficient number of 
strains of each species to become familiar with the extremes of 
variation within a given species. In criticizing these studies, Em- 
mons (1940) pointed out that, if Conant had examined more 
strains of Micros porinn fulvum and M. gypseum, he probably 
would have regarded them as specifically identical. At any rate 
it must be emphasized that the inherent tendency of all species to 
vary must never be lost sight of by students of this group nor of 
any other group of fungi. 

The difficulties attendant on making specific identification by 
clinical aspects are illustrated by the experiments of Dowding and 
Orr (1937). They isolated Trichophyton gypsevm from three 
clinically distinct diseases, namely kerion, tinea circinata, and vesi- 
cular lesions on feet. 



THE TRICHOPHYTONEAE 385 

Another technique for identification arises from the work of 
Davidson, Dowding, and Buller (1932). They observed that hy- 
phal fusions or anastomoses occur between hvphae of the same 
mycelium or between mvcelia of different origin but of the same 
species, and that no hyphal fusions form between mvcelia of dif- 
ferent species. If, then, the investigator has stock cultures whose 
identity is known, it becomes possible by suitable pairings to estab- 
lish the identity of unknown isolates. In their studies Davidson, 
Dowding, and Buller employed Micros porinn audoiiini, M. lano- 
sinn, and Trichophytum gypsenm. 

Relationship of ringworm fungi with other fungi. Evi- 
dence has been presented to show that the ringworm fungi are 
conidial forms of Gymnoascaceae and that they have lost their 
ability to produce asci. Some of this evidence includes the fact 
that Ctenomyces servants, growing naturally on feathers, possesses 
as peridial ornaments spiral hyphae that are like those of certain 
species of small-spored Trichophyton, and that it forms aleuro- 
spores and spindles in culture. Nannizzi (1926) is among those 
who would classify the ringworm fungi with the Gymnoascaceae. 
He maintained that they should be grown on hair, feathers, horn, 
or skin and that the morphologic structures developed on syn- 
thetic media are all abnormalities. When he cultivated Achorion 
gypsenm on these animal decidua, he reported the development 
of asci and ascospores like those of Gymnoascus. Tate (1929) 
was unable, however, to confirm Nannizzi's findings; moreover 
they remain without confirmation for other species, and hence it 
must be concluded that all these problems of relationships require 
further study. 

Pleomorphism. The phenomena of production of physiologi- 
cal species by fungi and of sectoring, saltation, and mutation were 
discussed in Chapter 7. The term pleomorphism is not to be asso- 
ciated with these phenomena; it applies to a peculiar and confus- 
ing change that is especially prevalent among ringworm fungi. 
When these fungi are grown on sugar-containing media and have 
reached their maximum development, which is usually attained 
after 4 to 6 weeks, white, downy tufts suddenly appear on the 
surface of the mature colonies, suggesting the presence of a sur- 
face contaminant. These tufts grow rapidly, enveloping the 
whole surface and spreading beyond the margin of the primary 
colony, until a mantle of pure white, downy mycelium envelops 



386 MEDICAL MYCOLOGY 

the entire surface. If a fragment of this pleomorphic mycelium 
is planted on a fresh medium, the cultures obtained are like the 
pleomorphic mycelium, and these characteristics are retained on 
subsequent repeated transfer. Pleomorphic forms do not revert 
to the normal once they have been isolated. 

The most striking feature of these pleomorphic colonies is that 
the majority of them remain completely sterile, whereas other 
species may form chlamydospores or may bear small, little differ- 
entiated, lateral spores. For this reason pleomorphic forms of the 
different species are very similar to each other, and identification 
has been difficult and very confusing. Many are so similar, in 
fact, that some workers question the plurality of species among 
ringworm fungi. 

Variation in this tendency to produce pleomorphic forms exists 
among these fungi. Pleomorphism is common among small- 
spored, animal-infecting species of Trichophyton and among spe- 
cies of Microsporon from animals but is rare or non-existent 
among species of Trichophyton attacking man. Tate (1929) 
states that it is not known to occur in Microsporon aiidoidni or in 
Trichophyton radians and T. denticulatum. 

According to Sabouraud, nutritional and temperature factors 
most favorable for growth are also most favorable for the develop- 
ment of pleomorphic forms. The presence of about 4% of carbo- 
hydrates in the medium and constant temperatures of 30° to 37° C 
induced pleomorphic changes, whereas media with 3% of peptone 
and no carbohydrates tended to prevent pleomorphic develop- 
ment. 

When Langeron and Milochevitch (1930) grew Sab our audit es 
asteroides ( the generic termination ites should be limited to genera 
of fossils), S. granulosus, S. lacticolor, and S. gypseus, all of which 
are pleomorphic on sugars, on cereals, straw, dung, or synthetic 
media enriched with dextrin or soluble starch, pleomorphic forms 
did not appear. They concluded that monosaccharides and disac- 
charides are toxic and that these sugars induce pleomorphic 
change, whereas polysaccharides and the colloidal complexes of 
the natural substrata are not toxic and may be used without first 
being cleaved. 

Emmons (1932) cultured Achorion gypseuni on horn, starting 
with a single aleurospore. Six distinct pleomorphic variants arose 
from the progeny, and all were so different that, if they had been 



THE TR1CHOPHYTONEAE 381 

isolated from lesions on patients, they might have been regarded 
as distinct species. When subcultures were isolated, using aleuro- 
spores or fuseaux from these variants, each produced a culture 
like those from the particular variant from which it originated. 
Furthermore none of the variants reverted to the parent form. 
Three different kinds of pleomorphic forms are also known for 
Micro sporum lanosum, and all are reversible to each other but 
not to the primary form. The three include a coarse, shaggy, 
downy form (the most common one), a white, downy form, and 
an immersed, glabrous, brown form. 

The most remarkable feature of pleomorphism is exhibited by 
the results of animal inoculations. When used as inoculum, the 
pleomorphic forms produce lesions that are indistinguishable from 
those arising from inoculum with the primary or normal form. 
When the fungus is reisolated from the infected hairs or scales, it 
invariably grows like the pleomorphic form. Langeron and Talice 
(1930) used the pleomorphic form of Sabonraudites felinus as 
inoculum, obtained a typical lesion on guinea pig, and were able 
to reisolate only pleomorphic mycelium. In its normal parasitic 
phase this fungus consists of an ectothrix sheath of spores sur- 
rounding the infected hair and of hyphae internal to the hair. In 
the pleomorphic form the ectothrix sheath was without spores. 
If the pleomorphic culture used as inoculum is completely pleo- 
morphic and quite sterile, the cultures reisolated from scales and 
hairs are likewise quite sterile. 

Mycides. In 1912 Jadassohn made the interesting observation 
that primary localized infections (mycoses) by species of Tricho- 
phytoneae may be accompanied by secondary lesions (mycides) 
on distant parts of the body in which no fungus can be found. 
These mycides have come to be designated as trichophytides, epi- 
dermophytides, microsporides, etc., depending upon the genus 
responsible for the primary lesions. Jadassohn [Gregory (1935)] 
explained this phenomenon as an allergic reaction, since he found 
that secondary lesions could be produced by rubbing the spores 
into the skin of other children. This external origin of mycides, 
however, has not been substantiated in subsequent investigations. 
Instead they have been determined to arise from spores or toxic 
products of the pathogen liberated in the primary lesions and dis- 
seminated by the blood stream. The reaction appears, therefore, 
to result from hypersensitivity to the fungus protein. Evidence 



388 MEDICAL MYCOLOGY 

in support of internal origin comes from the symmetrical distribu- 
tion of rashes or eruptions (the u id" lesions) on the body surface 
and from the isolation of spores from the circulating blood. Greg- 
ory (1935) summarized the findings of various workers regarding 
the isolation of fungi from the blood. He noted that among the 
fungi isolated are Trichophyton inter digit ale, T. granulosum, T. 
gypseinn, T. cerebrifonne, A chorion schoerleini, A. qiiinckeanuni, 
and Microsporia!! audouini. 

Wise and Wolf (1936) pointed out that the vesicular eruptions 
on the hands of patients with primary mycotic infections on the 
feet may not necessarily be ids. In their opinion, however, such 
eruptions, except in persons with occupational eczema or eczema 
of unknown cause nearly always occur coincidentally with in- 
fection of the feet. 

Species other than Trichophytoneae may evoke the formation 
of ids, as Monilia | Hopkins ( 1932) ] and Sporotrichum are known 
to do. Evidence presented by Hopkins shows that Monilia in the 
alimentary tract may produce substances which induce skin lesions 
in the sensitized person. 

Some persons possess a related allergy to such common fungi 
as Cladosporium, Penicillium, and Aspergillus, present in house- 
hold dust or in clothing. Consideration of this subject is outside 
the province of this book; the student may introduce himself to 
this problem by consulting the report by Rackemann, Randolph, 
and Guba (1937-38). They found that the tomato-mold fundus, 
Cladosporhnn fulvum, may irritate the nasal mucosa and eyes, 
producing asthma. 

Ids may also appear on sensitized animals. De Lamater and Ben- 
ham (1938) inoculated Trichophyton gypseinn through the skin 
and into the blood stream of guinea pigs, whereupon widely dis- 
seminated fungus-free lesions developed. 

Fluorescence. Margarot and Devese (1924-25) made the in- 
teresting observation that the affected hairs of patients with Alicro- 
sporum ringworm or with favus and also cultures of the causal 
fungi exhibit a greenish fluorescence if examined with ultraviolet 
light filtered through Wood's nickel oxide glass. This discovery 
has proved a useful tool in diagnosis. Others have confirmed and 
extended these observations and have sought an explanation of the 
source of these fluorescent properties. Kinnear ( 1931 ) concluded 
that fluorescence is resident in the fungus itself in the case of 



THE TRICHOPHYTON EAE 389 

Microsporwn audowni, Trichophyton crateri forme, T. acumina- 
tum, T. sulfureum, and T. polygonum and that it is retained in the 
hairs when treated with potassium hydroxide for indefinite 
periods. In endothrix trichophyta and in favus, however, fluo- 
rescence was attributed to keratin of the hairs. 

Davidson and Gregory (1932) noted that the ectothrix tricho- 
phyta, Trichophyton gypseum and T. album, do not exhibit 
greenish fluorescence, but with certain species with endothrix 
hvphae, such as Achorion schoenleinii fluorescence resides in the 
hair, as Kinnear (1931) maintained. They extended their obser- 
vations by defatting, in warm water or in ether, hairs infected by 
Microsporwn audpuini, M. felinum, or Achorion schoenleini and 
then extracting with potash and secured a fluorescent extract. The 
hairs so treated were no longer fluorescent. Normal hairs and 
Trichophyton-infected hairs, moreover, did not yield a fluores- 
cent substance by this same procedure. These results may be re- 
garded as proof that the invading fungus produces some hydro- 
lytic change in the hair substance and that this product has fluo- 
rescent properties. The exact nature of the substance still remains 
unknown. 

Physiologic activities. Both Tate (1929) and Dodge (1935) 
have briefly reviewed the publications dealing with the physiology 
of the Trichophytoneae that are peculiarly adapted to living on 
keratinized tissues. Since the early studies- of Verujsky (1887) on 
the activities of Trichophyton tonsurans and Achorion schoen- 
leini many investigators have been concerned with the physiology 
of this group of fungi. Verujsky found that both species grow 
best in neutral or slightly acid media, with 3 3 ° C the optimum tem- 
perature. Both produce proteolytic enzymes, as is evidenced by 
the liquefaction of gelatin. Trichophyton tonsurans can utilize 
glucose and maltose, but A. schoenleini does not possess the ability 
to ferment these sugars. 

A considerable number of these fungi have been grown in pure 
culture for periods varying from a few months to two years with- 
out loss of virulence, such substrates as feathers, horn, leather, silk, 
straw, and wood [Dodge (1935)] being employed. The organ- 
isms tested include Trichophyton flavum, T. floccosum, T. granu- 
losum, T. inter digit ale, T. mentagrophytes, Achorion gypseum, 
and A. muris. 



390 MEDICAL MYCOLOGY 

Roberts (1894) tried unsuccessfully to demonstrate a "kero- 
lvtic" enzyme bv growing certain species on hairs as a substrate. 
Later Tate (1929a) failed to demonstrate a keratin-cleaving 
enzvme in Trichophyton radiolatum, T. tonsurans, Microsporuiu 
lanosum, M. audouini, or Achorion schoenleini. All species were 
capable of utilizing maltose, starch, casein, and tributyin and, 
except for T. tonsurans, urea. None, however, produced peptase, 
invertase, lactase, zymase, and inulase. 

Goddard (1934), employing Trichophyton interdigitale and 
Microsporia!! lauosuiu, found that both showed increased growth 
in media containing glucose, mannose, fructose, and arabinose. 
There was a slight increase with sucrose, but not with lactose. 
Casein and peptone were hydrolyzed to amino acid and ammonia, 
with a sparing action in the presence of glucose. 

The production of pigments among Trichophytoneae and the 
properties of these pigments have been given consideration by 
Tate (1929a) and others. Such species as Trichophyton acumi- 
natum, T. magnini, T. vinosnm, Sabouraudites ruber, and 5. radio- 
latus form red to reddish brown pigments, which are soluble in 
dilute acids and acid alcohol. In these solvents the color is yellow, 
changing to a reddish hue if alkali is added. Reversal of 'color 
change may be accomplished repeatedly. Evidence indicates that 
these are anthracene pigments like those in Physcia and certain 
other lichens. 

IMPLICATIONS 

Medical mycology is still in its infancy. This conclusion is 
evident to staff members of hospitals where there are practitioners 
trained to recognize and diagnose mycoses. In hospitals not so 
staffed the etiologic role of fungi is not even suspected in many 
instances. This condition will continue to exist until this subject 
receives proper consideration in the curricula of medical schools. 

A "run-of-the-mine" mycologist could not expect to contribute 
materially to medical mycology. To become a medical mycolo- 
gist, he should supplement his training by the usual courses re- 
quired for a degree in medicine, with additional special training in 
bacteriology, biochemistry, pathology, and immunology. Finally, 
his laboratory should be so located as to insure ready contact 
with the clinical aspects of fungus diseases. 



LITERATURE CITED 391 



LITERATURE CITED 

Baker, E. E., E. M. Mrak, and C. E. Smith, "The morphology, taxonomy, 
and distribution of Coccidioides immitis Rixford and Gilchrist 1896," 
Farloivia, 1: 199-229, 1943. 

Benham, Rhoda W., "Certain Monilias parasitic on man, their identification 
by agglutination," /. Infectious Diseases, 49: 183-215, 1931. 
"The fungi of blastomycosis and coccidioidal granuloma," Arch. Der- 
matol. Syphiloid 30: 385-400, 1934. 

Benham, Rhoda W., and Beatrice Kesten, "Sporotrichosis, its transmission 
to plants and animals," /. Infectious Diseases, 50:437-^58, 1932. 

Beurmann, L., and H. Gougerot, Les Sporotrichosis. 825 pp. Felix Alcan, 
Paris, 1912. 

Brumpt, E., Precis de parasitologic Masson et Cie, Paris. 1935. 

Carrion, A. L., "Chromoblastomycosis," My col., 34:424-441, 1942. 

Conant, N. F., "Studies in the genus Microsporum. I. Cultural studies," 
Arch. Dennatol. Syphilol., 33: 665-683, 1936. 

II. "Biometric studies," Arch. Dermatol. Syphilol., 34: 79-89, 1936a. 

III. "Taxonomic studies," Arch. Dermatol. Syphilol, 35:781-808, 1937. 
"The occurrence of a human pathogenic fungus as a saprophyte in na- 
ture," My col, 29: 597-598, 1937a. 

"The taxonomy of anascosporous yeast-like fungi," My copathologia, 

2:255-266, 1940. 
"A cultural study of the life cycle of Histoplasma capsidatum Darling 
1906," /. Bact., 41: 563-578, 1941. 

Conant, N. F., and D. S. Martin, "The morphologic and serologic rela- 
tionships of the various fungi causing dermatitis verrucosa (chromo- 
blastomycosis)," Am. J. Trop. Med., 11: 553-577, 1937. 

Conant, N. F., D. S. Martin, D. T. Smith, R. D. Baker, and J. L. Calloway, 
Manual of clinical mycology. 348 pp. W. B. Saunders Co., Philadel- 
phia. 1944. 

Davidson, A. M., Eleanor S. Dowding, and A. H. R. Buller, "Hyphal fu- 
sions in dermatophytes," Can. J. Research, 6: 1-20, 1932. 

Davidson, A. M., and P. H. Gregory, "Note on an investigation into the 
fluorescence of hairs infected by certain fungi," Can. J. Research, 7: 378- 
385, 1932. 

Dodge, C. W., Medical mycology. Fungous diseases of man and other 
animals. 900 pp. C. V. Mosby'Co., St. Louis. 1935. 

Dowding, Eleanor S., and H. Orr, "Three clinical types of ringworm due 
to Trichophyton gypseum" Brit. J. Dermatol. Syphilis, 49: 298-307, 
1937. 

Emmons, C. W., "Pleomorphism and variation in the dermatophytes," Arch. 
Dermatol. Syphilol, 25:987-1001, 1932. 
"Dermatophytes : a natural grouping based on the form of the spores and 

accessory organs," Arch. Dermatol. Syphilol, 30:337-362, 1934. 
"Actinomyces and actinomycosis," Puerto Rico J. Pub. Health Trop. 
Med., 11:63-76, 1935. 



392 MEDICAL MYCOLOGY 

Emmons, C. W., u Hormodendron pedrosoi, an etiologic agent in chromo- 
blastomvcosis," Puerto Rico J. Pub. Health Trop. Med., 11: 639-650, 
1936. 
'Medical mycology," Botan. Rev., 5:474-514, 1940. 

"Isolation of Coccidioides from the soil and rodents," U. S. Pub. Health 
Rept.,51: 109-111, 1942. 

Emmons, C. \V., and A. L. Carrion, "Sporulation of the Phialophora type in 
Hormodendrum," Mycol., 29:327-333, 1937. 

Freeman, Walter, "Torula infection of the central nervous system," 
/. Psych. Neur., 43: 236-345, 1931. 

Goddard, D. R., "Phases of the metabolism of Trichophyton interdigitale 
Pricstlev," /. Infectious Diseases, 54: 149-163, 1934. 

Gregory, P'. H., "The dermatophytes," Biol. Rev., 70:208-233, 1935. 

Grigoraki, L., "Recherches cytologiques et taxonomiques sur les dermato- 
phytes et autres champignons parasites," Ann. sci. not. Botan., Ser. 10, 
7: 165^44, 1925. 
"Dermatophytes et dermatomycoses," Ann. Derm. Syphiligr., VI, 10: 18- 
53, 1929. 

Harz, C. O., "Actinomyces bovis, ein neuer Schimmel in den Gewebcn 
des Rindes," Dent. Z. Tiervied., Suppl. to Bd. 5 (Jahresber, Central- 
Tierarznei Schule in Mi'tnchen, 1877-78), pp. 125-140, 1879. 

Hopkins, J. G., ".Moniliasis and moniliids," Arch. Derm. SyphiloL, 25:599- 
614, 1932. 

Howeel, Arden, "Studies on Histoplasma capsulatum and similar form spe- 
cies. I. .Morphology and development," Mycologia, 31: 191-216, 1939. 

Kinnear, J., "Wood's glass in the diagnosis of ringworm," Brit. Med. /., 
7:791-793, 1931. 

I.vmater, E. D. de, and R. W. Benha.m, "Experimental studies with derma- 
tophytes," /. Investigative Dermatol., i: 451—488, 1938. 

Lamb, J. H., and .Margaret L., "A grouping of the Monilias by fermentation 
and precipitation reactions," /. Infectious Diseases, 56:8-20, 1935. 

Langeron, .M., "Travaux recents sur la classification des dermatophytes." 
Ann. parasitol. humaine comparee, 4: 193-198, 1926. 

Langeron, .M., and P. Guerra, "Xouvelles recherches de zymologie medi- 
cale," Ann. parasitol., 76:36-84, 162-179, 429-478, 481-525, 193S. 

Langeron, M., and S. Milochevitch, ".Morphologie des dermatophytes sur 
milieux naturels et milieux a base de polysaccharides. F.ssai de classifi- 
cation,"' Ann. parasitol humaine comparee, 8: 465-508, 1930. 

Langeron, M.. \\d R. Y. Taeice, "Noveau type de lesion pilaire experimen- 
tale produite par la culture purement pleomorphique du Sabouraudites 
felinus." Ann. parasitol. humaine comparee, 8: 419-421, 1930. 
"Xouvelles methodes d'etude et essai de classification des champignons 
levuriformes." Ann. parasitol. humaine comparee, 10: 1-89, 1932. 

Lentze, T. A., '"Zur Bakteriologie und Vakzintherapie der aktinomykose," 
Zentr. Bakt. Parasitenk., Orig., 141: 21-36, 1938. 

Lewis, Geori.e M., and Mary E. Hopper, An introduction to medical my- 
cology. 342 pp. The Year Book Publishers, Inc., Chicago. 1943. 



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MacKee, G. M., and G. M. Lewis, "Dandruff and seborrhea. I. Flora of 

normal and diseased scalps," /. Investigative Dermatol., 1: 131-139, 1938. 
Margarot, J., and P. Devese, "Aspect de quelques dermatoses en lumiere 

ultrapara-violette," Bull. soc. sci. vied. biol. Montpellier, 6: 375-378, 

1924-25. 
Martin, D. S., "The antigenic similarity of a fungus, Cadophora americana, 

isolated from wood pulp to Phialophora verrucosa isolated from patients 

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North Carolina," Am. J. Trop. Med., 16: 593-618, 1936. 
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in relation to medicine, Chap. I, pp. 11-70. H. M. Stationery Office, 

London. 1931. 
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"Generalities concernant les dermatophytes. II. La classification naturelle 

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27:31-54, 1929a. 



S94 MEDICAL MYCOLOGY 

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Dermatol. Syphilol., 34: 1-14, 1936. 



Chapter 17 
GEOGRAPHICAL DISTRIBUTION OF FUNGI 

Plant geography is admittedly a tremendously valuable branch 
of botanical knowledge, and its fundamentals, in relation to mosses, 
ferns, and especially seed plants, are now relatively well under- 
stood. Apparently, however, for reasons that will become mani- 
fest in the discussion which follows, any consideration of the geo- 
graphical distribution of fungi at this stage of mycological devel- 
opment has a limited usefulness, partly because to date this phase 
of inquiry has received little attention. Nevertheless it should 
eventually come to be recognized as having a very practical and 
very general interest. 

