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Marine Biological Laboratory Library

Woods Hole, Mas3achusetl5

Physiology of the Fungi

McGRAW-HILL PUBLICATIONS IN
THE BOTANICAL SCIENCES

Edmund W. Sinnott, Consulting Editor

ARNOLD An Introduction to Paleobotany

CURTIS AND CLARK An Introduction to Plant Physiology

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KRAMER AND KOZLOWSKi Physiology of Trecs

LILLY AND BARNETT PhvsiologV of the Fungi

MAHESHWARi An Introduction to the Embryology of the Angiosperms

MILLER Plant Physiology

POOL Flowers and Flowering Plants

SHARP Fundamentals of Cytology

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Vol. I. Algae and Fungi

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SMITH The Fresh-water Algae of the United States
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There are also the related series of McGraw-Hill Publications in the Zoological
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Frontispiece. Pilobolus showing phototropism. See page 339.

L -VI

Physiology
of the Fungi

VIRGIL GREENE LILLY

Professor of Physiology, Department of Plant Pathology

and Bacteriology, West Virginia University;

Physiologist, West Virginia Agricultural

Experiment Station

HORACE L. BARNETT

Professor of Mycology, Department of Plant Pathology

and Bacteriology, West Virginia University;

Mycologist, West Virginia Agricultural

Experiment Station

1951

McGRAW-HILL BOOK COMPANY
New York Toronto London

PHYSIOLOGY OF THE FUNGr

Copyright, 1951, by the McGraw-Hill Book Company, Inc. Prmtedinthe
United States of America. All rights reserved. This book, or parts thereof,
may not be reproduced in any form without permission of the pubUshers.

7 89 10 11 12-MAMM-l 09 8
37865

This book is dedicated to the mem-
ory of Leon H. Leonian and to Ernst A.
Bessey. The guidance and inspiration
of these men in directing our interests
to the study of fungi is gratefully
acknowledged.

PREFACE

Living fungi are being studied more intensively than ever before.
This may be attributed in part to increased interest in the potentiahties
of the fungi in industry as well as to the greater recognition of fungi as
important disease-producing agents of plants and animals and as destroy-
ers of fabrics and other cellulosic materials of commercial importance.
This has increased the interest in the cultivation of the fungi and has
shown the need for an adequate textbook covering the broad aspects of
physiology of the fungi, their growth requirements, and activities. It
was the intent of the authors to prepare a textbook which would fulfill
the needs of students desirous of some training in this field.

This book is primarily a text for the advanced student and assumes
some basic knowledge of the morphology of fungi and of organic chemistry.
It had its origin in the lectures and laboratory exercises used for three
years by the authors in a course in physiology of the fungi offered to
graduate students at West Virginia University. The authors have
contributed equally of their time and efforts in the preparation of this
text.

For those who are interested or are actively engaged in physiological
research on fungi, this textbook may serve as a reference book and as an
entry into the literature. The large ever-growing accumulation of liter-
ature has also made it desirable to bring together a summary and dis-
cussion of the information in this field. However, no attempt has been
made at complete documentation of the subjects discussed. Certain
particularly important references are marked with a star and are recom-
mended as required reading for students.

For the most part, the scientific names of the fungi are those which
were used by the investigators whose work has been cited. No attempt
has been made to reduce these names to synonomy. Because of the
close relation between fungus physiology and plant pathology, plant
pathogenic fungi have been used as examples whenever possible.

Several suggested laboratory exercises with suggested test fungi are
included at the end of the text, so that other teachers might profit by the
authors' experience in designing and conducting laboratory work in
fungus physiology.

All tables, graphs, and photographs not credited to other sources are
original.