Bisby (1933) has said, "Mycologists have been able to map with 
accuracy the geographic distribution of comparatively few fungi." 
The worker who turns his attention to this subject is early im- 
pressed with the fact that vast portions of the earth's surface re- 
main completely unexplored for fungi and hence are literally terrae 
incognitae fungornm. Such distributional data as are contained 
in monographs on special groups of fungi or in accounts of species 
of economic importance afford a basis for certain generalizations. 
Much additional pertinent information has been catalogued in 
herbaria but remains unpublished and hence quite unavailable. 

In a report Bisby and Ainsworth (1943) state that the exact dis- 
tribution of but few of the 3600 genera including 37,000 "good" 
species of known fungi has been determined. Distribution of 
genera by continents, as given by Bisby (1943), is as follows: 
Europe 1800, North America 1700, South America 1100, Asia 
1100, Africa 800, and Australia-New Zealand 600. 

On first thought it might appear that the nutritional factor 
should be all important in determining the distributional range of 
fungi for the reason that they are either saprotrophic or para- 
trophic in food habits. Food is not, however, the sole factor, for, 
just as with holophytic plants, natural distribution has been found 

395 



396 GEOGRAPHICAL DISTRIBUTION OF FUXGI 

to be governed bv the interaction of interrelated and interde- 
pendent factors, climatic, edaphic, and biotic. 

The validity of this conclusion becomes apparent if, for ex- 
ample, an attempt is made to deduce the probable distributional 
range of certain parasitic fungi from knowledge of the range of 
their hosts. The student may think that each parasite should be 
coextensive in range with its host (suscept), only to discover that, 
although such is the situation among certain species, it is not 
among others. Such observations lead to the conclusion that 
endemism exists, that is, certain fungi are indigenous on native 
species within particular areas where they have become dispersed 
by natural agencies. These certain species may eventually be 
spread to areas outside their natural range, but only as a result of 
"artificial" introduction, chiefly by man. In a very real sense 
man has interfered in no small measure with the natural factors 
that influence the distribution of fungi. To retard or prevent 
artificial dissemination of pathogenic species, quarantines have 
been instituted, eradication campaigns have been organized and 
conducted, inspection procedures have become compulsory in 
connection with shipment of plants or plant parts from one local- 
ity to another, and researches have been and are being made to 
produce hosts that are resistant to disease. Problems which have 
arisen as a result of disturbance of biological balance by man and 
of his attempts to rectify them, therefore, constitute an interesting 
and important phase of the geography of fungi. 

For a discussion of the structural features possessed by certain 
species that aid in their geographical distribution the reader is 
referred to Chapter 8, Spore Dissemination. In this chapter in- 
formation will also be found on such natural agencies as air cur- 
rents, rains, streams, floodwaters, and insects and various other 
animals as factors in distribution. In a very real sense fungi tend 
continually to extend their range, some behaving as settlers and 
others as tourists. 

In the account that follows, greatest emphasis will be placed 
on the geographic distribution of fungi as modified by man 
through the introduction and cultivation of exotic plants of eco- 
nomic importance. The presentation will not follow the logical 
arrangement based on the distribution of fungi as governed by 
climatic, edaphic, and biotic factors, but will be artificial and will 
be based on fungus groupings. 



DISTRIBUTION OF MYXOMYCETES 391 

DISTRIBUTION OF MYXOMYCETES 

Collectors of Mvxomycetes are quite universally inclined to 
the opinion that this group is among the most ubiquitous and cos- 
mopolitan of organisms. Intensive collecting, even in restricted 
areas at widelv distant points, has yielded for each locality only 
from one-third to one-half of all the species known throughout 
the world. Nevertheless the geographical distribution of slime 
molds is not fortuitous but depends upon such dominant factors 
in each locality and for each species as temperature, moisture, 
kind of substrate and its acidity or alkalinity, and other factors. 

More species of slime molds have been recorded for temperate 
regions than for the tropics, but this phenomenon appears to be 
causally related to the greater interest in collecting in the tem- 
perate zones. Some species, however, are limited to temperate re- 
gions; others, such as Trichamphora pezizoides and Alwisia bom- 
bar da, to tropical or subtropical regions [Martin (1940)]. This 
observation need not necessarily be interpreted as proof that tem- 
perature is the primary and controlling factor in determining the 
range of slime molds in general. Otherwise it becomes difficult 
to explain numerous observations like those of Smith (1931), who 
noted that in Colorado species of Badhamia prefer decaying aspen 
or cottonwood logs, whereas species of Cribraria are restricted 
to coniferous wood. As a result of several years' experience in 
collecting slime molds, Smith (1931) concluded that moisture, 
especially adequate rainfall for considerable periods, is the primary 
desideratum for their growth, the proper kind of decaying vege- 
table matter being; secondary. He correlated the Greater rainfall 
at elevations of 8000 to 9000 feet in Colorado with the greater 
abundance of species. Even though he collected Stemonitis fusca, 
Comatrichia nigra, and several species of Cribraria and Arcvria on 
dry exposed slopes, they invariably were found only on the lower 
side of logs kept moist by melting snow. The fact that the lower 
side of logs is preferred by slime molds is not regarded as a re- 
sponse to gravity, an opinion on which there is general accord. 
Smith (1931) and MacBride (1914) do not contend that any of 
the species they collected in the high mountains near the timber 
line are alpine. 



398 GEOGRAPHICAL DISTRIBUTION OF FUNGI 

The constant occurrence of lime granules as a constituent part 
of the fructifications among species of Badhamia, Craterium, 
Diderma, Diachea, Didvmium, Fuligo, Leocarpus, Alucilago, and 
Phvsarum and their absence in others, for example, among jComa- 
trichia and Stemonitis, are not without significance. Carr (1939) 
reported that 90% of the species on sandstone soils in a region in 
Virginia are "non-lime species" and 88% of those on limestone 
soils in this same region are "lime species." From comparison of 
collections made in Sweden with those made along the border 
between Bolivia and Argentina, Fries (1903) concluded that lime- 
containing species predominate over non-lime-containing species 
in the tropics, but that the reverse is true in temperate regions. 
That regional distribution is not determined entirelv by the cal- 
careousness of soils is borne out by the findings of other collectors, 
as Martin (1940) has indicated. 

Thorn and Raper (1930) found that numerous species may be 
isolated from arable soils, where they occur in the plant debris 
and litter. Abundant evidence is at hand to show that they sub- 
sist upon various fungi and bacteria that decompose plant re- 
mains. The influence of food in distribution is further evidenced 
by the rather constant occurrence of certain species among mosses, 
of others on decaying coniferous leaves, and of others on decaying 
leaves of hardwoods. 

Some slime molds may develop well above the ground. Smith 
(1931) collected Lycogala fusco-flavum and Mucilago spongiosa 
var. solida 8 to 10 feet up on exposed, heart-rotted trunks of Cot- 
tonwood. The plasmodium of some species, such as Phy sarin n 
cinereum, may climb upon blades of grass or other vegetation 
immediately before becoming transformed into sporangia. 

Plasmodiophora brassicae is now essentially world-wide in dis- 
tribution. Its wide host range among cruciferous species and its 
preference for acid soils constitute the important factors that have 
contributed to this broad range. Evidence indicates that for over 
200 years market gardeners have contended with club-root disease, 
which it causes on cabbage, radishes, and turnips. In Europe it is 
most destructive in the northern portions of the continent. It has 
been reported from nearly all parts of the United States and from 
Alaska and Canada. A monograph on Plasmodiophorales by 
Karling (1942) contains an extensive bibliography on the range 
of this organism and on other features. 



DISTRIBUTION OF PHYCOMYCETES 399 

Spongospora subterranea, causing powdery scab of potato, ap- 
pears to be endemic to Equador and Peru. It has become estab- 
lished throughout the British Isles, continental Europe, .Madagas- 
car, the area bordering the Mediterranean Sea on the east and 
south, South Africa, New Zealand, and Tasmania. The shipment 
of infected tubers from one region to another undoubtedly is the 
primary means of dispersal of this organism. 



DISTRIBUTION OF PHYCOMYCETES 

The lists of Bisby and his associates (1929) and of Bisby 
(1933) indicate that 85% of all Phycomycetes present in Mani- 
toba and 40% of those in India occur also in Europe. Of 35 
species, mostly Mucorales, present in soil in North America 26 are 
also European. The significance of nutrition as a factor in distri- 
bution among Phycomycetes is apparent when the Peronosporales 
are considered in contrast to other phycomycetous orders. The 
distribution of Peronosporales, all obligate parasites, is definitely 
limited by that of the hosts. 

Less is known regarding the distributional range of saprophytic 
species of Phycomycetes in general than that of pathogenic spe- 
cies, but Rhizopus nigricans and Mucor mucedo, both cosmopoli- 
tan species, are notable exceptions. Seemingly both can thrive 
wherever man lives, and both utilize the remains of numerous 
kinds of plants as food. 

Students of soil fungi have shown that species of Mucor are 
universally present in arable soils and also in many virgin soils. 
Another soil-borne genus is Allomyces, which is peculiarly suited 
to wet sites, its members being commonly regarded as "water 
molds." Allomyces arbusciila, representative of this genus, has 
been collected in wet soil on all continents. Since water molds 
do not thrive in the oceans and since A. arbuscula is unable to 
tolerate salinity, no explanation of its wide geographical range is 
forthcoming. 

The distribution of coprophilous Phycomycetes, such as species 
of Pilobolus, is conditioned, not by climate and soil, but only by 
the migration of the herbivor. Browsing animals eat the sporangia 
that are lodged on vegetation near dung piles. The spores germi- 
nate when voided with the feces, and within a few days Pilobolus 
will mature a crop of sporangia and discharge them. 



400 GEOGRAPHICAL DISTRIBUTION OF FUNGI 

Endemic species, artificially dispersed. The existence of 
endemism among pathogenic Phvcomycetes can be shown by 
numerous examples. Among them is Physodcnua zeae-maydis } 
which is known to have existed for about 40 years in the south- 
eastern United States, throughout the area south of the Ohio River 
and east of the Mississippi River. It is, however, found sparingly 
in the Corn Belt north and west of this range, but here it occurs 
sporadically. It produces serious losses within its normal range, 
especially in low, poorly drained lands during seasons of abun- 
dant and frequent rains. This fungus has been dispersed widely 
outside the United States, as is shown by records of collections in 
India, China, Japan, Rhodesia, Sierra Leone, Guatemala, and Mex- 
ico. It would be expected to occur wherever corn has been in- 
troduced, provided that moisture and temperature are favorable. 

Another example of this kind is the organism that causes potato 
wart, Synchytrium endobioticum, first described from Hungary 
in 1896. Soon thereafter it was encountered in other portions of 
central Europe, where it is presumably indigenous, for example, in 
Czechoslovakia, Poland, Silesia, Austria, and Germany. In 1902 
it was reported in the British Isles, in 1912 in Canada, in 1918 in 
the United States, in 1922 in South Africa, and in 1929 in Peru 
and Russia. Meantime stringent quarantines were established in 
many countries to prevent the introduction and spread of this 
organism. 

Among the classic examples of a pathogenic species that has 
been artificially dispersed is Fhytophthora infestans. This fungus, 
native of the northern Andes, home of the potato, was introduced 
into Europe and North America between 1830 and 1840. In 1845 
and 1846 an cpiphvtotic so severe as to cause failure of the potato 
crop occurred throughout northern Europe, especially in Ireland, 
where famine resulted. This pathogen was introduced into India 
between 1870 and 1880 and into Australia and South Africa be- 
tween 1900 and 1910. 

North America has contributed an organism, Plasmopara viti- 
colii, which becomes notoriously destructive when introduced 
into new areas. This downy mildew was first described in 1834 
and was transplanted into France with grape nursery stock early 
in the I870's. There it produced an epiphvtotic in 1879 and 
rapidly spread throughout the vineyards of France and Italy. Ef- 
forts to check this grape disease led to the discovery and use of 



DISTRIBUTION OF PHYCOMYCETES 401 

Bordeaux mixture as a fungicide. By 1907 the malady had reached 
South Africa, and in 1917 it caused the first severe outbreak in the 
vineyards of Australia. 

The tobacco downy mildew, Peronospora tabacina, endemic to 
Australia, seemingly has been introduced both into North America 
and South America, and there seems no reason for supposing it 
will not spread eventuallv to other continents or countries. Evi- 
dence indicates that it has been known in Australia for more than 
50 years. It first appeared in Florida in 1921 and in Rio Grande 
do Sul, Brazil, in 1938. Apparently it was eradicated from Florida 
in the first season of its introduction, but it reappeared in 1931. 
Since then it has gradually spread northward in the United States, 
reaching Connecticut and Massachusetts in 1937 and southern 
Ontario, Canada, in 1938. Clayton and Stevenson (1943) are of 
the opinion, however, that this fungus is native to all temperate 
regions having an indigenous Nicotiana flora. 

Influence of latitude. Meager data are available on latitude 
as a factor in limiting the range of Phycomycetes, but it is ap- 
parent that climatic zonation occurs. Phytophthora parasitica var. 
nicotianae, for example, is regarded as tropical and subtropical 
and was first recorded on tobacco from the East Indies in 1896. 
Since then it has been found to occur on this crop in India, Japan, 
Indo-China, the Philippine Islands, Nyasaland, Cameroons, 
Uganda, Rhodesia, Puerto Rico, Jamaica, and Guatemala. More 
recently the disease appeared in Florida, North Carolina, Virginia, 
and Kentucky and in Greece, Rumania, and Bulgaria, all of which 
are in the North Temperate Zone. In tropical regions tobacco 
plants of all ages are subject to attack, whereas in more northerly 
areas infection does not occur until the warmest weather, at which 
season the plants are essentially mature. 

Choanephora cucurbitamm grows most abundantly in tropical 
and subtropical regions but extends into adjacent temperate zones. 
Collections of it have been reported throughout the East Indies, 
Malaya, Burma, India, the Gold Coast, Sierra Leone, the West 
Indies, and the southern United States, commonly on fading flow- 
ers of cotton, okra, althea, squash, watermelon, cowpea, chili, 
cassava, papaw, peanut, hibiscus, and dahlia. The closely related 
Blakeslea trispora is quite restricted to tropical and subtropical 
areas having abundant and frequent rains. During 1942, which 



402 GEOGRAPHICAL DISTRIBUTION OF FUNGI 

was a very wet season, however, this fungus was noted on tobacco 
flowers in the vicinity of Durham, North Carolina. 

DISTRIBUTION OF ASCOMYCETES 

This discussion of the distribution of Ascomycetes must of 
necessity be fragmentary and is in no sense proportional to the 
vast bodv of data on this group that has been accumulated. 
Nevertheless the material is believed to be representative for the 
group as a whole. Many species of great economic importance 
have been spread by man to the extent that they now occur in all 
countries where the hosts are cultivated. The severity of the dis- 
eases which thev cause is modified, to be sure, by latitude, by 
seasonal differences in climate, or by application of palliative or 
control measures. Fungi of this kind include Taphrina deformans 
and Sclerotinia jracticola on peach and Venturia inaeqitalis on 
apple. Venturia inaequalis occurs througout the United States, 
Mexico, and Canada, but it rarely is found in the Coastal Plains 
of the southeastern United States. In Europe it has been noted in 
the British Isles, Belgium, Netherlands, Portugal, France, Switzer- 
land, Norway, Sweden, Denmark, Germany, Austria, Czecho- 
slovakia, Russia, Greece, and Bulgaria. Moreover it is reported 
from India, New Zealand, Tasmania, Rhodesia, Morocco, Argen- 
tina, and Peru and mav therefore be presumed to be global in 
distribution. 

The observations of Fawcett and Lee (1926) show that Dia- 
porthe citri on citrus is another in the same group of organisms. 
This fundus was first studied in Florida in 1892 and was subse- 
quently found in Brazil, Argentina, Mexico, the West Indies, 
China, Japan, Palestine, Algeria, South Africa, and South Aus- 
tralia. Its symptom-complex includes dying bark of twigs, stem- 
end rot of ripe fruits, and melanose markings on leaves, twigs, and 
fruits. The melanose form of the disease does not occur in Cali- 
fornia or at least is very rare, whereas it is always very abundant 
in central Florida. 

The fact that pathogenic species, especially, are capable of 
maintaining themselves for indefinite periods saprogenically, as is 
Thielavia basicola (Tknelaviopsis basicola), constitutes a compli- 
cating factor in distribution. Thielavia basicola attacks many spe- 
cies of legumes, especially beans, clovers, lupins, peas, soybeans, 



DISTRIBUTION OF ASCOMYCETES 403 

and vetches, but may also seriously involve tobacco, flax, cotton, 
and watermelon. Collection records indicate its presence in Cen- 
tral Asia, the Philippine Islands, Queensland and New South Wales 
in Australia, the British Isles, Russia, Turkey, Rumania, Hungary, 
Czechoslovakia, Germany, France, Switzerland, Italy, Puerto Rico, 
the United States, and Canada. 

Distribution of powdery mildews. Of the 60 species and 
varieties of Erysiphaceae listed by Salmon (1900), 22 are confined 
to the Old World and 19 to the New World, leaving 19 that are 
common to both hemispheres. This situation is accounted for in 
part by the limitation of certain mildews to particular hosts and 
to the seemingly almost complete lack of specialization in other 
species. Erysiphe tortilis, confined to Cormis sanguinea, Uncinula 
geniculata, to Morns rubra, and Podosphaera biuncinata, to Hania- 
nielis virginiana, are examples of highly restricted species. Unci- 
nula aceris is limited to species of Acer, U. flexitosa to Aesculus, 
and Spaerotheca lanestris to Quercus. Less restriction is exhibited 
by Erysiphe graminis, which occurs only on various Gramineae, 
by Uncinula salicis on Salicaceae, and by Sphaerotheca pannosa on 
Rosaceae. On the other hand, Erysiphe cichoracearum, E. poly- 
gon! , and Phyllactinia cor y lea are world-wide and attack a wide 
range of hosts. Phyllactinia corylea, for example, is recorded on 
48 host-genera in 27 families of plants. Continental distribution 
of powdery mildews has been summarized by Salmon (1900) as 
shown in Table 29. 





TABLE 29 




JTION OF Po 


WDERY 


Mildews 


BY CONTINI 






Total 


Number of 


Country 




Number 


Endemics 


Europe 




32 


12 


Africa 




8 


. . 


Asia 




28 


5 


Australia an 


d 






New Zealand 


5 


1 


America 




28 


19 



DlSTRBUTION OF PyrENOMYCETES AND DlSCO.MYCETES. Bisby 

(1933) indicates that about half of the species of Pyrenomycetes 
listed from Manitoba [Bisby et al. (1929)] are known to occur 
in Europe. Moreover, only about 12% of those in the list from 



404 GEOGRAPHICAL DISTRIBUTION OF FUNGI 

India [Butler and Bisbv (1931)] occur in Europe. Seemingly 
this group contains members that are restricted in range, and many 
of them are confined to the tropics or subtropics. 

Of the operculate discomycetes listed in Seayer's monograph 
(1928) 35 ° are limited to North America and 61 % are common 
to both North America and Europe. This group, which is almost 
wholly saprophytic, is thus quite cosmopolitan. 

Distribution of exotics. Endothia parasitica, the chestnut- 
blight fungus, is the best known and also the most destructive 
ascomycete introduced into the United States. It was first noted 
by Merkel in the New York Zoological Park in 1904 and thence 
spread with alarming rapidity throughout the entire Appalachian 
region where Castanea dent at a is native. In 1913 Meyer found 
that this organism is endemic on Castanea mollissima in northern 
China. 

Ceratostomella ulmi, causing the so-called Dutch elm disease 
[Clinton and McCormick (1936)], appeared in Holland in 1919, 
spread throughout continental Europe and the British Isles, and 
was first found in the United States in 1930. It is presumed to 
have been introduced into the United States on burl elm logs. 
Now it is gradually spreading on American elms in northern New 
Jersey and other localities in the vicinity of New York City. 

Dasyscypha ellisiana, widely prevalent in the eastern United 
States on the bark of pines, is a much less spectacular exotic. It 
was first collected by de Schweinitz in 1931 and does not injure 
pines in any way. Only recently, how ever, it was found capable 
of attacking Fsendotsnga taxifolia [Hahn and Avers (1934)], a 
species that does not grow in the natural range of D. ellisiana. 

Species with erratic distribution. Plausible explanations are 
lacking to account for the peculiar distributional range of many 
Ascomvcetes. For example, Urnula geaster, first collected near 
Austin, Texas, in 1893, was known only from that location until 
1938, when it was found in Japan. So large and so striking a disk 
fungus could scarcely have escaped observation elsewhere had 
it been present. A similar opinion is held regarding Sarcoscypha 
minuscula, occurring on dead cedar foliage, which is known only 
from Portugal, Bermuda, and the Yosemitc National Park [Seaver 
(1942)]. Furthermore, Poronia leporina, which is abundant on 
rabbit dung in Bermuda [Seaver (1942)|, has been collected in 
North America only three times during a period of over 50 years. 



DISTRIBUTION OF BASIDIOMYCETES 405 

Also, Ophionectria cylindrothecia is abundant on sterns of pal- 
metto palm in Bermuda [Seaver (1942)] and has been found on 
cornstalks in Ohio. 

Many fungi are limited to particular substrata, but the under- 
lying reasons for this limitation are unknown. None of the spe- 
cies of Melanconis, Pseudovalsa, Prosthecium, and Titania occurs 
on coniferous wood [Wehmeyer (1941)], whereas each species 
of Keithia is limited to a certain conifer. Keithia tetraspora occurs 
on Juniperus communis, K. jnniperi on Jwiiperus virginiana, K. 
tsugae on Tsuga canadensis, K. thujina on Tlnija occidentalism and 
K. chaviaecyparissi on Chamaecyparis thyoides. 

Until more is known about the reasons for differences between 
host species and varieties in susceptibility to a given fungus and 
about the influence of environment on the aggressiveness or viru- 
lence of pathogenic fungi, no one can prophesy the probable out- 
come of their introduction into new areas. Lophoderminm 
pinastri, for instance, has long been known in Europe as a serious 
menace in pine-seedling nurseries, but, although this fungus is 
not uncommon in the United States, it is as yet nowhere a major 
problem. 

DISTRIBUTION OF BASIDIOMYCETES 

Obligate parasitism, as correlated with host range, is a primary 
factor in accounting for the distribution of the smuts and rusts. 
This is not so, however, among Hymenomycetes and Gastro- 
mycetes generally, even among those that are not saprophytic. 

Distribution of smuts. The monograph by Clinton (1906) 
contains 206 species of smuts, of which 114 are strictly North 
American. Several of the smuts, including Ustilago zeae on maize 
and the stinking smuts of wheat, Tilletia foetans and T. tritici 
[Holton and Heald (1941)], are now and have been for a con- 
siderable period essentially coextensive in range with that of their 
host. Certain others are less widely dispersed. These include 
Tilletia horrida on rice, which is endemic in China and is known 
also from Indo-China, Burma, the Philippine Islands, and adjacent 
tropical and subtropical areas. It was first introduced during the 
late 1890's into South Carolina with seed rice sent from China 
[Anderson (1899)]. Subsequently it has spread to Louisiana and 
Arkansas. Another smut, alien to the United States, is Urocystis 
tritici, causing flag smut of wheat. In all likelihood this smut is 



406 GEOGRAPHICAL DISTRIBUTION OF FUNGI 

endemic to the Mediterranean area. It was first noted in the 
United States in 1919 and was early studied by Tisdale, Dugan, and 
Leighty (1923) and Griffiths (1924). It has been recorded from 
China, Japan, India, Australia, Tasmania, South Africa, Egypt, 
Tunis, Italy, Cyprus, and Spain. Dissemination in Australia is 
attributed to horses that are permitted to forage on wheat straw; 
the smut spores pass intact through the alimentary tract and then 
grow in the droppings. 

Distribution of rusts. More is known about the distribution 
of rusts than that of any other basidiomycetous group. Arthur 
(1929) considers Europe and North America as the best-explored 
regions of the earth for rusts, and he states that, as far as their 
rust flora is concerned, yast areas of other continents remain 
almost wholly unknown. The Genus Melampsora, according to 
Cunningham (1931), constitutes the only member of the Melamp- 
soraceae that is world-wide, most other rusts of this family being 
confined to Europe, Asia, and America. Alilesia also is regarded as 
world-wide by Faull (1932). Another distributional peculiarity 
noted by Cunningham (1931) is that species of Milesia and Puc- 
ciniastrum, but none of Cronartium, are found in New Zealand, 
whereas species of Cronartium, but none of Milesia nor of Puc- 
ciniastrum, thrive in Australia. In connection with Uredinopsis, 
attacking ferns and firs, Faull (1938) states that 13 species occur 
in the Western Hemisphere north of Mexico, 3 in Europe, 12 in 
Asia, and 1 in Africa. Uredinopsis m&crosperma is the most widely 
dispersed one, but strangely it is entirely absent in many regions 
where its fern host, Pteridium latiusculum, thrives. Some species 
have very limited ranges, such as U. adianti in northeastern Asia 
and U. investita in the mountains of Guatemala, both on Adian- 
tum. However, although U. mayoriana is known from Colombia, 
far from the range of fir, it is capable of producing aecia when 
artificially inoculated on fir. 

Of the 33 species and 2 varieties of Milesia, also fir-fern rusts, 
recognized by Faull (1932), only one, Milesia vogesiaca, is com- 
mon to both the Old World and the New World. Nine species 
have been taken in the United States and Canada, 7 in Central 
America, northern South America, and the West Indies, 13 in 
Asia, 1 1 in Europe, 2 in Africa, and 1 in Australia. Many species 
of .Milesia have the ability to perpetuate themselves for years in 
the entire absence of species of Abies, which are the aerial hosts. 



DISTRIBUTION OF BASIDIOMYCETES 401 

Furthermore many of them produce new crops of uredinospores 
in the spring before the old fern leaves die. 

Among the Pucciniaceae, the distribution of Puccinia, Uro- 
myces, and Phragmidium is global [Cunningham ( 193 1 ) ] . Trans- 
chel'ia pruni-spinosae occurs throughout the world wherever 
peaches and plums are grown, and Phrag7mdhtm disciflorum is 
found wherever cultivated roses can flourish. Other limitations 
imposed by host are exhibited by Uromycladium, confined to 
acacias in Australia and the East Indies, and by Phragmidium, con- 
fined to Rosaceae. Except for a few, all species of Gymnosporan- 
gium have Rosaceae as aecial hosts. Ravenelia typically occurs 
on leguminous hosts, but a few of its approximately 100 species 
attack Euphorbiaceae and Tiliaceae. 

Influence of climate on rust distribution. From the many sur- 
veys that have been made of whether the short-cycled rusts and 
long-cycled ones are proportionally alike in all regions of the 
world, it appears that short-cycled species are relatively more 
abundant in mountainous regions and in northern areas than they 
are in lowlands and in the tropics. This fact indicates that, just as 
temperature is a limiting factor in latitudinal and altitudinal dis- 
tribution of seed plants, so it is similarly operative among rusts. 
In his summary of this subject as it pertains to North America, 
Arthur ( 1929) divided the continent into the boreal zone, in which 
23% of the total rust population is short-cycled, the temperate 
zone, in which 19% is short-cycled, and the tropic zone, in which 
15% is short-cycled. 

Latitudinal zonation of rusts is strikingly indicated by Arthur 
(1929) as shown in Table 30. Some of these genera are plainly 
more northern than others, and Ravenelia and Uropyxis are to be 
considered tropical and subtropical. In fact, only 3 species of 
Ravenelia, namely, R. opaca, R. cassiaecola, and R. epiphylla, 
range north of 40° north latitude. 

Endemism among rusts. Since so many rusts attack plants of 
economic importance, it would be anticipated that each area into 
which alien plants or plant parts have been introduced would con- 
tain non-native species of rusts. That such is the case is shown 
by the work of Arthur (1929). Of approximately 1000 species of 
North American rusts, only about 600 are held to be endemic. 
McAlpine (1906) regards 31 of the 161 rusts in Australia as 
aliens. 



408 GEOGRAPHICAL DISTRIBUTION OF FUNGI 

TABLE 30 

Latitudinal Zonation of North American Rust Genera 

Boreal Temperate Tropical 



Genus 


Area 


Area 


Area 


Coleosporium 


1 


21 


16 


Melampsora 


8 


12 


3 


Pucciniastrum 


7 


10 


2 


Cronartium 


4 


6 


2 


Uredinopsis 


3 


6 


1 


Hyalospora 


1 


4 





Milesia 


2 


2 


3 


Puccinia 


130 


358 


261 


Uromyces 


28 


108 


70 


Ravenelia 





22 


44 


Gymnosporangium 


5 


33 


2 


Phragmidium 


13 


16 


2 


Uropyxis 





6 


5 



Total 202 604 411 

The list of rusts introduced into North America, in the account 
by Arthur (1929), contains such important species as Cronartium 
ribicola, Uromyces appendiciilatus phase oil, U. appendiculatus 
vignae, U. betae, U. caryophyllinus, U. trifolii, Puccinia arachidis, 
P. asparagi, P. chrysanthemi, P. glumarwn, P. malvacearum, P. 
rubigo-vera secalis, and P. rubigo-vera tritici. 

Cronartium ribicola was first known from collections made in 
Russia before 1856. In 1861 it was noted in Finland; in 1871, in 
East Prussia; in 1880, in Sweden; in 1885, in Norway; in 1889, in 
France; in 1892, in the British Isles; and in 1906, in the United 
States. 

Puccinia malvacearum is endemic in Chile, where it was first 
noted in 1852. It did not reach North America until 34 years 
later. Meantime it spread to Australia in 1857, to Spain in 1869, 
to France in 1872, to Germany and the British Isles in 1873, to 
Italy in 1874, to Switzerland in 1875, to Greece in 1877, to Sweden 
in f887, and to Finland in 1890. 

Distribution of Septobasidium. The symbiotic relationship 
between Septobasidium and scale insects, clarified by the work of 
Couch (1938), serves as the most potent factor in accounting for 
the distribution of members of this genus. If, for example, the 
symbiotic scale insect is limited to the tropics, then the particular 



DISTRIBUTION OF BASIDIOMYCETES 409 

species of Septobasidium is likewise restricted to the tropics. 
Couch (1938) found that species occurring in the southeastern 
United States are entirely different from those in the West Indies. 
Moreover, some West Indian species are not found in Central 
America and northern South America. Again, all those in Cuba 
are distinct from those in Jamaica, except for 5. rhobarbarinum. 
This species is indicated to be widely dispersed in Central Amer- 
ica, tropical Africa, and the Orient. 

There is evidence that 5. pseudopedicellatum and S. curtisii, 
common on many native species of trees and on cultivated ones 
as well throughout the southeastern United States, may have been 
introduced into other lands with shipments of trees. 

Distribution of other Basidiomycetes. Some impressions have 
been recorded of comparative distribution of agarics in North 
America and Europe by Lange (1934) and of the polypores in 
these countries by Overholts (1939). Lange (1934) states that 
70% of the species that he encountered on a tour across North 
America were known also in Europe. He mentions certain species 
that are common to both continents, such as Psalliota silvicola (P. 
arvensis), Lactarius deliciosns, Pamis stipticus, Amanita muscaria, 
A. caesarea, Hypholoma fascicular e, Inocybe geophylla, Laccaria 
lac cat a, Stropharia psathyroides and Lepiota cygnea. The differ- 
ence between the agaric floras of the two continents is in no wise 
as striking as are differences on each continent due to latitude. 
Stropharia depilata is a boreal species ranging from the Rocky 
Mountains to the Scandinavian subarctic zone [Lange (1934)]. 
Amanita caesarea is a temperate species and extends northward to 
southern Denmark and northern Germany. Lange (1934) also 
states that certain species in Europe are limited to the Mediter- 
ranean region and rarely, if ever, extend beyond the Alps. Spe- 
cies of Marasmius, some of which cause thread blights, and Lenti- 
nus abound in the subtropics and tropics. 

Several well-known agarics, such as Clitocycbe ilhidens, Armil- 
laria mucida, Colly bia radicata, and Lepiota procera, were noted 
by Bisby (1933) as being absent from iManitoba for some un- 
known reason. 

Certain agarics and Boletaceae are mycorrhizal, and some of 
them are known to be restricted to certain species of trees. In 
such cases the range of the tree is the factor which governs the 
distributional range of the particular fungus. (See Chapter 13.) 



410 GEOGRAPHICAL DISTRIBUTION OF FUNGI 

Another feature reeardins the distribution of Basidiomvcetes 
that has impressed every mycologist who has intensively collected 
in a given area for a period of years is that certain species found 
one season mav be entirely absent in succeeding years. 

The extensive studies of Overholts (1939) led him to conclude 
that of the 227 species of North American pileate Polyporaceae 
at least 43% occur in the Eastern Hemisphere. Of the more com- 
mon genera, he found that 54 0/ of Fomes and of Trametes species, 
50% of Daedalea and of Lenzites species, and 44% of Polyporus 
species are common to North America and the Old World. Cer- 
tain of them, such as Polyporus conchifer and P. texam/s, are 
limited in range to that of their hosts, Uhmis americana and 
Prosopis jttliflora, respectively. Fomes applanatus is a cosmopoli- 
tan species. Polyporus abietinus can utilize all species of conifers; 
P. versicolor, P. pargamemis, and Lenzites bctid'nw, many kinds of 
hardwoods; and all are widely distributed. Fomes pint and Poly- 
poms schweinitzii, both capable of causing heart rots of conifers, 
are widely present throughout the United States and Canada. 

The Gastromycetes are widely dispersed, with little evidence 
of being affected by latitude. An exception is the Phallales, which 
are mostly tropical, whereas the Lycoperdales are temperate. 

DISTRIBUTION OF DEUTEROMYCETES 

The imperfect fungi of most economic importance are either 
seed-borne or soil-borne or else are dispersed with nursery stock. 

Distribution of seed-borne species. Many pathogenic im- 
perfect fungi, particularly those of cultivated plants, have been 
demonstrated to be seed-borne [Orton (1931)]. This fact ac- 
counts for the wide distribution and establishment of such fungi 
as Ascochyta pisi, leaf and pod blight of pea; Cercospora beticola, 
leaf spot of beet; Cercospora daizu, frog-eye leaf spot of soybean; 
Collet otrichum gossypii, cotton anthracnose; C. lagenarium, water- 
melon anthracnose; C. I'mdemuthiamivi, bean anthracnose; Clado- 
sporinm fulvum, leaf mold of tomato; Diplodia zeae, ear rot of 
corn; Kabatiella caidivora, anthracnose of clover; Helvnntho- 
sporhim graminenm, barley stripe; Phoma I'm gam, cabbage black- 
leg; Polyspora Urn, flax-stem break; Septoria apii, celery blight; 
Septoria ly coper sic'i, leaf spot of tomato. Presumably each of 
these fungi occurs wherever its hosts are cultivated, and com- 



DISTRIBUTION OF DEUTEROMYCETES 411 

petent collectors would no doubt find them all in regions from 
which there are now no collection records. To mention a few 
ranges, Cercospora beticola is known to be present in Korea, 
Japan, Czechoslovakia, Hungary, Ukraine, Jugoslavia, Rumania, 
Germany, Austria, Italy, Poland, Latvia, Lithuania, France, Spain, 
Holland, Belgium, Ireland, Morocco, Mauritius, Bermuda, Cuba, 
Dominican Republic, the United States, and Canada. Polyspora 
lirii has been noted on flax in Ireland, Holland, Sweden, Denmark, 
Germany, Poland, Latvia, Russia, Italy, New Zealand, Canada, and 
the United States. Septoria ly coper sici is known to occur in the 
United States, Canada, Great Britain, France, Denmark, Germany, 
Norway, Esthonia, Lithuania, Russia, Rumania, middle Asia, Cey- 
lon, southern Australia, Fiji, Mauritius, Kenya, Morocco, east 
Africa, Rhodesia, Argentina, Brazil, Trinidad, Guatemala, Ber- 
muda, and Hawaii. 

Distribution of species dispersed with nursery stock. Some 
very important imperfect fungi have been widely disseminated 
with shipments of nursery stock, for example, Cladospor'mm car- 
pophiliimi, causing scab and freckle of stone fruits, Phyllosticta 
solitaria, causing canker, blotch, and leaf spot of apple, and Sphace- 
loma fawcetti, causing citrus scab. Data on the occurrence of 
Cladosporhim carpophilium outside the United States and southern 
Canada are not abundant; nevertheless Keitt (1917) is of the 
opinion that this species is present in all countries where peach, 
nectarine, and cherry are grown. Austria, Germany, Bulgaria, 
Holland, South Africa, New South Wales, and Brazil are among 
the regions where C. carpophilium is known to occur. 

Phyllosticta solitaria has not been given serious attention out- 
side the central and eastern United States. Guba (1925) suspected 
that wild crabapple, Pyrus coronaria, is the original host and 
source of inoculum. This fungus has been reported from Argen- 
tina, Rhodesia, Spain, and Holland. 

Sphaceloma jauccetti is believed [Fawcett (1926) ] to have been 
present in Japan since ancient times, but it was given little atten- 
tion until its discovery in Florida about 1886. It occurs also in 
China, India, the East Indies, Australia, New Zealand, Hawaii, 
Brazil, and Argentina. 

Distribution of soil-borne species. Many of the Moniliales 
are soil-borne. The outstanding representative of this group of 
imperfect fungi is Phymatotrichum omnivorum, commonly called 



412 GEOGRAPHICAL DISTRIBUTION OF FUNGI 

the Texas root-rot fungus. The appropriateness of its specific 
name is indicated by the fact that it is known to attack more than 
1 700 species of flowering plants, more than does any other known 
pathogen. An appreciation of the destructiveness of P. omni- 
vorum can be gained from the fact that a complete bibliography 
of it would include about 300 titles, and annual losses which it 
occasions are estimated to approximate one hundred million 
dollars. 

Its range extends throughout the greater part of Texas and con- 
tiguous parts of Arkansas, Oklahoma, New Mexico, and .Mexico. 
It also occupies areas in Arizona, California, Nevada, and Utah. 
Its existence outside this range has been noted in the Dominican 
Republic, Hawaii, and (doubtfully) Russia. 

Active dissemination is accomplished largely bv growth of the 
fungus through the soil, where it may hibernate by means of 
sclerotia. 

Various species of Fusarium that live saprophytically in the soil 
for long periods and cause wilt diseases when the appropriate 
crop is'planted on such a soil are also included in this group. 
Among them are Fusarium vasinfectum on cotton, F. cubense on 
banana, F. oxysporum on potato, F. lycopersici on tomato, F. 
niveum on watermelon, and F. lini on flax. All are widely dis- 
persed in any region where these crops are grown. 

IMPLICATIONS 

It is quite apparent that no comprehensive information regard- 
ing fungus floras throughout the world is available at this time. 
Many additional monographic studies of fungal groups must first 
be made, and also many more lists of the type of the Host Index 
of the Fungiof North America [Seymour ( 1929)], The Fungi of 
Manitoba [Bisbv, Buller, and Dearness (1929)], The Fungi of 
India [Butler and Bisby (1931)], and British Stem and Leaf Fungi 
[Grove (1935, 1937)] must be prepared. Seymour's book in- 
cludes about half of all the known species of fungi, and about 
60 c of the Canadian species listed by Bisby et al. (1929) are also 
known to occur in Europe. 

Students of the geographic distribution of fungi seem agreed 
that climate has a controlling effect [Bisby ( 1943) ], Diehl (1937), 
Lind (1934). Diehl (1937) concluded that the life zones of 



IMPLICATIONS 413 

fungus vegetation are bounded or delimited by climatic lines or 
factors. These climatic factors operate by controlling the distri- 
bution of the particular substrata, for both endemic and exotic 
species. The provinces of fungi are delimited by such natural 
barriers as climate, oceans, mountains, deserts, wind direction, and 
vectors, but man has operated to break down these barriers and 
to carry the fungi over them into new sites. 

Lind (1934) has also emphasized the influence of climate as a 
factor in distribution. 

Of the 422 species collected in the Arctic, Lind indicates that 
many occur also in the Alps and are otherwise widespread and 
that no genus in these collections is endemic to the northern polar 
region. 

Bisby's (1943) opinion is: "There are perhaps three times as 
many [species of] phanerogams as fungi on earth." Moreover 
saprophytic species generally have a wider distribution than do 
parasitic ones, although distribution of substrata and hosts is of 
primary importance as a control factor. 

The natural ranges and habitats of fungi tend toward the estab- 
lishment of stability and biological balance. Man has always up- 
set this stability by intensive cultivation of a given species of host 
in a limited area, bv constructing artificial environments such as 
cold frames and greenhouses, in which to grow plants, by attempts 
to grow crops in new areas, and by introducing fungi into areas 
where the environment unfortunately has too frequently proved 
more favorable for the fungi than did their natural range. In re- 
gard to the results of man's activities upon the distribution of 
fungi, it is apparent that he has indeed made his own difficulties 
and problems; nevertheless he seems to thrive in spite of his 
tendency to learn things the hard way. 

There are good reasons for believing that some so-called new 
diseases of cultivated plants are not caused by new species of fungi 
but by old ones long present in a particular locality. As a result 
of the conditions that obtain under cultivation, the host may 
succumb to attack, whereas it might be immune in its natural or 
native habitat. Of course, it must always be remembered that 
both the susceptibility of the host and the aggressiveness of the 
parasite are influenced by environmental factors which may 
eventuate in a modification of the distributional range both of the 
host and of the parasite. 



414 GEOGRAPHICAL DISTRIBUTION OF FUNGI 

Because of the enormous financial outlay that has become 
necessary in connection with quarantines and with the control and 
eradication of fungi already introduced, all exotics should per- 
force be regarded as potentially undesirable aliens and should be 
so treated. Some fungi are closely restricted in range and are 
very complacently provincial, some can be widely transplanted 
without becoming obnoxious, some are exceedingly noisome when 
transported to new environments, and some are naturally cosmo- 
politan and international and, in consequence, have become widely 
established. 

LITERATURE CITED 

Anderson, A. P., "A new Tilletia parasitic on Oryza sativa" Botan. Gaz., 
21: 461-411, 1899. 

Arthur, J. C, et al., The plant rusts (Uredinales) . 446 pp. John Wiley 
and Sons, New York. 1929. (See Chap. V, pp. 161-205.) 

Bisby, G. R., "The distribution of fungi as compared with that of phanero- 
gams," Am. J. Botany, 20:246-254, 1933. 
"Geographical distribution of fungi," Botan. Rev., 9: 466-482, 1943. 

Bisby, G. R., and G. C. Ainsworth, "The numbers of fungi," Trans. Brit. 
Mycol. Soc, 26: 16-19, 1943. 

Bisby, G. R., A. H. R. Buller, and J. Dearness, The fungi of Manitoba. 
viii + 194 pp. Longmans, Green, and Co., London. 1929. 

Butler, E. J., and G. R. Bisby, The fungi of India, Imper. Counc. Agr. Re- 
search, Sci. Monograph I. xviii + 237 pp. 1931. 

Carr, L. G., "A comparison of Mycetozoa found in sandstone and limestone 
regions of Augusta County, Virginia," Mycol., SI: 157-160, 1939. 

Clayton, E. E., and J. A. Stevenson, u Peronospora tabacina Adam, the or- 
ganism causing blue-mold (downy-mildew) disease of tobacco," Phyto- 
pathology, 55: 101-113, 1943. 

Clinton, G. P., "Ustilaginales," North Am. Flora, 1: 1-82, 1906. 

Clinton, G. P., and F. A. McCormick, "Dutch elm disease, Graphium uhn'u' 
Conn. Agr. Expt. Sta. Bull., 389: 707-752, 1936. 

Couch, J. N.,The genus Septobasidium. 480 pp. University of North 
Carolina Press. 1938. 

Cunningham, G. H., The rust fungi of New Zealand, together with the 
biology, cytology, and therapeutics of the Uredinales. xx + 261 pp. 
J. .Mclndoe, Dunedin, New Zealand. 1931. 

Diehl, W. W., "A basis for mycogeography," /. Washington Acad. Sci., 
21: 244-254, 1937. 

Faull, J. H., "Taxonomy and geographical distribution of the genus Milesia," 
Contrib. Arnold Arboretum, 2:5-138, 1932. 
"Taxonomy and geographical distribution of the genus Uredinopsis," 
Contrib. Arnold Arboretum, 11:5-120, 1938. 



LITERATURE CITED 415 

Fawcett, H. S., and H. A. Lee, Citrus diseases and their control, xii +582 

pp. McGraw-Hill Book Co., New York. 1926. 
Fries, R. E., "Myxomyceten von Argentinien und Bolivia," Ark. Bot., 1: 57- 

70, 1903. 
Griffiths, Marion A., "Experiments with flag smut of wheat and the causal 

fungus, Urocystis tritici Kcke," /. Agr. Research, 27:425-449, 1924. 
Grove, W. B., British stem and leaf fungi (Coelomycetes), 1: xx + 488 pp., 

1935; 2: xi + 406 pp., 1937. Cambridge University Press. 
Guba, E. F., "Phyllosticta leaf spot, fruit blotch and canker of the apple: 

etiology and control," ///. Agr. Expt. Sta. Bull., 255:481-557, 1925. 
Hahn, G. G., and T. T. Ayers, "Dasyscyphae on conifers in North Amer- 
ica II. Dasyscypha ellisiana," My col., 26: 167-180, 1934. 
Holton, C. S., and F. D. Heald, Bunt or stinking smut of wheat (a world 

problem). ii + 211pp. Burgess Publishing Co., Minneapolis. 1941. 
Karling, J. S., Plasmodiophorales. ix + 144 pp. Published by the author, 

New York. 1942. 
Keitt, G. W., "Peach scab and its control," U. S. Dept. Agr. Bull., 395: 1-66, 

1917. 

Lange, J. E., "Mycofloristic impressions of a European mycologist in Amer- 
ica," My col, 26: 1-12, 1934. 

Lind, J., "Studies on the geographical distribution of arctic circumpolar 
micromycetes," Kgl. Danske Videnskb. Selskab Biol. Medd., 11 (12): 1- 
152, 1934. 

MacBride, T. H., "Mountain Myxomycetes," My col., 6: 146-149, 1914. 

Martin, G. W., "The Myxomycetes," Botan. Rev., 6: 356-388, 1940. 

McAlpine, D., The rusts of Australia. 349 pp. 1906. 

Orton, C. R., "Seed-borne parasites, a bibliography," West Va. Agr. Expt. 
Sta. Bull., 245: 3-47, 1931. 

Overholts, L. O., "Geographical distribution of some American Polypo- 
raceae," My col, 31: 629-652, 1939. 

Salmon, E. S., "A monograph of the Erysiphaceae," Mem. Torrey Botan. 
Club, 9. 292 pp. 1900. 

Seaver, F. J., The North American cup fungi (Operadates) . 284 pp. Pub- 
lished by the author, New York. 1928. 
"The mvcoflora of Bermuda," Science, 96: 462-463, 1942. 

Seymour, A. B., Host index of the fungi of North America, xiii + 732 pp. 
Harvard University Press. 1929. 

Smith, E. C, "Ecological observations on Colorado Myxomycetes," Tor- 
reya, 37:42-44, 1931. 

Thom, C, and K. B. Raper, "Myxamoebae in soil and decomposing crop 
residues," /. Wash. Acad. Sci., 20: 362-370, 1930. 

Tisdale, W. H., G. H. Dugan, and C. E. Leighty, "Flag smut of wheat with 
special reference to varietal resistance," ///. Agr. Expt. Sta. Bidl, 242: 511- 

537, 1923 
Wehmeyer, L. E., "A revision of Melanconis, Pseudovalsa, Prosthecium, and 
Titania," Univ. Mich. Studies, 14. 161 pp. 1941. 



Chapter 18 
MYCOLOGY IN RELATION TO PLANT PATHOLOGY 

Plant materials constitute the substrate on which nearly all 
fungi thrive in their natural habitats. Relatively few utilize ani- 
mals or animal tissues as substrates of first choice. Furthermore 
many fungi, whether saprogenic or pathogenic, are quite closely 
restricted to a particular plant species. The fundamental reasons 
for these idiosyncrasies in the choice of food are not without sig- 
nificance, but they remain quite unknown beyond the point that 
there is a correlation between the enzyme-producing abilities of 
each fungus and the kind of substrate on which it grows. 

The idea that all fungi are either parasitic or saprophytic has 
had far-reaching consequences. It has had a deleterious effect 
primarily on understanding the activities of fungi and secondar- 
ily on appreciating the intimate interdependence of mycology 
and plant pathology. It is not uncommon for a plant pathologist 
to remark that parasitic fungi are of interest to him but that 
saprophytic species are of no concern. He chooses to entrust 
saprophytic species to the tender care of a mycologist! In so do- 
ing he may overlook the fact that a particular species may have 
both a parasitic and a saprophytic phase. Perhaps the terms para- 
sitic and saprophytic have outlived a measure of their usefulness. 

A4uch information regarding the natural habitats of fungi has 
come from studies, not of saprogenic species, but of pathogenic 
ones and has therefore been contributed by plant pathologists. In 
so far as such studies have emphasized the disease aspect, including 
disease prevention and control, they properly constitute the sub- 
ject matter of phytopathology. On the other hand, in so far as 
such studies pertain to the etiologic agent itself, they belong to 
mycology. The two fields are therefore closely interrelated, as 
may be brought out by consideration of their parallel develop- 
ment, but they have grown to be quite distinct. In fact, some 
workers regard mycology as the parent science and phytopathol- 

416 



EARLY CONCEPTS OF DISEASE IN PLANTS 411 

ogy as the offspring. The purpose of this discussion is to bring 
these interrelations into perspective. To anyone who attempts 
to do this properly, it soon becomes apparent that the task is 
herculean, for the reason that the subject matter of each field of 
science is dispersed in a bewildering array of books, technical re- 
ports, and bulletins. Manifestly it is impossible to accomplish 
such a task within the scope of a single chapter. Moreover, to 
date no one has attempted a comprehensive interpretative history 
of mycologic and phvtopathologic development. Little more can 
be attempted in this discussion than to point out a few of the land- 
marks along the pathway, beginning with the completely un- 
scientific era from which both mycology and plant pathology 
emerged and ending with present-day concepts. Both fields, as 
was briefly indicated in Chapter 1, Vol. I, had their beginnings 
in the dim, distant past, long before the period of recorded his- 
tory. The development of each has been dependent, as would 
be expected, upon advances in such fields as bacteriology, medi- 
cine, animal pathology, physics, and chemistry, and especially 
upon the improvisation of new methods or techniques. 

EARLY CONCEPTS OF DISEASE IN PLANTS 

Some appreciation of the ideas concerning disease in plants that 
prevailed before 1807 may be gained from a treatise by Re (1807). 
Later Smith (1902, 1929), Arthur (1906), and Whetzel (1918) 
sketched the background against which present-day ideas can be 
interestingly evaluated. As these writers point out, man long 
recognized the existence of disease, especially among cultivated 
plants, but from earliest times such diseases were uniformly inter- 
preted as supernatural phenomena and ascribed to offended deities. 
Later came the belief, generally accepted among scientists, that 
fungi were generated by the host or suscept on which they oc- 
curred. The works of Unger, Meyen, and Hallier [Whetzel 
( 1918) ] are based on this concept. 

Certain other contemporary writers, however, held a different 
opinion, as is shown by the observations of Fontana, published in 
1767, in which he made the following statement regarding grain 
rust: "We are dealing with a great number of hungry and in- 
satiable plants that live by violence, feeding at the expense of the 
tender green plant; they grow rapidly, thanks to the food that 



418 MYCOLOGY IN RELATION TO PLANT PATHOLOGY 

they steal from the grain, feeding in a great number of places, 
stopping entirely the flow of the already prepared and digested 
juice, which is to nourish the grain and to be converted into pulp 
and flour." 

The mystical and ethereal nature of the cause of disease in plants 
was also refuted by Fabricius in a treatise published in 1774, in 
which he maintained that smut is caused by "something origan- 
ized," that is, something living, and by Prevost in a dissertation 
published in 1807, in which he concluded that rust and smut dis- 
eases are produced by "internal parasitic plants." These ideas did 
not gain acceptance among scientists, however, and the real turn- 
ing point in progress on the nature of disease in plants came with 
the publication in 1853 of Die Brand Pilze, based on experimenta- 
tion by de Barv. He showed that the rust and smut fungi are 
entities that induce disease by growth within the host tissues, with 
resultant modification of the structure and the function of the in- 
fected plants. 

CONTRIBUTORY ADVANCES IN BACTERIOLOGY 

The impact of such conclusions from the work of de Barv upon 
mycology and plant pathology can be appreciated only if con- 
sidered in connection with discoveries that had already been made 
or were made soon thereafter in other fields, especially bacteriol- 
ogy. It should be remembered that for a long time scientific 
thought was permeated with the concept that many kinds of liv- 
ing things, especially those of microscopic proportions, originated 
by spontaneous generation. Using goose-necked flasks containing 
fermentable fluids, Pasteur demonstrated with finality that fermen- 
tations may be induced by air-borne bacteria and that during 
fermentation these bacteria generate other bacteria like them- 
selves. This discovery led to his subsequent studies, which served 
as the basis for the establishment of the germ theory of disease 
in animals, a theory that soon came to pervade the entire field of 
medicine. Concurrently came the development of laboratory 
methods for the isolation and cultivation of organisms in pure cul- 
ture, notably (1) the use of semisolid media, originating with the 
work of Koch on the anthrax bacillus; (2) the use of cotton 
stoppers, interposed between the medium and the open air to 
strain out organisms floating in the air, first employed by 



SIGNPOSTS ALONG THE PHYTOPATHOLOG1CAL PATH 419 

Schroder and Dusch; and (3) the establishment of pathogenicity 
by compliance with axiomatic rules of proof, called Koch's rules. 
Gradually other techniques from procedures developed in bac- 
teriology were adapted for use in studying fungi. These tech- 
niques involve the influence of such environmental factors as tem- 
perature, food requirements, and hydrogen-ion concentration of 
the medium and the complex reactions involved in studies of 
antigenic properties of fungi. 

SIGNPOSTS ALONG THE PHYTOPATHOLOGICAL PATH 

Certain outstanding events and discoveries indicate the course 
of development in any field of science. Those in phytopathology, 
as has been stated, have been very directly and quite uniformly 
related to mycology. The most significant are categorically listed 
as follows: 

1. The epiphytotics of late blight of potatoes in 1843, 1844, 
and 1845 in northern Europe and the British Isles. The destruc- 
tion of the potato crop was so complete that in Ireland alone 
approximately a quarter of a million persons died of famine. As 
a secondary consequence of the catastrophe, attempts were made 
to determine the cause and control of this potato disease, and 
plant pathology, as a science, may properly be concluded to have 
originated with these studies. For the first time the public appre- 
ciated the significance and the necessity of plant pathological 
investigations. 

2. The publication in 1853 of the first textbook of plant 
pathology by Julius Kiihn, who is generally regarded as the 
father of plant pathology. In this book considerable emphasis 
is placed on the disease itself rather than on its cause. This is 
true also of important books that followed, such as those by 
Berkeley, Cooke, Hartig, Sorauer, W. G. Smith, Tubeuf, Kirch- 
ner, Ward, Comes, Prillieux, Massee, and Viala; all of these, how- 
ever, are preponderantly mycologic. 

3. The establishment of proof of the heteroecism of rusts by 
de Bary in 1864 to 1865. The relationship between rust on wheat 
and that on barberry had long been suspected by farmers. In 
fact, they had compelled the enactment of legislation providing 
for the eradication of barberry as early as 1660 in France and as 
early as 1726 in the state of Connecticut. 



420 MYCOLOGY IN RELATION TO PLANT PATHOLOGY 

4. Outbreaks of downy mildew on grapes in Europe, espe- 
cially in the vineyards of France and Italy. The causal fungus, 
Plamwpora viticola, indigenous in the United States, had been in- 
troduced into Europe in 1878. In order to prevent pilfering of his 
grapes, a grower sprinkled his vines with a mixture of lime and 
copper sulphate. Millardet noted that the grapes so treated re- 
mained free from downy mildew, and as an eventual result the 
world's best-known fungicide, Bordeaux mixture, was developed. 

5. The establishment of the Office of Vegetable Pathology in 
the United States Department of Agriculture and the organization 
of the state agricultural experiment stations under the Hatch act, 
both in 1887. Provision was thus made for the first time for the 
initiation of organized research on diseases of crop plants. In 
the beginning only meager financial support was forthcoming 
for this work, but the appropriation has increased throughout the 
years in proportion to needs and to growing appreciation of the 
importance of such studies. 

6. The publication of Saccardo's Sylloge Fimgorinn, a com- 
pendium containing descriptions of all known species of fungi. 
This monumental work, the first volume of which appeared in 
1882, now contains twenty-five volumes. It is truly a requisite 
for the mycologist and phvtopathologist. 

7. The introduction of two species of alien fungi, Endothia 
parasitica, causing chestnut blight, and Cronartium ribicola, caus- 
ing blister rust of five-needle pines. Endothia parasitica was first 
noted in the United States in 1904 and Cronartium ribicola in 
1906. These two organisms became widely dispersed with rapid- 
ity, and their ravages stimulated the general public to an apprecia- 
tion of the destructiveness of plant diseases and to an interest in 
problems of disease prevention and control. 

8. The establishment of the Federal Plant Quarantine Law in 
1912. The enactment of this law was the outgrowth of experi- 
ences with chestnut blight and with blister rust of white pines. 
Moreover it was the first legalized effort by a nation to exclude 
foreign pests and plant diseases. 

9. The organization of departments of plant pathology at 
Cornell University in 1907 and at the University of Wisconsin in 
1909 for the training of specialists in research and the teaching of 
plant pathology. The emphasis on instruction in so-called plant 



DEVELOPMENTS IN TERMINOLOGY 421 

pathology up to that time had been largely taxonomic mycology, 
and in fact it remains all too much so to this day. 

10. The organization of the American Phytopathological So- 
ciety in 1909 and the publication of Phytopathology, the official 
organ of this society, beginning in 1911. The charter member- 
ship included 130 names, but the membership has now grown to 
well over 1000 persons. These two agencies, the society and the 
journal, have been potent factors in stimulating interest and in 
directing the trend of phytopathologic development not only 
throughout the United States but also throughout the world. 

11. The initiation of the abstract journal, Review of Applied 
Mycology, at the Kew Gardens in 1922. This journal, published 
at regular intervals throughout the year, contains complete refer- 
ences to all current publications on plant pathology, together with 
a summary of the content of each report. It is absolutely indis- 
pensable as a tool in keeping abreast of developments in mycology 
and plant pathology. 

DEVELOPMENTS IN TERMINOLOGY 

Correct terminology is essential properly to express concepts 
in any field of learning. Certain terms have been used both in 
mycology and in phytopathology without regard to precision 
of expression, and, as a consequence, confusion and inaccuracies 
have appeared. Fortunately some of these inaccuracies have been 
rectified, as inevitably occurs during the course of the normal 
development of a science. The terminology in both fields could 
be expected to have much in common, especially during their 
formative periods. In fact, in the beginning the terminology of 
mycology and phytopathology reflected the influence of animal 
pathology and medicine, since many of the early workers in the 
newer fields were medical practitioners or at least had been 
trained in medicine. This fact is demonstrated by the use in 
Fabricius' treatise of 1774 of such terms as anasarca, gangraena, 
tabes, exulceratio, polysarcia, and carcinoma. Moreover, in the 
period before 1850, the employment of such names in connection 
with diseases of plants as icterus, anemia, phlegmasia, fluxion or 
bleeding, verrucosis, and exanthema is further confirmation of the 
influence of medical terminology. 



422 MYCOLOGY IN RELATION TO PLANT PATHOLOGY 

During the latter half of the nineteenth century the overwhelm- 
ing interest in plant diseases centered around etiology. In the 
textbooks of this period, as in nearly all recent textbooks, plant 
diseases are arranged on the basis of the classification of the etio- 
lo^ic agent. The reasons for this situation are numerous and in- 
clude the following: 

1. The period of 50 years after the overthrow of the theory of 
spontaneous generation, now regarded as the "golden age of dis- 
covery" in bacteriology, was also the golden age of discovery in 
fields pertaining to bacteria and fungi as causes of disease in plants. 
In this period the cause of a disease and the disease itself were all 
too commonly regarded as synonymous. The connotation host- 
parasite, which indicates a food relationship, became a common- 
place and was used instead of pathogen-suscept, which indicates 
the disease relationship. Writers spoke of "spread of disease" and 
"spread of infection" when they meant spread of inoculum or of 
pathogenic agents. These examples indicate the confusion of 
ideas that have been carried over from mycology to phyto- 
pathology. 

2. The emphasis in studies of plant-pathogenic fungi has re- 
mained so overwhelmingly etiologic that even at the present time 
too little recognition is being given to the influence of "predispos- 
ing factors," as stressed by Sorauer, and to the morbid anatomy 
of diseased plants, as stressed by Kiister (1925) in his first edition 
of Phytopathologische Pflanzenanatomie, which appeared in 1903. 
That plant diseases should be classified on the basis of the disease 
processes themselves is cogently argued by Whetzel (1929). It is 
becoming increasingly apparent that instead of stating that a 
given fungus is the cause of a particular disease one should state 
that it is one of the causes, because environmental factors may 
exert a controlling influence. It is also apparent that the classifica- 
tions of disease by Kiister are fundamental, and future develop- 
ments must be built on his scheme. 

3. The investigators of this period lacked training in phyto- 
pathology, and in consequence their attention was centered pri- 
marily on the pathogen, with only passing consideration being 
given the diseased plant. In their scientific writings they em- 
ployed terms from fields of knowledge with which they were 
familiar. As soon as interest shifted, a distinctive terminology, 
applicable only to plant pathology, began to develop, as exempli- 



FUNGI AS ANTIGENS AND PLANT PATHOLOGY 423 

fied by such common terms as wilt, scorch, blight, scald, stripe, 
die-back, shot hole, leak, damping-ofT, chlorosis, stunt, dwarf, 
drop, russet, intumescence, curl, gall, and scab, all of which indi- 
cate characteristic symptoms of disease. With the increase in 
knowledge of changes in cellular structure and function induced 
by pathogenic fungi, technical terms have been and are being 
introduced, just as they were in the field of animal pathology. 
Also there is an increasing tendency among plant pathologists to 
classify diseases as root-rot diseases, fruit diseases, leaf diseases, 
seedling diseases, etc., terms analogous to respiratory diseases, gas- 
trointestinal diseases, skin diseases, etc., as used by the medical 
worker. There is now a growing tendency to clarify terminol- 
ogy as belonging to mycology or to phytopathology and to em- 
ploy terms that are distinctive in each field. 

FUNGI AS ANTIGENS AND PLANT PATHOLOGY 

A very extensive literature on studies of resistance to disease 
among plants exists and has been recently reviewed by Wingard 
(1941). Nearly all such studies deal with natural immunity, as 
opposed to acquired immunity. Experimental evidence that plants 
may acquire immunity after being "vaccinated" and that anti- 
body formation results was first submitted approximately 40 years 
ago. Plant pathologists generally have not reacted favorably to 
this type of research and have given it little credence for the rea- 
son that plants lack a tissue system comparable with the circula- 
tory system in animals. Nevertheless additional reports have ap- 
peared from time to time of studies that tend to support the possi- 
bility of acquired immunity in plants. An excellent monographic 
review of such studies, together with a summary of their own 
work, was prepared by Carbone and Arnaudi (1930). The "vac- 
cines" used were either injected into plants or applied to the 
surface of seeds before planting. Arnaudi (1933) prepared vac- 
cine of Thielaviopsis basicola from dried powdered mycelial mat 
or from fresh mycelial mat mixed with sand and triturated in a 
mortar. These vaccines were applied to the tobacco seed or to 
the soil with apparent protection of the seedlings. 

Series of studies on immune reactions in plants were conducted 
by Chester (1932) and by Chester and Whitaker (1933), which 
snowed that the so-called "plant precipitins" are in fact non- 



424 MYCOLOGY IN RELATION TO PLANT PATHOLOGY 

protein precipitates arising from a reaction between oxalates and 
calcium. Their results led them to conclude, "The published im- 
munological reactions in plants are rendered untenable because 
of lack of homology between the animal and plant reactions, and 
because of the wide-spread occurrence in plants heretofore used 
of simple non-protein reactions." Even though the weight of 
evidence is against the existence of acquired immunity against 
pathogenic fungi in plants, ample evidence has been accumulated 
to show that fungi pathogenic to man and animals have antigenic 
properties. 

PRESENT TRENDS IN MYCOLOGIC AND 
PHYTOPATHOLOGIC WORK 

Since the turn of the present century marked changes have 
taken place in the prescribed disciplines for the training of teach- 
ers and investigators of mycology and plant pathology and in the 
kinds of research involving plant-pathogenic fungi. It is difficult 
at this time to determine or to decide whether these changes have 
always tended in the direction of improvement over previous 
studies, mainly for the reasons that not enough time has elapsed 
to appraise them disinterestedly and without bias and to view 
such matters in perspective. Improvement should have been made 
because, as must be admitted, present-day students of fungi are 
better trained for their tasks than were their elders. Additional 
support for this conclusion is found in the fact that during the 
first quarter of the present century undue attention was devoted 
to projects involving "spray schedules" and "spray calendars." 
This kind of project was not sponsored by so-called plant pathol- 
ogists and mycologists alone, but also by horticulturists, agron- 

* -I'll 

omists, entomologists, and botanists, all of whom vied with each 

other to acquire direction of such projects. Sprays were all too 
commonly applied, not at critical times in the development of the 
pathogen, but on planned and prearranged dates. Indeed, basic 
knowledge about the pathogens involved was extremely meager, 
and efforts to gain such know ledge were regarded by some work- 
ers as a "not practical" expenditure of time. Determination of 
not only the most effective times to spray but also the proper 
fungicidal concentrations was sought by empirical methods. 
Needless to say, a body of contradictory and inexplicable data was 



MYCOLOGIC AND PHYTOPATHOLOG1C WORK 425 

assembled from such experimentation, and it is not surprising that 
the epithet "squirt-gun pathologists" came to be applied to such 
workers. 

Although plant pathologists have gradually assumed charge of 
studies on the prevention and control of plant diseases, some still 
fail to acquire or to utilize knowledge of the seasonal cycle of 
development of the pathogen, of its epiphytology, and of agencies 
of its dissemination as a basis for instituting experiments on how 
best to control the given disease. Two obvious reasons may be 
offered for this situation. It may arise from lack of adequate 
mycological training or else from pressure exerted by administra- 
tive officials for the publication of experimental findings. In any 
event the net result is reflected in the content of published reports 
and of papers presented at conferences or meetings. It is apparent 
in many cases that too little cognizance has been taken of exist- 
ing knowledge of the disease and that the materials presented are 
preliminary and are fragmentary rather than comprehensive in 
scope. For these reasons they are intrinsically limited in applica- 
tion and in usefulness. The validity of these criticisms is sup- 
ported by the fact that many papers presented at meetings are 
not deemed of sufficient merit for publication. 

At present, plant pathologists do not occupy positions of re- 
spect and honor in society comparable with those held by medi- 
cal practitioners. Of course, the difference in age of the two 
professions is a causal factor, but several other reasons, such as 
the following, seem equally plausible and more fundamental in ac- 
counting for this state of affairs. 

1. Remuneration for services rendered by plant pathologists is 
made from funds raised by taxation. Plant pathologists are there- 
fore public servants whose help and advice on the problems of 
diagnosis and treatment of plant diseases must be given gratuitously 
to all who request aid. The public has ironically come to feel 
that the cost of things and their* real value to them as individuals 
are either identical or at least closely correlated. 

2. Reports, both those dealing with very meritorious research 
on pathogenic fungi and those having little or no value, are alike 
published and distributed free of charge. The public is not al- 
ways able to differentiate between these two types of reports 
nor to evaluate them, and they are, in consequence, appraised as 
though of equal value. It is unfortunate that they should be simi- 



426 MYCOLOGY IN RELATION TO PLANT PATHOLOGY 

larlv publicized or perhaps that either should be given any popular 
publicity, for they thereby partake all too much of the nature of 
nostrums for the cure of human ailments, as advertised in news- 
papers and popular magazines or over the radio. It should be 
remembered that reputable physicians never sponsor the dissemi- 
nation of panaceas for human diseases. Neither do they make 
diagnoses nor prescribe treatment without first-hand knowledge 
of the patient. It seems altogether probable, therefore, that the 
plant pathologist could raise the esteem and respect with which he 
is regarded by emulating the medical practitioner in these respects. 

3. It is patently a mistake for the student of fungi to confine 
himself to his armchair or to the four walls of his laboratory or 
greenhouse. It is equally fatal for best results if he depends en- 
tirely upon observations made in the field. Laboratory experi- 
ments with fungi and observations on them in the field each have 
a limited usefulness, but they can be used to complement each 
other. Results of laboratory experiments are intended to serve 
as a basis for field trials but should never be translated into definite 
recommendations for field practice until after they have been ade- 
quately tested under conditions that obtain in the field. To do 
otherwise might cause the reputation of the plant pathologist to 
suffer a serious decline; furthermore the mistakes of an individual 
sometimes reflect discredit to his associates and colleagues as well. 
Unfortunately scientific theory, as developed from experiments 
under controlled conditions existing in the laboratory, and field 
practice may prove to be miles apart. All in all, there clearly 
exists a real need among plant pathologists and mycologists for 
better acquaintance with both saprogenic and pathogenic fungi 
as they occur in garden, orchard, field, and forest. Such meetings 
with fungi in their natural haunts would serve the same function 
to students of mycology as does the holding of clinics to the 
physician. 

Gradually the interests of students of fungi have become more 
sharply delimited, one group being concerned primarily with 
taxonomic problems and the other with disease problems. This 
specialization has been carried to the extent that it is unusual for 
a mycologist to do research in plant pathology and for a phyto- 
pathologist to do research in mycology. The underlying reasons 
are not difficult to discern. They may most charitably be at- 
tributed to the frailties and limitations of the human mind and to 
the fact that specialization in training and interest has become 



IMPLICATIONS 421 

compulsory as a consequence of competition and the desire to gain 
recognition in a chosen field. 

The control of plant diseases is based mainly upon ( 1 ) preven- 
tion and (2) natural resistance. Prevention may be accomplished 
by attention to sanitary measures, rotation of crops, seed treat- 
ment, establishment of quarantines, application of fungicidal 
sprays and dusts, and other means. Natural resistance is sought 
and isolated by selection and hybridization. Little has been done 
in the field of chemotherapy, or the cure of plant diseases bv 
chemical agencies, especially by the use of vaporous substances, 
although this field of inquiry seems to offer inviting possibilities 
for development. The principle involved in the use of chemicals 
as therapeutic agents is the existence of a differential between 
pathogen and suscept in tolerance for the chemical or drug. That 
such studies have merit is indicated by results from the use of 
benzol and related compounds in the cure of tobacco downy mil- 
dew. Similarly the vapors of ethyl mercury chloride and ethyl 
mercury phosphate have been found effective against Glomerella 
gossypii in cotton-seed treatment [Lehman ( 1943) ]. It is of more 
than passing interest to note that studies of chemotherapy in hu- 
man diseases, beginning with the work of Ehrlich, resulted in the 
discovery of only a few specifics until the recent introduction of 
the use of sulphonamides and antibiotics oroduced by certain 
fungi and bacteria. 

IMPLICATIONS 

Mycology and phytopathology, parent and offspring, respec- 
tively, have not always worked together harmoniously. ' Seem- 
ingly, parents have not learned to accept gracefully the counsel 
and dictation of their children! The offspring have become 
numerically larger than the parent, and as an outcome the irritat- 
ing question of their relative importance has been raised. If their 
relation were to become synergetic rather than antagonistic, both 
mycology and phytopathology would profit. It is indicated that 
such a development is in process of accomplishment. This end 
could be attained most effectively and most rapidly if teachers 
earnestly strove to impart instruction that not only embodied all 
tradition, theories, and useful truth about fungi but also indicated 
the relation of such knowledge to a balanced, well-rounded edu- 
cation. Moreover, teachers with this viewpoint are true scientists 
and cannot be nationalistic, for science, like literature, music, and 



428 MYCOLOGY IN RELATION TO PLANT PATHOLOGY 

art, is international. Scientists contribute their efforts and find- 
ings for the betterment of mankind everywhere, without regard 
to race, creed, or political and social affiliations. Such scientists, 
as teachers, are not unduly concerned with the degree of esteem 
and respect accorded them by the public. They are true human- 
ists, servants of their times, and this in itself is their all-sufficient 
and soul-satisfying reward. 

LITERATURE CITED 

Arnaudi, C, "On the vaccination of the tobacco plant against Thielaviopsis 

basicola" Bull. Torrey Botan. Club, 60:583-597, 1933. 
Arthur, J. C, "History and scope of plant pathology," Congr. Arts Sci., 

St. Louis, 5: 149-164, 1906. 
Carbone, D., and C. Arnaudi, Vimmuuita nelle piante. Monographic dell 

Inst. Sieroterapico Milanese. 271 pp. 1930. 
Chester, K. S., "Studies on the precipitin reaction in plants. I. The specifi- 
city of the normal precipitin reaction," /. Arnold Arboretum, 13: 52-74, 

1932. 
Chester, K. S., and T. YV. Whitaker, "Studies on the precipitin reaction in 

plants. III. A biochemical analysis of the 'normal' precipitin reaction," 

/. Arnold Arboretum, 14: 118-197, 1933. 
Fabricius, J. C, "Attempt at a dissertation on the diseases of plants," 

Pbytopath. Classics, 1. 66 pp. 1926. (Translated by Margaret K. 

Ravn.) 
Fontana, Felice, "Observations on the rust of grain," Pbytopath. Classics, 

2. 40 pp. 1932. (Translated by P. P. Pirone.) 
Kuster, Ernst, Phytopatbologiscbe Pflanzenanatomie, 3rd ed. xii -+- 558 pp. 

G. Fischer, Jena. 1925. 
Lehman, S. G., "Vapor action of certain fungicidal materials prepared for 

dusting cotton," Phytopathology, 35:431^148, 1943. 
Prevost, Benedict, "Memoir on the immediate cause of bunt or smut of 

wheat, and of several other diseases of plants, and on preventives of 

bunt," Pbytopath. Classics, 6. 94 pp. 1939. (Translated by G. YV. 

Keitt.) 
Re Fillipo, Saggio teorico-pratico sulle malattie delle piante. 1-437. 

Venezia. 1807. 
Smith, E. F., "Plant pathology: a retrospect and prospect," Science, 75: 601— 

612, 1902. 
"Fifty years of pathology," Proc. Intern. Congr. Plant Sci. Ithaca, 1: 13-46, 

1929. 
Whetzel, H. H., An outline of the history of phytopathology. 130 pp. 

W. B. Saunders Co. 1918. 
"The terminologv of phytopathology," Proc. Intern. Congr. Plant Sci. 

Ithaca, 2: 1204-1215, 1929. 
Wingard, S. A., "The nature of disease resistance in plants," Botan. Rev., 

7:59-109, 1941. 



Chapter 19 
SOIL FUNGI 

In studies of soil fertility much emphasis has been placed upon 
mineral composition, and all too little attention has been given to 
microbial composition of soils. Indeed Boussingault and Lew y 
(1853) long ago showed that the nitrate content of soils, if left 
fallow, increased, but the causal relation of biologic factors was 
not recognized at that time, nor was it definitely established until 
1877. Then Schloesing and Miintz (1877), in epoch-making 
studies involving the purification of sewage, established the foun- 
dations of nitrification and soil fertility, and their findings consti- 
tute the basis for present-day knowledge of relationships between 
biologic factors and soil fertility. An appreciation of these mat- 
ters can best be gained from reading The Microorganisms of the 
Soil [Russell et al. (1923)], Principles of Soil Microbiology 
[Waksman (1927)], and Die microscopischen Boden-Pilze 
[Niethammer (1937)]. 

A proper appraisal of the composition of soil must take into 
account its content of microbes, including bacteria, protozoa, 
blue-green algae, green algae, and fungi. It appears that Adametz 
(1886) was the first to isolate fungi from the soil. No real interest 
in the fungus flora of the soil was manifest, however, until nearly 
20 years later, when Oudemans and Koning (1902) isolated and 
described 45 species of soil fungi. Subsequent studies on this sub- 
ject may be grouped into three essential types: (1) taxonomic, 
those concerned with the kind and number of fungi in soils; (2) 
biochemic, those dealing with the physiological activities of soil 
fungi; and (3) epidemiologic, those dealing with soil-borne plant 
and animal pathogens. 

TAXONOMIC STUDIES 

Methods. As might be anticipated, various techniques for iso- 
lating and culturing soil fungi have been employed. Oudemans 

429 



430 SOIL FUNGI 

and Koning (1902) placed a fragment of humus in a small vessel 
containing 1 ml of sterilized water. After the humus has been 
thoroughly triturated, a platinum loopful of suspension was intro- 
duced into 10 ml of sterilized water. A small quantity of this di- 
lute suspension was then poured upon the surface of poured plates 
of media, consisting of agar 1.5%, gelatin 10 o/ o , and sucrose 2%, 
with an added quantity of wort. 

HaQ-em (1910) sprinkled small amounts of soil on the surface 
of poured plates in attempts to isolate Alucorales. By repeated 
transfer of mycelium and spores to new substrates he secured pure 
cultures. Lendner (1908) employed tubes or flasks of wort 
gelatin or of moist bread on which small amounts of soil were 
planted. Several other workers have used a filtrate of soil, sus- 
pended in water for 24 hours, to enrich the media. Matters in- 
volving media and methods of sampling and of isolating soil fungi 
are discussed in an outline bv Waksman and Fred (1922). They 
recommend the use of sodium albuminate agar, sodium caseinate 
agar, or soil-extract agar and gelatin. 

Since fungi are tolerant of acid substrates, whereas bacteria and 
actinomycetes grow best on neutral or alkaline media, a reaction 
of pH 4.0 to 5.0 is preferable in the isolation of fungi. 

Conn (1922) proposed the use of a technique by means of which 
the presence of fungus hvphae in soils could be demonstrated by 
direct microscopic examination. 

In isolating "water molds," a small quantity of soil, along with 
some sterilized water, is first placed in a Petri dish, and then 
boiled hemp seeds are introduced as "bait." Many workers, be- 
ginning with Harvey (1925), have employed this technique. 

Kinds of fungi isolatfd. Opinion was divided among earlier 
students on whether funQ-i are normal inhabitants of the soil. 
The weight of evidence, however, has gradually favored the ex- 
istence of a true fungus flora of the soil. The isolations of 
Adametz (1886) yielded 11 species of fungi, among which were 
Aspergillus gluteus, Penicillium glaucum, Mac or vmcedo, M. 
raceinosus, and M. stolonifer. These species have been quite com- 
monly found by all whose interest has centered on the problem 
of kinds of soil fungi. These investigators have included Oude- 
man and Koning (1902), Lendner (1908), Hagem (1910), Beck- 
with (1911), Dale (1912, 1914), Jensen (1912)" Goddard (1913), 
Werkenthin (1916), Paine (1927), Gilman and Abbott (1927), 



TAXONOM1C STUDIES 431 

LeClerg and Smith (1928), Jensen (1931), Cobb (1932), and 
Gillman (1944). 

The list of Oudeman and Koning (1902), from Netherlands 
includes 45 species, 9 of which are Alucorales. Lendner (1908) 
described 9 new species of Mucorales among the fungi which he 
isolated from soils in Switzerland. Hagem (1910) isolated 18 
species of Alucorales from field, meadow, forest, and garden soils 
in Norway, 9 of them being new species. 

Dale (1912, 1914) isolated more than 100 species of fungi from 
soils in England. Jensen (1912) isolated 35 species in New York 
state. Waksman (1917) obtained from different sections of the 
United States and Hawaii 25 soil samples, from which he isolated 
more than 200 species, 137 of which he was able to identify. 
Among the genera represented were Absidia, Alucor, Rhizopus, 
Zygorhynchus, Saccharomyces, Hypoderma, Sordaria, Sphaero- 
nema, Monilia, Oidium, Papulospora, Aspergillus, Penicillium, 
Scopulariopsis, Rhinotrichum, Sepedonium, Botrytis, Verticil- 
lium, Acrostalagmus, Cephalothecium, Stachybotrvs, Dematium, 
Cladosporium, Alternaria, Macrosporium, Helminthosporium, 
Stysanus, and Fusarium. The summary by Brierley (1923) in 
1923 indicated that up to that time there had been recorded from 
isolations from soils 56 species in 11 genera of Phy corny cetes, 12 
species in 8 genera of Ascomy cetes, and 197 species in 62 genera 
of Fungi Imperfecti, including Actinomycetes. This did not in- 
clude, of course, the startling multitude of species of Basidiomy- 
cetes that grow especially in forest soils. Later the report by 
Gilman and Abbott (1927) listed a total of 61 genera, including 
242 species from Iowa soils. A later, more comprehensive report 
by Gilman (1944) contained a list of 198 species of Phycomycetes, 
30 Ascomycetes, and 383 Fungi Imperfecti. Paine (1927) de- 
scribed as new 5 among the 31 species isolated. 

Beginning with the studies of Harvey (1925), there has been a 
lively interest in the occurrence of Phycomycetes, especially water 
molds in soils. Harvey isolated the following species: Brevilegnia 
diclina, Geolegnia inflata, G. septisporangia, Leptolegnia subter- 
ranea, Saprolegnia ferax, hoachlya eccentrica, and Achlya caro- 
liniana. Among other soil-inhabiting species are Allomyces arbus- 
cula, A. javanicus, A. cysto genus and A. moniliformis. These spe- 
cies, especially A. arbuscula, appear to be widely distributed 



432 SOIL FUNGI 

throughout the world, according to Emerson (1941) and Wolf 

(1940. 

Number of fungi in soils and factors influencing preva- 
lence. The quantitative determinations of fungi in soils have been 
made by use of dilution-poured plates, and the results obtained do 
not constitute an entirely satisfactory estimation. Anions the 
factors that are known to influence the results are: (1) dilution 
of soil suspension, (2) kind of culture medium, (3) reaction of 
medium, (4) kind of soil, (5) soil reaction, (6) depth at which 
sample was taken, (7) moisture, (8) season of the year, (9) till- 
age, (10) manuring practices. 

If the soil fungi are sporulating in the sample being examined, 
the number of colonies will be lar^e; if they are merely ves;etatin<j;, 
the investigator may get a small count and as a result may infer 
that few fungi are present. 

Data presented by Brierley (1923) show that portions of the 
same soil suspension plated on different media yield strikingly dif- 
ferent numbers of colonies! Furthermore, when the same soil 
suspension is plated on the same medium, adjusted to different 
initial hydroeen-ion concentrations, the number of colonies de- 
veloping is very different. Brierley 's observations from monthly 
plate counts of fungi in soils at the Rothamsted Experiment Sta- 
tion led him to conclude that there is a seasonal rhythm in the 
number of soil fungi, ranging from approximately 200,000 to 
1,600,000 per gram. Jensen (1931), using European soils from 
fields, meadows, forests, heaths, moors, and marshes, secured 
counts ranging from 24,300 to 46,000 per gram of soil. 

Accord exists among all investigators that fungi are most abun- 
dant near the surface of the soil and that the number decreases 
with depth. LeClerg and Smith (1928) found Aspergillus mger 
and Trichoderma lignorum in Colorado soils only at the surface. 
Russell (1923) isolated 30 species at a depth of 1 in. from the 
surface of an unmanured grass plot, 19 species at 6 in., and 1 1 spe- 
cies at 12 in. Goddard (1913) in .Michigan and Werkenthin 
(1916) in Texas found quite the same uniform distribution of 
species to a depth of approximately 4 in. Waksman (1916) found 
Z,y gorhynchus vuilleminii most often in subsoil at depths of 12 
to 20 in. Cobb (1932) recorded that fungi are 10 times as abun- 
dant in the top soil under hemlock trees as in the subsoil. The 
data of Takahashi (1919) showed 590,000 fungi per gram of soil 



TAXONOMIC STUDIES 433 

at a depth of 2 cm and 160,000 at 8 cm. He found Zygorhynchns 
molleri and Trichoderma koningii at the lower depths. 

Since the soil is such a complex environment, there are abun- 
dant reasons for differences of opinion regarding kinds of soil 
fungi in different soils. Goddard (1913) and Werkenthin (1916) 
found a constant and characteristic fundus flora of soils, regard- 
less of tillage, soil type, and manuring. Dale (1912, 1914) found 
certain species common to chalky, peaty, and black earth soils in 
England. Waksman (1916) obtained the same species from culti- 
vated and uncultivated soils in New Jersey but concluded that 
each soil possesses a more or less characteristic fungus flora. 
Brown (1917) expressed a similar opinion by stating that different 
soils have different fungus floras. Hagem (1910) isolated Alucor- 
ales from the soil of meadows, gardens, forests, and cultivated 
fields but observed that they are most abundant in forest soils. 
On the other hand, Jensen (1931) found that Mucorales are most 
abundant in field and garden soil, whereas species of Trichoderma 
are most common in virgin soils, such as those of forests, moors, 
and heaths. 

Cobb (1932) was led to conclude that species of Mucor and 
Aspergillus are scarce in forest soils. She also reported differences 
in abundance between soils under hemlock trees and under de- 
ciduous trees, there being twice as many in the top soil under 
hemlocks as in that under deciduous species. Other observations 
on factors that modify the presence of specific fungi in soils in- 
clude those of LeClerg and Smith (1928). Their evidence showed 
that Rhizopns nigricans and Trichoderma lignornm occur most 
abundantly in soils of low mineral and low moisture content and 
that Fenicillium expansum is not limited by soil moisture and oc- 
curs abundantly, as does P. lilacimim, in soils of high mineral 
content. 

Experimentation involving the influence of each of the several 
factors that modify the activities of soil fungi has been limited. 
Coleman (1916) employed sterilized soils, with Aspergillus niger, 
Trichoderma koningii, and Zygorhynchus vuilleminii among the 
test organisms on which to study the effects of temperature, aera- 
tion, and food supply. All grew best at approximately 30° C, 
but the species differed in their oxygen and food requirements. At 
any rate, there was no interaction of one species with another nor 
with the numerous species of soil microorganisms that occur in 



434 SOIL FUNGI 

unsterilized soil, so that the application of Coleman's findings to 
conditions in the field is difficult or even impossible of accom- 
plishment. 

Waksman (1922) applied several treatments to soils to determine 
their influence upon the numbers of fungi and obtained the results 
shown in Table 31. 



TABLE 


31 




ence of Soil Amendments upon the Ni 


jmbers of Soi 




Soil 


Number of 




Reaction 


Fungi per 


Substance Applied 


(pH) 


Gram of Soil 


Minerals only 


5.6 


37,300 


Heavy supply of manure 


5.8 


73,000 


Sodium nitrate 


5.8 


46,000 


Ammonium sulphate 


4.0 


110,000 


Minerals and lime 


6.6 


26,000 



Ammonium sulphate and lime 6.2 39,100 

In general, it would be expected that soils rich in organic matter 
would support the most abundant fungus population. Jensen 
(1931) is among those who hold this belief, for he concluded that 
the application of barnyard manure to soils results in increased 
numbers of fungi. 

The kind of organic matter, through its correlation with the 
kind of cleavage products resultant from decomposition, may 
well be a factor of consequence in determining the kind of fungi 
that predominate. Species of Penicillium and Trichoderma were 
noted by Jensen (1931) to prevail in acid soils. In this instance 
carbohvdrates may have constituted the source from which the 
acids were derived. On the other hand, Jensen (1931) also made 
the observation that My co gone nigra and Coccospora agricola 
prevailed in alkaline soils that may be assumed to have derived 
their alkalinity bv ammonification of proteins. 

BIOCHEMICAL ACTIVITIES OF SOIL FUNGI 

The purpose of this discussion is to stress the role that soil fungi 
play in the transformation of organic matter into humus and into 
other material necessary for the nutrition of green plants. The 
impact of bacteriologic study and teaching has resulted in estab- 
lishing the impression that bacteria constitute the organisms most 



BIOCHEMICAL ACTIVITIES OF SOIL FUNGI 435 

concerned in these important changes, when, as a matter of fact, 
soil fungi are also vitally concerned in these processes. An at- 
tempt will be made to show that these fungi function in three in- 
terrelated ways: (1) in decomposing carbohydrates, (2) in am- 
monifying proteins, and (3) in producing mineral transformations. 

Decomposition of carbohydrates. Both simple and complex 
carbohydrates are now known to be fermented by various fungi. 
It may be recalled that Hoppe-Seyler (1886) long ago secured 
evidence that filter paper is digested in the presence of a little 
sewage slime. He placed 25.773 grams of filter paper in a flask, 
so constructed that he could lead off the gases for analyses. After 
4 years 15 grams of the cellulose had been digested, with the pro- 
duction of 3281 cc of carbon dioxide and 2571 cc of methane. 
This decomposition was established to be induced by anaerobic 
bacteria. Evidence that fungi can also function in the decomposi- 
tion of cellulose was first presented by van Iterson (1904) in 
1904. His experiments were performed not with pure cultures 
but with soil as inoculum. The medium consisted of filter paper 
moistened with tap water in which small amounts of ammonium 
nitrate and monopotassium phosphate had been dissolved. By this 
procedure evidence was secured to show that certain fungi, includ- 
ing Chaetommm kunzeanum, Trichocladium asperum, Stachy bo- 
try s altemans, Sporotrichum bomby cinum, S. roseolum, S. griseo^ 
lum, Botrytis sporoideiim^ My co gone pnccinioides, and Clado- 
sporhim herbarum, digest cellulose. Van Iterson's observations 
initiated a series of studies on cellulose digestion by fungi, among 
them those by Kellerman and AIcBeth (1912), Daszewska (1913), 
Scales (1916), Waksman (1918), and Henkelekian and Waksman 
(1925). Kellerman and McBeth (1912) made use of cellulose 
agar, the preparation of which they describe, and established that 
many species of Aspergillus, Fusarium, Penicillium, and Sporo- 
trichum utilize cellulose as nutrient in pure cultures. Daszewska 
(1913) found that Sporotrichum olivaceum, Verticillium glau- 
cum, V. celhdosae, and various other Hvphomvcetes are more 
important in cellulose decomposition than are bacteria and that 
the color of the humus formed is related to that of mycelium and 
conidia. Among 22 species of soil fungi tested by Waksman 
(1916), 15 were able to decompose cellulose. 

Henkelekian and Waksman (1925) have shown that Tricho- 
derma and Penicillium possess the ability to decompose cellulose 



436 SOIL FUNGI 

completely, with carbon dioxide as the only waste product. 
Moreover a considerable proportion of the carbon in cellulose 
may be reassimilated by the fungus in building protoplasm. This 
observation on the utilization of carbon dioxide is elaborated by 
Foster et al. ( 1941) in their recent studies on this subject. 

Abundant evidence, some of which is summarized in Chapter 3, 
has been secured that many Basidiomycetes, especially wood- 
rotting species, are capable of utilizing cellulose. Phycomycetes 
are generallv regarded as incapable of digesting cellulose. The 
work of Whiffen (1941), however, shows that certain chytrids 
possess this ability. 

Manv fungi are known to be capable of utilizing starch. Among 
22 species of soil fungi tested by Waksman (1916) for diastatic 
abilitv, 6 proved capable of using starch. The Mucorales have 
been shown to utilize manv monosaccharides, disaccharides, and 

also pectins. 

Decomposition of proteins. That fungi differ in ability to use 
elemental nitrogen and nitrogen complexes was given considera- 
tion in Chapter 2, where it was pointed out that some few species 
can assimilate atmospheric nitrogen but that most of them prefer 
amino acids, nitrate nitrogen, or else ammonium salts. That soil 
funoi have the power of ammonifying proteins was first demon- 
strated in 1893 by Miintz and Coudon (1893), using Mucor race- 
mosus and Fusarium miitzii, and by Marchal (1893), using As- 
pergillus terricola and Cephalothechtm roseum. Numerous in- 
vestigations of this problem followed, including those of McLean 
and \vilson (1914), Waksman (1916), and Henkelekian and 
Waksman (1925). 

McLean and W T ilson (1914) employed members of the Mucor- 
aceae, Aspergillaceae, Dematiaceae, and Moniliaceae, finding that 
all could produce ammonia either from dried blood or from cot- 
tonseed meal. It was observed that some species are more active 
than others, but of much more interest was the finding that soil 
fungi exceed bacteria in ammonifying power. Waksman (1916) 
showed that Trichoderma koningii is an especially potent ammoni- 
fier. Evidence is lacking that any species of soil fungi takes part 
in nitrification. 

Henkelekian and Waksman (1925) observed a direct correla- 
tion between the amount of nitrogen transformed into ammonia 



IMPLICATIONS 437 

by species of Penicillium and Trichoderma and the amount of 
cellulose decomposed. 

The abundance of studies on protein decomposition by soil 
fungi has yielded data on the various factors that modify the 
accumulation of ammonia. These factors are known to include 
aeration, soil moisture, soil type, soil reaction, duration of the 
incubation period, temperature, nature of the protein complex, 
and presence of soil minerals, especially phosphates. 

SOIL-BORNE PATHOGENS 

No attempt can be made adequately to summarize the vast liter- 
ature on the relation of soil-inhabiting fungi to disease in plants. 
Species of Pythium, Phvtophthora, Aphanomyces, Thielaviopsis, 
Fusarium, Sclerotinia, Colletotrichum, Gloeosporium, Botrytis, 
Rhizoctonia, Sclerotium, and Phymatotrichum are among those 
well-known to be soil-borne and to cause serious destruction of 
crops. Some of them occur in virgin soils, and others are intro- 
duced with the culture of the host species. Unfortunately many 
of them, when once introduced into a field, persist for years, even 
when susceptible hosts are not planted in these fields for long 
periods. Pratt (1918) isolated Fusarium radicicola, F. tricho- 
thecioides, and Rhizoctonia solani from soils in southern Idaho 
that had never been cropped to potatoes. Rathbun (1918) found 
Fusarium, a cause of damping-ofl of coniferous seedlings, in vir- 
gin seed-bed soils. Soils that are "crop sick," on the other hand, 
may contain a variety of species capable of producing infection 
[Beckwith (1911)]. 

Infection by soil-inhabiting fungi has been shown to be con- 
trolled by such factors as temperature, reaction, and interaction, 
subjects given consideration in Chapters 5, 7, and 12. 

Few cases involving soil-borne human pathogens have been 
proved. Emmons (1942) determined that Coccidioides immitis, 
the cause of "valley fever," may be isolated from the soil in re- 
gions where this disease is endemic. 

IMPLICATIONS 

As a result of the transformation of organic materials into 
humus by soil fungi, organic acids are produced, and these acids 



438 SOIL FUNGI 

have properly been assumed to account for soil acidity. Hagem 
(1910) concluded that inorganic soil constituents containing such 
minerals as calcium, magnesium, and phosphorus are dissolved 
bv these organic acids and thereby made available for green plants. 
Soil fungi are therefore to be regarded as important in soil fertil- 
ity. Much remains to be determined, however, concerning the 
indirect role of fungi in making available iron, sulphur, and the 
many other elements that green plants require in small amounts. 

.Many soil fungi, as grown on artificial media or on sterilized 
soil, should be studied intensively to increase our knowledge of 
their biochemical activities. Similarly two or more species, if 
grown in association in the same culture, might yield valuable 
data. The application of these findings in explaining the activities 
of fungi in normal soils would require the exercise of incisive 
thinking and well-balanced judgment. Success would be most 
likely attained if such studies were undertaken by a corps of work- 
ers, including microbiologists, chemists, and physicists, working 
in collaboration. 

Means for measuring soil fertility continue to be sought because 
in the future an adequate supply of food and feed crops will come 
more and more to depend upon a better knowledge of soil fertility. 
Partly for this reason the use of Aspergillus niger to test the soil- 
potassium needs of a given crop, as was proposed by Mehlich 
etal. (1933), has intriguing possibilities for application to require- 
ments for other minerals. 

Undoubtedly soil fungi perform an important role in produc- 
ing growth-promoting substances that are utilized by green plants. 
It is a well-established fact that crop plants do not grow as well 
on infertile soil if the fields are enriched with mineral fertilizers 
as if they are enriched with manure or organic material containing 
equivalent amounts of minerals. The relationship of soil fungi to 
the production of growth regulators should be further elucidated. 

The results of researches on soil fungi, if viewed in perspective, 
emphasize that soils are not static, but dynamic. The ever-chang- 
ing balance between each kind of soil microbe and the mineral 
and non-living organic content of soils still remains largely un- 
known. A concise summary of these subjects, together with an 
excellent bibliography, is to be found in a paper by YVaksman 
(1944). 



LITERATURE CITED 439 



LITERATURE CITED 

Adametz, L., "Untersuchungen iiber die niederen Pilze der Ackerkrume," 

Inaugural dissertation. 78 pp. Leipzig. 1886. 
Beckwith, T. D., "Root and culm infections of wheat by soil fungi in North 

Dakota," Phytopathology, 7:169-176, 1911. 
Boussingault, J. B., and Lewy, "Sur la composition de Fair confine dans 

de terre vegetale," Ann. chim. phys., Troisieme Serie, 57:5-50, 1853. 
Brierley, W. B., "The occurrence of fungi in the soil." In E. J. Russell, 

Microorganisms of the Soil, pp. 118-146. 1923. 
Brown, P. E., "Importance of mold action in soils," Science, 46: 171-175, 

1917. 
Cobb, Mary Jo, "A quantitative study of the microorganic population of a 

hemlock and a deciduous forest soil," Soil Sci., 33: 325-345, 1932. 
Coleman, D. A., "Environmental factors influencing the activity of soil 

fungi," Soil Sci., 2: 1-65, 1916. 
Conn, H. J., "A microscopic method for demonstrating fungi and actinomy- 

cetes in soil," So/7 Set., 14: 149-152, 1922. 
Dale, E., "On the fungi of the soil," Ann. My col, 10:452-477, 1912; 

72:33-62, 1914. 
Daszewska, W., "Etude sur la desagregation de la cellulose dans la terre de 

bruyere et la trube," Bull. soc. botan. Geneve, Ser. 8, fasc. 8: 255-316, 

1913. 
Emerson, Ralph, "An experimental studv of the life cvcles and taxonomy of 

Allomyces," Lloydia, 4: 77-144, 1941. 
Emmons, C. W., "Isolation of Coccidioides from soil and rodents," U. S. 

Pub. Health Rept., 57: 109-111, 1942. 
Foster, J. W., S. F. Carson, S. Ruben, and M. D. Kamen, "Radioactive car- 
bon dioxide utilization. VII. The assimilation of carbon dioxide bv 

molds," Proc. Nat. Acad. Sci., 27:590-596, 1941. 
Gilman, J. C, A manual of soil fungi. 392 pp. Iowa State College Press, 

Ames, Iowa. 1944. 
Gilman, J. C, and E. V. Abbott, "A summary of the soil fungi," Ioiva State 

Coll. J. Sci., 1: 225-343, 1927. 
Goddard, H. M., "Can fungi living in agricultural soil assimilate free nitro- 
gen?" Botan. Gaz., 55:249-305, 1913. 
Hagem, O., "Untersuchungen iiber Norwegische Mucorineen. I," Viden- 

skapsselskapets-Skrifter Mat.-natiirv. Klasse Kristiania, 7: 1-50, 1907; 

II, 4: 1-152, 1910. 
"Neue Untersuchungen iiber Norwegische Alucorineen," Ann. Mycol., 

8:265-286, 1910a. 
Harvey, J. V., "A survey of the water molds and Pythium occurring in the 

soils of Chapel Hill?' /. Elisha Mitchell Sci. Soc, 41: 151-164, 1925. 
Henkelekian, H., and S. A. Waksman, "Carbon and nitrogen transforma- 
tion in the decomposition of cellulose by filamentous fungi," /. Biol. 

Chem., 66: 323-342, 1925. 



440 SOIL FUNGI 

Hoppe-Seyler, F., "Uber Gahrung der Cellulose mit Bildung von Alethan 
und Kohlensaure," Hoppe-Seylefs Z. physiol. Chem., 70:201-217; 401- 
440, 1886. 

Iterson, C. van, "Die Zersetzung von Cellulose durch aerobe Mikroorganis- 
men," Zentr. Bakt. Parasitenk., II Abt., 77:689-698, 1904. 

Jensen, C. N., "Fungus flora of the soil," Cornell Agr. Expt. Sta. Bull., 
575:415-501, 1912. 

Jensen, H. L., "The fungus flora of the soil," Soil Sci., 31: 123-158, 1931. 

Keller.man, K. F., and I. G. McBeth, "The fermentation of cellulose," 
Zentr. Bakt. Parasitenk., II Abt., 5-^:485-494, 1912. 

LeClerg, E. L., and F. B. Smith, "Fungi in some Colorado soils," Soil Sci., 
25:433-^41, 1928. 

Lendner, A., Les Mucorinees de la Suisse. 180 pp. Berne. 1908. 

.Marchal, E., "Sur la production de rammonique dans le sol par les 
microbes," Bull. acad. sci. Belg., 25:727-771, 1893. 

McLean, H. C, and G. AY. Wilson, "Ammonification studies with soil 
fungi," N. J. Agr. Expt. Sta. Bull., 210. 39 pp. 1914. 

Mehlh ii. A., E. Truog, and E. B. Fred, "The Aspergillus niger method of 
measuring available potassium in soil," Soil Sci., 55:259-276, 1933. 

.Muntz, A., and H. Coudon, "La fermentation ammoniacale de la terre," 
Covipt. rend., 116: 395-398, 1893. 

Xiethammer, A., Die microscopischen Boden-Pilze, ihr Leben, ihre r cer- 
breitung, soivie ihre oeconomische und pathogene Bedeutmig. 193 pp. 
W. Junk, The Hague. 1937. 

Oudemans, C. A. J. A., and C. J. Koning, "Prodrome d'une flore my- 
cologique, obtenue par la culture sur gelatin preparee de la terre humeuse 
du Spanderswould pres de Bussum," Arch, neerland. sci., 7: 266-298, 1902. 

Paine, F. S., "Studies of the fungous flora of virgin soils," My col., 19: 248- 
267, 1927. 

Pratt, O. A., "Soil fungi in relation to diseases of the Irish potato in south- 
ern Idaho," /. Agr. Research, 13: 73-100, 1918. 

Rathbun, Annie E., "The fungous flora of pine seed beds. I," Phyto- 
pathology, 8: 469-483, 1918. 

Russell, E. J., et al. The microorganisms of the soil. 188 pp. Longmans, 
Green and Co., London. 1923. 

Scales, F. M., "Studies on the decomposition of cellulose in soils," Soil Sci., 
7:437-487, 1916. 

Schloesing, T., and A. Muntz, "Sur la nitrification par les ferments or- 
ganises," Compt. rend., 84: 301-303, 1877; 85: 1018-1020, 1877. 

Takahashi, R., "On the fungus flora of the soil," Ann. Phytopath. Soc. 
japan, 12: 17-22, 1919. 

Waksman, S. A., "Soil fungi and their activities," So/7 Sci., 2: 103-155, 1916. 
"Is there any fungus flora of the soil?" Soil Sci., 3: 565-589, 1917. 
"The importance of mold action in the soil," So/7 Sci., 6: 137-155, 1918. 
"The growth of fungi in the soil," So/7 Sci., 14: 153-158, 1922. 
Principles of soil microbiology, xix + 897 pp. Williams and Wilkins 
Co., Baltimore. 1927. 



LITERATURE CITED 441 

Waksman, S. A., "Three decades with soil fungi," So/7 Set., 58: 89-114, 1944. 

Waksman, S. A., and E. B. Fred, "A tentative outline of the plate method 
for determining the number of microorganisms in the soil," So/7 Sci., 
14: 27-28, 1922. 

Werkenthin, F. C, "Fungus flora of Texas soils," Phytopathology, 6: 241- 
253, 1916. 

Whiffen, Alma J., "Cellulose decomposition by the saprophytic chytrids," 
/. Elisha Mitchell Sci. Soc, 51: 321-329, 194L 

Wolf, Fred T., "A contribution to the life history and geographical distri- 
bution of Allomyces," MycoL, 33: 158-173, 1941. 



Chapter 20 

FUNG US-INSECT INTERRELA TIONSHIPS 

The studies to date on the interrelationships of fungi and in- 
sects may be placed largely in one or the other of five categories: 

( 1 ) those dealing with insects as vectors of plant-pathogenic fungi, 

(2) those concerned with fungi that produce diseases of insects, 

(3) those involving fungi as agencies in the biological control of 
insects injurious to crops, (4) those dealing with insects that make 
it possible for certain species of fungi to undergo their cyclical 
changes or developmental processes, and (5) those involving fungi 
that are cultivated by insects for food. These studies deal with a 
laro-e number of species in each group of organisms. For this 
reason any account that attempts a complete review of the litera- 
ture and a discussion of it would of necessity be voluminous, and 
such an undertaking is beside the present purpose. Instead an 
attempt will be made by the use of representative examples to 
introduce each of these important fields of interest. A much more 
comprehensive account of these subjects, to which the student is 
referred, is contained in a volume by Leach ( 1940). 

INSECTS AS VECTORS OF PLANT-PATHOGENIC FUNGI 

This subject was briefly considered in Chapter 8, and to avoid 
repetition some details are omitted at this point. Attention may 
well be directed, however, to certain general features of this 
phase of the fungus-insect relationship. It should be appreciated, 
first of all, that a background of related evidence facilitated ac- 
ceptance of the fact that insects are instrumental in dispersing 
certain pathogenic fungi and in implanting them within host 
tissues. Before 1900 it had been established that certain mos- 
quitoes are vectors of the nematode worm, Wuchereria bancrofti, 

442 



INSECTS AS VECTORS OF PLANT-PATHOGENIC FUNGI 4i3 

causing elephantiasis of man, that the malaria-producing protozoa 
are transmitted by mosquitoes, and that ticks transmit the Texas 
cattle-fever organism. Furthermore it had been established that 
the bacterium responsible for fire blight of pears and apples may 
be dispersed by bees and wasps. From such basic observations on 
insect transmission of nematodes, protozoa, and bacteria respons- 
ible for animal and plant diseases, interest in insects as vectors 
increased. As an outcome, instances were found and convincing 
proofs were submitted that viruses and plant-pathogenic fungi 
may also be dispersed by insects. 

The dispersal of plant-pathogenic fungi by insects is accom- 
plished quite fortuitously. Spores that adhere to the body of the 
vector may become dislodged on a near-by host. Leaf-eating in- 
sects quite generally consume diseased and non-diseased tissues 
indiscriminately, and the spores may pass intact through the ali- 
mentary tract. Such spores in the fecal matter can then serve as 
inoculum. Again, spores may be introduced or may gain entrance 
into plant tissues, especially of fruits and twigs, through incisions 
made in connection with oviposition. 

In general, all the dispersal of spores of a given fungus is not 
accomplished by one species of insect. Rather several species of 
insects serve as vectors; they may belong to entirely different 
groups. Among the kinds of insects known to be vectors of plant- 
disease-producing fungi are grasshoppers, crickets, aphids, scale 
insects, beetles, true bugs, flies, wasps, and bees. 

As might be anticipated, abundance of a given vector may be 
directly correlated w T ith the severity of an outbreak of disease 
among plants. For this reason insect control and plant-disease 
control sometimes become mutually interdependent. 

Among important plant-pathogenic fungi known to be trans- 
ported by insects are the following: Endothia parasitica, the chest- 
nut-blight fungus, Claviceps purpurea, the ergot-producing organ- 
ism, Phoma lingam, the cause of cabbage blackleg, Nematospora 
phaseoli, the cause of pod spot of Lima bean, Septoria ly coper sic'u 
the cause of a leaf spot of tomato, Sclerotinia fnicticola, the 
brown-rot fungus of stone fruits, Botrytis anthophila, the cause of 
clover-blossom blight, Ceratostomella uhni, the Dutch elm patho- 
gen, and C. pilifera and related blue-stain-producing species on 
coniferous wood. 



444 FUNGUS-INSECT INTERRELATIONSHIPS 

FUNGI OCCURRING OX OR WITHIN INSECTS 

The entomogenous fungi, or fungi that naturally occur on or 
within the bodies of insects, vary greatly in food habits. Some 
utilize only living insects, whereas others subsist entirely as scav- 
engers. Some exhibit a high degree of specialization; others are 
quite generalized. Furthermore, with the exception of the La- 
boulbeniales and nearly all the Entomophthorales and species of 
Cordvceps, the entomogenous habit is not a characteristic pos- 
sessed by any large group of closely related species. All seem 
markedly influenced in their food habits both by biotic and en- 
vironmental factors. 

.Much of our knowledge of these fungi comes from the investi- 
gations of Thaxter (1888, 1896, 19o{ 1924, 1926) and Petch 
0914, 1921, 1924, 1925, 1926, 1931, 1932, 1935, 1939). Thaxter 
devoted his attention to the Entomophthorales and Laboulbeniales; 
Petch, to various others, principally to Ascomycetes belonging to 
Cordvceps, Hypocrella, Sphaerostilbe, Myriangium, Podonectria, 
and Xectria, and to members of the Fungi Imperfecti belonging 
to Aspergillus, Penicillium, Spicaria (Isaria), Aschersonia, Alicro- 
cera, Yerticillium, Acremonium, Cephalosporium, Rhinotrichum, 
Cladobotryum, and Beauveria. The student of entomogenous 
fungi should also acquaint himself with the check lists by Seymour 
(1929, pp. 698-718) and Charles ( 1941) to gain some appreciation 
of the large number of species of fungi and insects involved. In 
the following account mention will be made only of a few of the 
better-known ones. 

In 1921 Keilin (1921) described as Coelomy ces stegomyiae an 
organism parasitizing the larvae of the mosquito, Stegoviyia scu- 
tellaris. Later Couch (1945) found additional species of Coelo- 
myces in larvae of other mosquitoes, Culex and Anopheles, in 
Georgia, and determined that the parasites belong among the 
Blastocladiales. 

Among the better-known species of Entomophthora may be 
mentioned E. vmscae on houseflies, E. grylii on crickets, and E. 
sphaerosperma on the caterpillars of cabbage butterflies. Ento- 
vwphthora sphaerosperma was reported by Sawyer (1929) as para- 
sitizing Rhopobota vacciniaua, which attacks cranberry vines. 
Speare (1912) described E. psendococci as parasitic on mealy bugs, 



FUNGI OCCURRING ON OR WITHIN INSECTS 445 

Fsendococciis calceolariae, on sugar cane in Hawaii. Petch 
(1926b) lists, from Mysore, E. (Empusa) lecanii as the first mem- 
ber of this genus found attacking a scale insect. 

The "seventeen-year locust," Tibicina septendecem, is very 
commonly attacked by a peculiar fungus, Massospora cicadina 
[Speare (1921)]. This organism grows within the insect's body 
and causes the posterior segments to drop off while the cicada is 
still alive. The conidia and resting spores are then dispersed as 
the cicada crawls or flies. 

In the American tropics Metarrhiziinn anisopliae, called the 
green muscardine fungus, is known to be destructive to approxi- 
mately 60 species of insects. Some of these insects are of im- 
portance, including the sugar-cane froghopper, Tomaspis varia, 
and May beetles, because they normally cause appreciable damage 
to crops. 

Beauvaria bassiana is another generalized species, best known 
from its occurrence on chinch bug, Blissiis leucopterus, and corn 
borer, Fyransta nubilalis. It was first described as a parasite of 
silk-worm larvae and given the name Botrytis bassiana [Petch 
( 1914) ] . Previously another species, Beauveria globulifera, which 
has been confused with B. bassiana, was described from France 
and from South America. In South America it was identified as 
Sporotrichum globulifernm. However, Lefebvre (1931) regards 
B. bassiana an'd B. globulifera as distinct species, and his evidence 
indicates that B. globulifera is the more virulent as a pathogen 
on corn borer. 

Sorosporella uvella, a hyphomycete pathogenic to certain cut- 
worms and to larvae of the sugar-beet curculio, Cleonus pancti- 
ventris, is of peculiar interest because there is no external evidence 
of its presence in host larvae [Speare (1920)]. The fungus is an 
obligate parasite. Its resting spores are formed internally to the 
body wall and come to fill the body cavity with a brick-red 
powdery mass. This organism was first described from Russia, 
where it was given the name Tarichhmi uvella. 

The generic name Isaria has come to be widely known among 
mycologists in connection with conidial stages of Cordyceps that 
parasitize various beetles and other insects. Petch (1934) pro- 
posed, however, that the name Isaria be discarded in favor of 
Spicaria. Then the name Isaria farinosa, as the type, becomes 



446 



FUNGUS-INSECT INTERRELATIONSHIPS 




Fig. 74. Various entomogenous fungi. A. Simple conidiophores of Ccph- 
alosporium lecanii on Lecanium viride on coffee. B. Branched conid o- 
phore of C. lecanii. C. A head of conidia of C. lecanii. D. Conidiophore 



FUNGI OCCURRING ON OR WITHIN INSECTS 441 

Spicaria jarinosa. Spicaria javanica attacks the cottony cushion 
scale, I eery a purehasi, in Puerto Rico. 

According to Petch (1921), there are about 50 valid species of 
Hvpocrella, most of them parasitic on scale insects. A consider- 
able number possess a pvenidial stage belonging to Aschersonia. 
The first Aschersonia to be described was A. aleyrodis on Aley- 
rodes citri, collected in Florida, in 1894. 

Other parasites of scales are mostly species of Myriangium, 
Sphaerostilbe, Nectria, and Podonectria. Of the 15 species of 
Myriangium recognized by Petch (1924a), 4 are entomogenous, 
namely M. duriaei, M. curtisii, M. montagnei, and M. thueakesii. 

Apparently the first entomogenous fungus on scales was col- 
lected in Normandy and given the name Microcera coccophila by 
Desmazieres in 1848 [Petch (1921)]. This is a conidial stage, and 
soon thereafter the Tulasne brothers wrongly attached this name 
to Sphaerostilbe coccophila. Petch, however, maintains that 5. 
flammea is the correct perithecial-stage name for Microcera cocco- 
phila, which is a widely distributed fungus on scales in North 
America. The next scale parasite to be recognized was collected 
on orange twigs in Ceylon and identified as Nectria aurantiicola. 
Later Luttrell (1944) studied the development of this species, 
using the name Sphaerostilbe aurantiicola, which is widely present 
in the Orient and in the southeastern United States. Like 5. 
-flammea, it possesses a similar conidial (Microcera) stage. 

Perhaps the most remarkable of the fungi that attack insects 



of Spicaria javanica with phialides and conidia. E. Conidia of S. javanica. 

F. Conidiophore and conidia of V erticillhim heterocladium, parasitic on 
Aleyrodes. G. Botrvoid clusters of conidiophores of Beanveria bassiana, 
bearing conidia. H. Mature conidia of B. bassiana. I. Germinating conidia 
of B. bassiana. J. Flask-shaped phialides terminating conidiophore branches 
of B. bassiana. K. Colony of young resting spores of Sorosporella uvella 
from diseased cutworm, showing budding. L. Mature resting spores of 5. 
uvella with remains of walls of cohering spores. M. Mature conidia (sec- 
ondary) of S. uvella. N. Verticillate conidiophore of 5. uvella, bearing 
secondary conidia. O. Conidia (Microcera) of Sphaerostilbe aurantiicola. 
P. Ascus of S. aurantiicola. (A, B, C, D, and E after Petch, F after Fawcett, 

G, H, I and / after Lefebvre, K, L, M and N after Speare, and O and P 

after Luttrell.) 



448 FUNGUS-INSECT INTERRELATIONSHIPS 

are species of Septobasidium, a genus monographically treated by 
Couch (1938). Its members live in mutualistic association with 
colonies of scale insects, using some individuals for food and 
giving shelter and protection to others. 

BIOLOGICAL CONTROL OF INSECTS 

That competition between organisms exists everywhere 
throughout nature is clearly appreciated by biologists. This con- 
cept was crystallized from observations and incisive analyses by 
Darwin, and he expressed it by the connotation "the struggle for 
existence." The discussion that follows is intended merely to 
direct attention to man's efforts to intervene in a struggle between 
fungi and insects in order to suppress epidemics of insect pests, 
at least to the extent of bringing them under control. 

The basic principles of biological control of noxious insects by 
microorganisms (fungi, bacteria, viruses, and protozoa) have been 
given consideration by Sweetman (1936). He indicates that the 
following factors should be given particular attention: (1) the 
differences in receptivity or susceptibility of the insect at different 
stages of development; (2) the environment most favorable to the 
pathogenic agent; (3) the virulence of the pathogen as modified 
by environment; and (4) the necessity of having the optimum 
conditions for attack by the pathogen coincide with the occur- 
rence of favorable abundance and developmental stage of the 
insect to be controlled. 

Upon contemplation of these factors it will become apparent 
that little hope of success should be expected in controlling a par- 
ticular insect pest by use of a given entomogenous fungus unless 
and until an understanding has been gained of the aggressiveness 
or virulence of the fungus. For example, some fun^i, such as 
species of Penicillium, Alternaria, and Cladosporium, whose mem- 
bers rarely attack living organisms, may be presumed when pres- 
ent to have invaded the bodies of insects after they have died. At 
the opposite extreme in intergradation of parasitism are such obli- 
gate parasites as Entomophthora and Sorosporella, which thrive 
only while the insect remains alive. Such fungi produce spores 
during a brief period before the death of their victim or immedi- 
ately thereafter, and the spores remain dormant or fail to germi- 
nate unless they come in contact with another living insect. 



BIOLOGICAL CONTROL OF INSECTS 449 

Furthermore weather factors are known to influence the adores- 
siveness of entomogenous fungi. Most of them, especially species 
of Sphaerostilbe, Aegerita, Aschersonia, and Beauveria, are favored 
by wet weather or periods of high humidity coupled with high 
temperature. If dense populations of insects occur at such times, 
these fungi spread with great rapidity, and the insects become dis- . 
eased in epidemic proportions. These factors may therefore be- 
come limiting in man's efforts toward artificial control. 

Beauveria bassiana, first observed in 1835 by Bassi di Lodi as 
pathogenic to silk-worm larvae, is among the better-known spe- 
cies that have been used in efforts to secure control of insects. 
Attempts extending over several seasons were made to control 
flea beetles (Haltica) in Algeria, with the result that the adult 
stage readily became infected, but the larvae seemed quite resistant. 
Attempts were also made over the period 1888 to 1896 to control 
chinch bug, Blissus leucoptenis, in Illinois by use of the related 
Beauveria globulifera. A measure of success was obtained in these 
trials but only when the insects were present in excessive abun- 
dance and when hot, wet weather prevailed. 

Extensive attempts have been made in Florida to utilize naturally 
occurring entomogenous fungi against white flies and scale insects 
in citrus groves [Fawcett (1907, 1908), Berger (1909, 1910), Mor- 
rill and Back (1912)]. In Florida citrus groves, several species, 
including Aegerita ivebberi, Aschersonia aleyrodis, A. goldiana, 
V erticilliinn cinnamomeum, Sphaerostilbe aurantiicola, and Po- 
donectria coccicola, are of importance and have been used arti- 
ficially. Increase of white flies and scale insects has been stimu- 
lated there by the use of Bordeaux mixture to control citrus scab, 
Sphacelovm fawcetti, and citrus melanose, Diaporthe citri. 

The inoculum for these entomogenous species consisted of spore 
suspensions sprayed upon insect-infested trees or of fungus-bear- 
ing leaves or twigs tied to such trees. In some instances the spore 
suspensions were prepared from pure cultures and in others from 
fungi removed from infested leaves. 

Under some conditions the results in Florida and elsewhere 
show that the artificial introduction of fungi has very materially 
aided in the destruction of insects [Picard (1914), Berger (1921, 
1932), and Watson and Berger (1937)]. 

Morrill and Back (1912) concluded, however, that Aegerita 



450 FUNGUS-INSECT INTERRELATIONSHIPS 

ivebberi is so effective in controlling white fly in low-lying ham- 
mock groves that artificial measures are unnecessary. 

Aspergillus parasiticus has been found effective against various 
mealy bugs in Hawaii [Speare (1912)], Puerto Rico [Johnston 
(1910)1, and California [Smith and Armitage (1931)]. 

Species of Entomophthora are not easily cultivated in pure 
culture but, if artificially disseminated, may aid in bringing epi- 
demics of plant lice under control. Entomophthora sphaero- 
spervia, for example, caused considerable reduction in the popu- 
lation of apple sucker, Psyllia mali [Sweetman (1936), p. 71], 
in Nova Scotia and in parts of Europe. Another species, E. 
clorojiiaphidis, was found very destructive to walnut aphis, Chro- 
viaphis juglandicola, in California. In some seasons in Florida E. 
fresemi becomes an important factor in the control of Aphis 
spiraecola, especially on tangerines. 

Metarrhiziwn anisopliae, when artificially applied in some lo- 
calities to corn leaves, has been found very destructive to corn 
borer [Sweetman (1936), p. 75]. 

It becomes apparent to anyone who critically reads accounts 
dealing with attempts to use fungi to control insect pests that the 
results are not always in accord, and the conclusions are often con- 
tradictory. Petch (1921) summarized his pertinent experiences as 
follows: "The problem which has yet to be solved by those who 
wish to control insects by means of fungi is to create an epidemic 
at a time when such an epidemic would not occur naturally." On 
the basis of the relatively few cases in which outstanding control 
has been accomplished Fawcett (1944) suggests that more atten- 
tion should be given to the artificial spreading of entomogenous 
fumn and to more efficient ways of increasing their use. 

INSECTS IX RELATION TO REPRODUCTION OF FUNGI 

It has long been known that insects carry pollen and that the 
setting of seed and the development of certain fruits, for example, 
clovers, apples, and peaches, is conditioned by insect pollination. 
Similarly insects disperse reproductive elements (spermatia) 
among fungi. In support of this conclusion, Brodie (1931 ) found 
that flies are agents of diploidization of Coprimts lagopi/s, a hetero- 
thallic species. The fruit bodies of this mushroom do not form 
unless oidia from the plus mycelium are transported to the minus 



FUNGI CULTIVATED BY INSECTS 451 

mycelium, or vice versa, whereupon they germinate, the hyphae 
fuse, diploidization results, and mushrooms are developed. 

Craigie (1931) showed that Puccinia helianthi and P. graminis 
may be diploidized by the agency of insects. The pycniospores 
of these rusts are haploid. Diploidization occurs only if pycnio- 
spores from one pycnium are transferred to another of opposite 
sex, whereupon the process is initiated by fusion of a germinating 
pycniospore with a receptive (flexuous) hypha that projects from 
the pycnium. Insects may be essential agents in the transfer of 
pycniospores, and such transfer is an essential condition in the 
development of dicaryotic aeciospores. Subsequent findings with 
other rust fungi substantiate these observations. Spermatization 
of certain ascomycetes also is known to result from insect trans- 
fer of spermatia. 

FUNGI CULTIVATED BY INSECTS 

Much of our knowledge on this topic involves "ambrosia" 
beetles (timber-boring Scolytidae, including engraver beetles and 
bark beetles that tunnel and breed in bark and sapwood), leaf- 
cutting ants, and termites. Such relationship of insect and fungus 
is termed an ectosvmbiotic one bv Buchner (1930). By ectosym- 
biosis, in this instance, is meant an association in which the fungus 
occurs chiefly outside the body of the insect. 

Beetles and fungi. Many species of Scolytid beetles are asso- 
ciated with fungi; the better-known ones belong to Scolytus, 
Dendroctonus, Ips, and Hvlurgopinus. Their relationship with 
specific fungi seems none too well understood in most cases, al- 
though the phenomenon of fungus-insect association was first ob- 
served about a hundred years ago. As indicated in Buchner's 
treatise (1930), Thomas Hartig in 1844 recognized that the am- 
brosia of Xyle bonis dispar in Ahms cor data is a fungus, which he 
named Monilia Candida. Subsequent investigations have shown 
that there are many other species of ambrosia fungi. Leach 
(1940) emphasizes, however, that ". . . taxonomic studies of am- 
brosia fungi are conspicuous by their absence." 

Ambrosia fungi in general permeate the wood and enter into 
the burrows and brood galleries made by the beetles. The my- 
celium and spores that protrude into the galleries are eaten by the 



452 FUNGUS-INSECT INTERRELATIONSHIPS 

beetles, and spores either may be regurgitated or may resist diges- 
tion and then be yoided in the excrement. 

A great deal of interest in ambrosia fungi has centered on those 
species associated with the staining of wood, since such wood 
staining is of so much economic importance to the lumber indus- 
try. The work of Rumbold (1936) shows that Ceratostomella 
ips, C. pilifera, and C. pint occur in pines, C. piceaperda in spruce 
in eastern Canada, C. pseudotsugae in Douglas fir and larch in the 
Northwest, and C. pluriannulata in hardwoods. In white fir 
damaged by seyeral species of Scolytus, Wright (1935, 1938) 
noted two wood-staining species, Trichospor'nim symb'wt'icum 
and Spicaria anomala. 

Ceratostomella ulvii, which attacks elms, is always associated 
with galleries produced by Scolytus scolytus and 5. vmltistriatus. 

Seyeral other species of fungi are associated with wood staining, 
but their relationship to insects remains unknown. These species 
include Endocon'uiiophora coemlescens, E. moniliformis, Diplodia 
natalensis, Graphiinn rigidum, Lasiosphaeria peziztda, Pemcill'unn 
divaricatum, P. roseum, P. aureum, Chlorospleminn aeriig'nwsinih 
Fusarium moniliforme, F. viride, F. roseum, Demathnu pulli/lans, 
and seyeral species of Cadophora, Hormodendrum, and Alternaria. 
It seems probable that all of them are not cultivated by wood- 
boringr beetles. 

Ants and fungi. Approximately a hundred species of tropical 
and subtropical myrmicine ants have the remarkable habit of culti- 
vating and feeding upon fungi. These ants live in large colonies 
in underground nests. They cut out bits of leaves and carry them 
into these nests. The plant tissues are then built into spongy 
masses that serve as a culture medium upon which the ants im- 
plant spores and mycelium. Such inoculum is transported within 
the infrabuccal pouch, especially by the queens. The fungi grow 
in these "ant gardens" and produce bromatia, swollen, roundish 
hvphal tips, which are consumed by the ants. New crops of bro- 
matia continue to replace those that have been eaten. 

Divergent opinions have been expressed on the identity of the 
fungi involved. Moller (1893) found in the nests of Acromyrex 
disciger a gill fungus that he named Rozites gonglyophora (the 
termination "ites" should be reserved for fossil fungi). Xylaria 
mi crura was identified in the nest of Acromyrex lundi by Spegaz- 
zini. Cladosporimn myrmecophilwn is cultivated by Lasius fiilig- 



FUNGI CULTIVATED BY INSECTS 



453 



inosus, and Hormisciinn pithy ophilum by Lashis umbratus. The 
fungus cultivated by Atta cephalotes was identified by Weber 
(1938) as Lentinus atticohis. Spores of several unidentified species 
from infrabuccal pouches are represented in the illustrations that 
accompany the report by Bailey (1920). 




Fig. 75. Fungi used as food by insects. A. Globular hyphal tips (bromatia) 
of a fungus cultivated by ants. (Adapted from Wheeler.) B. Aloniliod 
fungus in artificial culture. The ambrosia beetle, Trypodendron betnlae, 
uses this fungus. (Adapted from Leach, Hodson, Christiansen, and Chilton.) 

Some workers have maintained that the associated fungus oc- 
curs in "pure culture," a claim which is denied by Goetsch and 
Stoppel [Uphof (1942), p. 584]. These investigators isolated the 
following fungi from the nests of Atta sexdens: Hypomyces ipo- 
moeae, Fusarium oxysporinn, F. angustum, F. equiseti, Verticil- 
lium candidum, and Clonostachys araucariae; from nests of Acro- 
myrex they isolated Mucor racemosus, Actinomiicor re pens, 
Moniliopsis aderholdii, Rhizopns nigricans, Trichoderma sp., and 
Penicillium sp. 



454 FUNGUS-INSECT INTERRELATIONSHIPS 

Interest in such problems involving ants as growers of fungi 
should be stimulated bv perusal of the reports of Aloller (1893), 
Wheeler (1907), Elliott (1915), Bailey (1920), Spegazzini (1922), 
and Weber (1938). 

Termites and fungi. One group of the termites cultivates 
fungi in "gardens" for use as food. Such termites are colonial and 
live either in lar^e nests (termitaria) built underground or in 
mounds above ground. Within these nests are compartments in 
which the fungi are cultivated on termite excrement. The fungi 
grown are eaten by the young and constitute essentially the only 
food used. In spite of the fact that naturalists found fungi in ter- 
mite nests nearly 200 vears ago, little is yet known regarding the 
identitv of such fungi, as is indicated bv the accounts of Holter- 
man (1898),Petch (i906, 1913), Brown (1918), Bose (1923), and 
Uphof (1942). Uphof states that Berkeley in 1869 described a 
fungus taken from white-ant nests as Agaricas terviitigina, prob- 
ably identical with Lentimts cartilagineus. He further states that 
Cesati in 1870 regarded Tricholoma snbgavibosnm, which occurs 
in Cevlon, Java, Singapore, and Borneo as a termite fungus. 
Fungi identified as Pint ens tennitus and Xylaria m gripes have been 
taken from termite nests in Brazil. In India Bose (1923) found 
that termites cultivate Colly bia albnminosa but "weed out" the 
stromata of Xylaria nigripes. Petch (1906) made the observation 
that Agaricus sp. does not grow in the soil surrounding the nest 
but onlv on the "comb" in compartments while the nests are in- 
habited. After the nests have been abandoned, Peziza epispadia, 
Podaxon sp., and other fungi develop on the comb. 

In addition to the cases of ectosvmbiosis involving fungi and 
insects which have been enumerated, attention may be directed 
to the existence of endosvmbiosis. The best known of these endo- 
biotic relations involve termites that digest wood but are able to 
do so onlv through the agency of svmbiotic protozoa that live 
within their intestines. The claim has been made by Koch ( 193 1 ) 
that an unnamed fundus, which is endobiotic, lives in the fat 
bodies of the saw-tooth grain weevil, Oryzaephilus snrinamensis, 
and in some manner contributes to fat metabolism. This relation- 
ship is maintained from generation to generation by invasion of 
the eggs. 



IMPLICATIONS 455 

IMPLICATIONS 

It appears that problems of interrelationship of fungi and insects 
are basically ecological, and more emphasis should be placed on 
approaching them from this viewpoint. To this end closer co- 
operation between mycologists and entomologists is required. 
The usefulness of these scientists' findings should be enhanced if 
natural rather than artificial environments can be employed for 
their experimentation. The resistance of seed plants to attack bv 
the noxious insect should in any event be regarded as an essential 
phase of such ecologic problems. 

LITERATURE CITED 

Bailey, I., "Some relations between ants and fungi," Ecology, 1: 174-189, 

1920. 
Berger, E. W., "White fly studies in 1908," Fla. Agr. Expt. Sta. Bull. 91: 43- 

71, 1909. 
"White fly control," Fla. Agr. Expt. Sta. Bull., 103: 1-28, 1910. 
"Natural enemies of scale insects and white flies in Florida," Fla. Sta. 

Plant Bd. Quart. Bull., 5: 141-154, 1921. 
"The latest concerning natural enemies of citrus insects," Fla. State Hort. 

Soc. Proc, 45: 131-136, 1932. 
Bose, S. R., "The fungi cultivated by the termites of Barkuda," Rec Indian 

Mus., 25: 253-258, 1923. 
Brodie, H. J., "The oidia of Coprinus lagopus and their relation with insects," 

Ann. Botany, 45:315-344, 1931. 
Brown, W. H., "The fungi cultivated by termites in the vicinity of Manila 

and Los Banos," Philip. J. Sci., Ser. C. (Bot.), 75:223-231, 1918. 
Buchner, P., Tier und Pflanze in Symbiose. Gebruder Borntrager, Berlin. 

1930. 
Charles, Vera K., "A preliminary check list of the entomogenous fungi of 

North America," U. S. Dept. Agr., Bur. Plant Indus., Insect Pest Survey 

Bull, 21: 770-785, 1941. 
Couch, J. N., The genus Septobasidium. 480 pp. Chapel Hill, N. C. 1938. 
"Revision of the genus Coelomvces, parasitic in insect larvae," /. Elisha 

Mitchell Sci. Soc, 61: 124-136, 1945. 
Craigie, J. H., "An experimental investigation of sex in the rust fungi," 

Phytopathology, 21: 1001-1040, 1931. 
Elliott, J. S., "Fungi in the nests of ants," Trans. Brit. My col. Soc, 5: 138 

142, 1915. 
Fawcett, H. S., "Fungi parasitic on the Citrus white fly," Fla. Agr. Expt. 

Sta. Kept., 1901: 47-49, 1907. 



456 FUNGUS-INSECT INTERRELATIONSHIPS 

Fawcett, H. S., "Fungi parasitic upon Aleyrodes citri," Univ. Fla. Spec. 

Studies, 1: 1-41, 1908. 
"Fungus and bacterial diseases of insects as factors in biological control," 

Botan. Rev., 10: 327-348, 1944. 
Holterman, C, Mykologische Untersuchungen aus den Tropen. 107 pp. 

1898. 
Johnston, J. R., "The entomogcnous fungi of Porto Rico," Porto Rico 

Bd. Covnns. Agr. Bull., 10: 1-33, 1910. 
Keilin, D., "On a new type of fungus, Coeloviyces stegoviyiae, n.g., n.sp., 

parasitic in the body cavity of the larva of Stegomyia scutellaris 

Walker," Parasitology, 13: 225-234, 1921. 
Koch, A., "Die Svmbiose von Oryzaephilus surincnnensis L. (Cucujidae, 

Coleoptera)," Z. Morphol. Okol. Tiere, 23: 389-424, 1931. 
Leach, J. G., Insect transmission of plant diseases, ix + 615 pp. McGraw- 
Hill Co., New York. 1940. 
Lefebvre, C. L., "Preliminary observations on two species of Beauveria 

attacking the corn borer, Pyrausta nubilalis Hiibner," Phytopathology, 

21: 1115-1128, 1931. 
Luttrell, E. S., "The morphologv of Sphaerostilbe aurantiicola (B. and 

Br.) Petch," Bull. Torrey Botan. Club, 27:599-619, 1944. 
Moller, A., "Die Pilzgarten einiger Siidamerkanischen Ameisen," Schimper's 

Bot. Mitt, aus Trope?!., 6: 1-127, 1893. 
Morrill, A. W., and E. A. Back, "Natural control of white flies in Florida," 

U. S. Dept. Agr., Bur. Entom. Bull, 102: 1-73, 1912. 
Petch, T., "The fungi of certain termite nests," Ann. Roy. Botan. Garden, 

Peradeniya, 3: 185-270, 1906. 
"Termite fungi, a resume," Ann. Roy. Botan. Garden, Peradeniya, 5: 303- 

341, 1913. 
"The genera Hvpocrella and Aschersonia," Ann. Roy. Botan. Garden, 

Peradeniya, 5:52 1-537, 1914. 
"Fungi parasitic on scale insects," Trans. Brit. My col. Soc, 7: 18-40, 1921. 
"Studies in entomogenous fungi. I. The Nectriae parasitic on scale in- 
sects," Trans. Brit. Mycol. Soc, 7:89-167, 1921a. 
II. "The genera Hvpocrella and Aschersonia," Ann. Roy. Botan. Garden, 

Peradeniya, 7:167-278, 1921b. 

IV. "Some Cevlon Cordyceps," Trans. Brit. Mycol. Soc, 10: 28-45, 1924. 

V. "Mvriangium," Trans. Brit. Mycol. Soc, 70:45-80, 1924a. 

VI. "Cephalosporium and associated fungi," Trans. Brit. Mycol. Soc, 
10: 152-182, 1925. 

VII. "Spicaria," Trans. Brit. Mycol. Soc, 10: 183-189, 1925a. 

VIII. "Notes on Beauveria," Trans. Brit. Mycol. Soc, 70:244-271, 1926. 

IX. "Aegerita," Trans. Brit. Mycol. Soc, 11: 50-66, 1926a. 

XI. "Empusa lecanii Zimm," Trans. Brit. Mycol. Soc, 77:254-258, 1926b. 

"Notes on entomogenous fungi," Trans. Brit. Mycol. Soc, 16:55-75, 1931; 
76:209-245, 1932; 7£: 48-75, 1933; 7 9: 34-38, 1934; 20:161-194, 1935; 
23: 127-148, 1939. 
Picard, F., "Les champignons parasites des insects et leur utilization agri- 
cole," Ann. ecole nat. agr. Montpellier, 13: 121-248, 1914. 



LITERATURE CITED 451 

Rumbold, Caroline T., "Three blue-staining fungi, including two new spe- 
cies associated with bark beetles," /. Agr. Research, 53: 419^-37, 1936. 
Sawyer, W. H., "Observations on some entomogenous members of the 

Entomophthoraceae in artificial culture," Am. J. Botany, 16: 87-121, 

1929. 
Seymour, A. B., Host index of the fungi of North America, xiii + 718 pp. 

Cambridge, Alass. 1929. 
Smith, H. S., and H. M. Armitage, "The biological control of mealy bugs 

attacking citrus," Calif. Agr. Expt. Sta. Bull., 509: 1-74, 1931. 
Speare, A. T., "Fungi parasitic upon insects injurious to sugar cane," 

Hawaiian Sugar Planters Assoc, Expt. Sta. Bull., 12: 1-62, 1912. 
"Further studies of Sorosporella uvella, a fungous parasite of noctuid 

larvae," /. Agr. Research, 18: 399-440, 1920. 
u Massospora cicadina Peck, a fungus parasite of the periodical cicada," 

My col, 13:72-82, 1921. 
Spegazzini, C, "Description de Hongos Alirmecofilos," Rev. museo de la 

Plata, 26: 166-173, 1922. 
Sweetman, H. L., The biological control of insects. 461 pp. Comstock 

Publishing Co., Ithaca, N. Y. 1936. 
Thaxter, R., "The Entomophthoraceae of the United States," Mem. Boston 

Soc. Nat. Hist., 4: 133-201, 1888. 
"Contributions toward a monograph of the Laboulbeniaceae. I," Mem. 

Am. Acad. Arts Sci., 12: 189-429, 1896; II, 75:219-469, 1908; III, 74:313- 

414, 1924; IV, 75:431-580, 1926. 
Uphof, J. C. Th., "Ecological relations of plants with ants and termites," 

Botan. Rev., 8: 563-598, 1942. 
Watson, J. R., and E. W. Berger, "Citrus insects and their control," Univ. 

Fla. Agr. Ext. Bull., 88: 1-135, 1937. 
Weber, N. A., "The biology of the fungus-growing ants. III. The sporo- 

phore of the fungus grown by Atta cephalotes and a review of other 

reported sporophores," Rev. Entomologia, 8: 265-272, 1938. 
Wheeler, W. M., "The fungus-growing ants of North America," Bull. Am. 

Mus. Nat. Hist., 23: 669-807, 1907. 
Wright, Ernest, "Trichosporium symbioticum, n.sp.: a wood-staining fungus 

associated with Scolytus ventralis^ J. Agr. Research, 50:525-538, 1935. 
"Further investigations of brown-staining fungi associated with engraver 

beetles (Scolytus) in white fir," /. Agr. Research, 51: 759-774, 1938. 



Chapter 21 
MARINE FUNGI 

Among students of fungi and of marine biology generally, a 
knowledge of marine fungi is largely non-existent. The under- 
lying reasons for this strange state of affairs are not apparent in 
view of the enormous volume of work dealing with marine life 
that has been accomplished. Biologists quite generally concede 
that the ocean is the ancestral home of life and that the progenitors 
of present-day land animals and plants came from the ocean. 
With similar reasoning fungi may be assumed to have originated 
within the ocean. It might be anticipated, moreover, that marine 
fungi would constitute favorable materials for studies on phylog- 
eny and on the place which such organisms occupy in the 
economy of life in oceans. 

Terrestrial fungi and bacteria are well known to be responsible 
for the decomposition of organic debris of all sorts, and it may 
reasonably be assumed therefore that organisms of these types 
play a similar role in the ocean. This assumption is not supported, 
however, by any body of observational and experimental data of 
appreciable magnitude. Similarly, relatively little appears to be 
known about the activities and life histories of any species of 
marine fungi and bacteria, although marine bacteria have been 
studied somewhat intensively and extensively. 

Students of the phytogeny of the fungi regard the Archimy- 
cetes as the ancestral and the most primitive group. Among 
Archimvcetes the asexual spores and both kinds of gametes or 
those of one sex only may be motile, whereas among present-day, 
higher, terrestrial Phvcomvcetes and anions all Ascomvcetes and 
all Basidiomvcetes motility is lacking. This fact might be inter- 
preted to indicate that all these present-day, non-motile forms 
were derived in a monophyletic line from terrestrial progenitors 
after the land habit had once become established. It is conceiv- 
able too that the higher marine fungi of the present day may 

458 



HISTORICAL BACKGROUND 459 

have evolved on land and thereafter migrated from the land to 
the ocean. On the other hand, those who would derive the 
Ascomvcetes from Florideae regard the fungi as polvphyletic. 
They emphasize as a basis of relationship similarities between sex- 
ual reproductive structures rather than the phenomenon of mo- 
tility. Regardless of whether fungi are mono- or polvphyletic, 
there do not seem to be adequate explanations to account for the 
paucity of Phycomycetes and Ascomycetes within the oceans. 
There should be an abundant population of marine fungi, primar- 
ily because the ocean constitutes a relatively stable environment 
which should be favorable for the continuous maintenance cf spe- 
cies, without major adaptative modification, even of those whose 
origin dates back into remote ^eolomc time. This environmental 
stability may in itself be used to account for the lack of evolutional 
development of new or different species. If numerous kinds of 
marine fungi exist, the fact is not revealed by publications. In- 
stead the literature on marine fungi conveys the definite impres- 
sion that the oceans do not constitute the natural habitat of di- 
verse fungi, nor are they at any place densely populated by any 
given species. 

HISTORICAL BACKGROUND 

Evidently many of the early students of marine animals and 
plants failed to recognize the presence of fungi among their col- 
lections or else interpreted the fungi as structures possessed by the 
animals or plants themselves. Nevertheless occasional observers 
noted hyaline objects which were interpreted to be fungoid. In 
1858 Wedl [Bornet and Flahault (1889)] observed that corals 
from the littoral zone down to a depth of 1095 fathoms are fre- 
quently invaded by filaments that lack septations and are termi- 
nated by clavate cells resembling sporangia of the Saprolegniaceae. 
Kolliker (1859-1860) made similar observations in his examination 
of animals possessing calcareous shells. Stirrup (1872) observed 
fungoid growths within the shells of molluscs, and Duncan (1876- 
1877) identified as Achlya penetrans and Saprolegnia ferax two 
water molds within the canals of Caryophyllia smithii, one of the 
Aladreporia. Since these two species have not been found subse- 
quently in salt water, their identification must be questioned. The 
solvent action of carbon dioxide produced by the hyphal tips 
made possible the penetration of the shells. Bornet and Flahault 



460 MARINE FUNGI 

(1889) identified the fungi which they found in molluscan shells 
as Ostraco blade implexa, presumably a saprolegniaceous form, and 
Lithopytbiinn gangliiforme, a pythiaceous species. 

Evidently non-filamentous Phycomycetes are more abundant 
among marine species than are Saprolegniaceae and Pythiaceae. 
The work of Petersen (1905) in Denmark and the more recent 
studies by Sparrow in Denmark and along the New England coast 
(1934, 1936) on marine Chytridiales should be considered in 
orienting one's knowledge of this group. 

Barghoorn and Linder (1944) and Linder (1944) gave special 
consideration to marine fungi on wood and cordage. Nearly all 
of the 10 imperfect species and 18 Pyrenomycetes which they 
isolated had not been described previously. Seven of the Pyreno- 
mycetes tolerated well the salinity of sea water and were able to 
utilize cellulose, pectin, and starch. 

For a period of years no one seems to have devoted himself to 
a study of marine Ascomycetes. The reports by Reed (1902) 
from collections on the California coast and of Cotton (1907) 
and Sutherland (1914, 1915, and 1915a) on the English coast are 
among those of most importance. 

Knowledge of the imperfect fungi of the sea is very meager, as 
is that of the Alyxomycetes, except for a few species in the aber- 
rant order Labyrinthulales. The best known of these is Laby- 
rinthula viacroystis, associated with the "wasting disease" of eel 
grass, Zostera spp. 

In the account that follows each of these four major groups of 
fungi will be considered to a degree consonant with available 
knowledge and with its importance. 

MARINE PHYCOMYCETES 

One of the first chytrids to be studied is Ewychasma dicksonii, 
parasitic upon Ectocarpus. Wright (1879) named this parasite 
Rhizophydhmi dicksonii, a name which was subsequently changed 
to Olpidinm dicksonii by the algologist Wille and then to Enry- 
chasvia dicksonii by Mansmus. Information on its structure and 
parasitism appears in the accounts by Lowenthal (1905) and 
Dangeard (1934). Lowenthal (1905) states that at maturity the 
thallus contains a large vacuole with a peripheral segmentation 
of this layer. Petersen (1905) traced zoosporogenesis also and 



MARINE PHYCOMYCETES 461 

found that numerous vacuoles, separated bv thin layers of cyto- 
plasm, function in zoospore formation. Once formed, they are 
active for a brief period and then encyst within the sporangium, 
giving it a reticulate appearance. Encystment within the spo- 
rangium seems, however, to be abnormal. ScherfTel (1925) be- 
lieves that these methods of zoosporogenesis in Eurychasma are 
not those of true chytrids but of Saprolegniaceae, and he would 
therefore place it in this group. 

Reports of chytrids from the Pacific coast briefly describe 
Chy iridium alar him [Kibbe (1916)] on Alarm fistnlosa and C. 
codicola and Rhizophy diitm codicola on C odium miicronatum 
[Zeller (1918)]. In a brief note Martin (1922) calls attention to 
the fact that Polysiphonia sp. along the New Jersey coast is para- 
sitized bv Chytridhim (Rhizophid'mm) polysiphoniae, and Spar- 
row (1936) records the occurrence of this same chytrid on Poly- 
siphonia fibrillosa and Ceraminm rubrum in the vicinity of Woods 
Hole, Massachusetts. This pathogen is in turn parasitized by the 
chytrid Pleolpidhnn (Rozella) marinum [Sparrow (1936)]. All 
other known species of this genus occur in fresh water. 

Among the 15 species of chytrids collected by Sparrow (1936) 
in the waters near Woods Hole, 2 are especially noteworthy. 
One, Peter senia (Olpidiopsis) andreei, occurs on Ectocarpus sili- 
cidosiiSj upon which it may be pathogenic. Its zoospores are later- 
ally biciliate, as was first pointed out by Petersen (1905) and con- 
firmed by Sparrow (1936). The other species, Thransto chytri- 
dhim proltferum, occurring saprophytically upon Ceramium dia- 
phamim and Bryopsis plumosa, is described as a new generic type. 
Its sporangia are Thraustotheca-like in their discharge of zoo- 
spores and sporangial proliferation. At the time of discharge the 
zoospores lack cilia, but each may later come to have a single 
anterior flagellum. 

In Karling's (1943) account mention is made of an organism 
collected near Beaufort, North Carolina, which is parasitic on 
Ectocarpus mitchellae and E. siliculosns and is Olpidium-like in 
structure and development but possesses anteriorly uniflagellate 
zoospores. Karling described it as Amsolpidium ectocarpii and 
placed it in the family Anisolpidiaceae, which was to include 2 
other genera and 5 other species, each having zoospores with a 
single flagellum that rises anteriorly. Furthermore he believed 
that the members of this family and those of the Rhizidiomyce- 



462 



MARINE FUNGI 



taceae and Hyphochytriaceae should together be placed in the 
new order Anisochvtridiales. 

Among- the collections by Petersen (1905) and Sparrow (1934) 
from the coast of Denmark are listed 22 species of chytrids. Their 
studies indicate that chytrids are the most abundant members of 



the marine fungus flora. 



h <3 



'• • v'"< r 
i - ■ V ^W 




Fig. 76. Various marine chytrids. (After Sparrow.) A-D. Developmental 
stages of Thraustochytridhim proliferum on Bryopsis plumosa. E. Chytrid- 
ium polysiphoneae in thallus of Polysiphonia. F. Pleolpidium (Rozella) 
marimtm, discharging its spores within the thallus of Chytridium polysi- 
pkonieae. G. Sporangium of Petersenia andreei in Ectocarpus. H. P. 
andreei, mature resting spore with empty male cyst attached. 

Perhaps the most singular member of this group that has been 
described is Ichthyophomis hoferi, first mentioned in 1904 as a 
parasite of certain fishes by Hofer and later studied by Plehn and 
Alulsow (191 1) and Daniel (1933). This species causes enormous 
losses to marine fish, particularly herring and trout. Plehn and 
Mulsow (1911) described and named the organism, placing it 
anions the Chytridiales. Daniel (1933) made a rather detailed 
study of the pathogen as it occurs in the sea herring, Clupea 
harengus. The spores are non-motile and usually multinucleate 



MARINE PHYCOMYCETES 



463 



and escape from the apex of a thick exit-tube-like hypha. During 
transformation of the spore into a globular thallus nuclear division 
is accompanied by an increase in the volume of the thallus. 

An earlier account by Neresheimer and Clodi (1914) deals com- 
prehensively with the morphology, life history, and pathogenicity 
of Ichthy ophonus hoferi. The later study by Fish (1934) em- 




Fig. 77. Schematic life cycle of Icthyophomts hoferi, which parasitizes 

fishes. (Adapted from Daniel.) 

ploys the name Ichthy osporidhnn hoferi for the pathogen, which 
Fish encountered in sea herring, ale wife (Pomobohis pseudo- 
harengus), and flounder (Pseudoplenronectes americanus) 
throughout the Gulf of jMaine. He concluded that fishes become 
infected by way of the alimentary canal. Association of these 
species and cannibalistic food habits, especially of flounder, which 
eats herring, account for acquisition of the pathogen. 

Several saprolegniaceous parasites of marine animals have been 
observed. Apstein (1910) noted that Synchaeta monopus, a roti- 
fer occurring in brackish waters along the Baltic Sea, may be in- 
vaded by mycelia of an organism that he named Synchaetophagns 
balticus. The hvphae may more or less completely occupy the 
body cavity, destroying the organs and leaving only the outer 



464 MARINE FUNGI 

body membrane. Eventually an isolated branch or the entire 
mycelium is transformed into sporangia that liberate motile zoo- 
spores 5 to 8 /x in diameter. Apstein also observed structures 
which he doubtfully referred to as oogonia. 

In England Atkins (1929) found that pea crabs (Pinnotheres) 
are killed by one of the Saprolegniaceae. Infection is indicated 
by whitish patches that show through the body wall in the region 
of the gills and along the junctions of the abdominal segments. 
Intricately branched hyphae occupy the tissues of the gills and 
those between the gill chamber and the dorsal surface of the 
carapace. Hyphae do not appear at the exterior. The sporangia, 
which are confined to the gills and pleopods, form at hyphal tips 
and are cut off by septa. They are of the same diameter as the 
assimilatory hyphae. The zoospores are pyriform and bicilate. 
After a brief period of motility they encyst and may undergo a 
second motile phase. Atkins' evidence for diplanetism, however, 
is not conclusive. Its identity among Saprolegniaceae is not estab- 
lished beyond the fact that it differs from all other members of 
this family, mainly in its occurrence wholly within the body of 
the animal. 

Two other parasites of marine animals, described by Niezabitow- 
ski (1913), are of interest, Thalassoviyces spizakovii and T. batei. 
He placed them in the new family, Thalassomycetineae, among 
the Oomycetes. ThalassoiJiyces spizakovii occurs on the deep-sea 
decapod, Pasiphaea sivado, in the Mediterranean, and T. batei on 
P. cristata on the coast of the Fiji Islands. Evidence of infection 
is the presence of clusters of colorless hyphae on the underside 
of the crustacean's body. Apparently the assimilatory mycelium 
lives wholly within the interior of the body, and the reproductive 
hyphae constitute the external hyphal tufts. The non-septate, 
external hyphae consist of a stalk cell that divides dichotomously 
one or more times, and each tip eventually becomes segmented to 
form a row of three cells, which are liberated as conidia. Nieza- 
bitowski (1913) places Thalassomyces near the Saprolegniaceae 
and Monoblepharidaceae. 

In the vicinity of Beaufort, North Carolina, mud crabs, Pano- 
pens herbstii, and mole crabs, Emerita talpoida, are commonly 
parasitized by a species of Enterobryus, apparently unnamed. 
The organism consists of thick-walled, cvlindrical filaments of uni- 
form diameter that are about 2 to 3 mm long and 15 to 20 fx wide. 



MARINE PHYCOMYCETES 



465 



These filaments are straight or coiled and are non-septate. They 
are attached by disk-shaped holdfasts to the intestinal wall and 
project as a tuft of white hairs from the anal opening. At matur- 
ity a series of three or four cylindrical cells of the same diameter 
as the hypha is formed. These cells appear to be spores. When 




Fig. 78. An eccrinid, presumably a species of Enterobryus from mud crab, 
Pajiopeus herbstii. A. Apex of hypha, showing endogenously formed spore. 
B. Basal portion of hypha with disk, by means of which the eccrinid is 
attached to intestinal wall. C. Entire plant, unbranched and unsegmented. 

the apex of the hypha ruptures, the spores are freed seriatim by 
growth and pressure from below. All efforts to cultivate this 
organism on artificial media have failed. It is strictly parasitic, as 
are related species. 

Little is known about the taxonomy and systematic position of 
Enterobryus, and it is not included in Saccardo's Sylloge Fungo- 
rum, even though a considerable number of species have been 
described. The Genus Enterobryus was founded by Leidy (1849, 
1853) from observations of several entophytous species. 



466 MARINE FUNGI 

In 1895 Hauptfleisch (1895) described as a new genus and spe- 
cies Astreptonema longispora, occurring in the intestine of Gam- 
marus locust a. He regarded it as among the Saprolegniaceae, but 
Saccardo {Sylloge Fungomm, 14: 446) placed it among the chy- 
trids. In 1920 Thaxter (1920) found a closely related organism 
growing exposed on the anal plates of beetle, Passalus sp., and 
properly assigned it the name Enter obryus compressus. He was 
of the opinion that the organism described by Hauptfleisch is an 
Enterobryus and that it belongs among the Phycomycetes, near 
the Saprolegniaceae. 

The ordinal name Eccrinales has been employed to include 
Enterobryus and several related genera, all of which have the 
same growth habit and form endogenous non-motile spores. None 
of them is genuinely aquatic, although some species, such as 
Eccrinopsis hydropilontni, parasitize aquatic beetles. No accord 
has been reached on the relationship of the Eccriniales to other 
fungi, but presumably they are related to the Saprolegniales. The 
reports by Leger and Duboscq (1916) and Poisson (1929) will 
introduce the reader to the status of this strange order of fungi. 

Only one species of Pvthium having a marine habitat has been 
recorded. Sparrow (1934) obtained it from Ceramium rubruvi 
and described it as Pythium mar mum. 

MARINE ASCO.MYCETES * 

Representatives of the Sphaeriales, Dothidiales, and Hysteriales 
have been found on marine plants and animals. Most of the known 
species occur on marine algae. Among the 35 species of marine 
seed plants, included in 8 genera, all monocotyledonous, only 
Zostera and Posidonia are known to serve as hosts for ascomyce- 
tous fungi. Ophiobolus halimus on Zostera marina is associated 
with the'so-called "wasting disease," which has wrought so much 
havoc with this valuable marine species. Amphisphaeria posi- 
doniae has long been known on Posidonia Oceania. 

Of most interest, perhaps, are those species that are thought 
to be symbiotic. Reed (1902) found that the Guignar