It is a pleasure to acknowledge our indebtedness to the many individuals

X PREFACE

who have aided us with their suggestions. Among our colleagues at
West Virginia University who have read portions of the manuscript are:
J. G. Leach, C. R. Orton, M. E. Gallegly, R. P. True, W. T. Jackson,
B. G. Anderson, R. B. Dustman, and J. H. Hare. In addition, the
following have read one or more chapters: B. W. Henry, J. B. Routien,
W. D. Gray, and J. B. Conn. We are also indebted to Mrs. H. L.
Barnett, who typed most of the manuscript.

We wish to thank the many individuals, societies, and publishers who
have granted us permission to use data and illustrations. Particular
acknowledgment is made in connection with the material cited.

Virgil Greene Lilly
Horace L. Barnett

MORGANTOWN, W. Va,

May, 1951

CONTENTS

Preface ix

1. Introduction 1

Fungus Physiology in Relation to Other Sciences — Aims — Scope — Historical
Development.

2. Culture Media 8

ICinds of Media — Natural Versus Synthetic Media — Choice and Preparation

of Media — Ways of Expressing Concentration — Comparison of Media — -
Summary.

3. Growth 24

Phases of Growth — Rate of Growth — Ways of Measuring Growth — Methods

of Presenting Results — Factors Affecting Growth — Effect of External
Factors on Morphology — Summary.

4. Enzymes and Enzyme Action 45

Classification of Enzymes — Chemical Nature of Enzymes — Factors Affecting
Enzyme Activity — Mechanism of Enzyme Action — Adaptive Enzymes-
Energy and Energy Utilization by Fungi — Summary.

5. Essential Metallic Elements 65

Biological Essential Elements — The Essential Macro Elements — Essential
Micro Elements — Periodicity of Biologically Essential Elements — Summary.

6. The Essential Xonmetallic Elements Other Than Carbon 87

Hydrogen — Oxygen — Sulfur — Phosphorus — Nitrogen — Other Nonmetallic
Elements — Summary.

7. Carbon Sources and Carbon Utilization 116

Monosaccharides and Related Compounds — Organic Acids — Glycosides —
Oligosaccharides — Polysaccharides — Heterotrophic Utilization of Carbon
Dioxide — Utilization of Carbon — Summary.

8. Hydrogen-ion Concentration 149

Ionization of Compounds — The Meaning of pH — Buffers and Buffer Capacity

— Methods of Determining pH Values — Effects on Fungi — Summary.

9. Vitamins and Growth Factors 171

Part I. General Considerations — Synthesis of Vitamins by Fungi — Vitamin

xii CONTENTS

Deficiencies in Fungi — Inhibitory Effects of Vitamins — Vitamers — Unidenti-
fied Growth Factors, Part II. Specific Vitamins — Thiamine and Its
Moieties — Biotin — Inositol — Nicotinic Acid — Pantothenic Acid — Pyridoxine —
p-Aminobenzoic Acid — Riboflavin — Summary.

10. Fungi AS Test Organisms 208

General Procedures — Vitamin Assays — Amino-acid Assays — Assays for
Essential Elements — Sugars — Tests for Certain Metabolic Products — Testing
Fabric Protectants — Summary.

11. Metabolic Antagonists 226

Antivitamins — Amino-acid Antagonists — Development of Fastness — Sum-
mary.

12. The Action of Fungicides 245

Copper — Mercury — Sulfur — Organic Fungicides — Evaluating Fungicides —
Summary.

13. Metabolic Products 266

Decomposition of Organic Materials — Fungi as Food — Cultivation of Fungi
for Food — Fat Production — Production of Vitamins — Enzyme Production —
Alcoholic Fermentation — Organic Acids — Esters — Antibiotics and Drugs —
Toxins — Pigments — Summary.

14. Factors Influencing Sporulation op Fungi 304

Environmental Factors — Other Physical Factors — Nutritional Factors —
Other Factors — Summary.

15. Spore Discharge and Dissemination 338

Methods of Spore Discharge — Influence of External Conditions — Spore
Dissemination — Summary.

16. Spore Germination 355

Physical Factors — Nutrients and Stimulants — Longevity of Spores — Sum-
mary.

17. The Physiology of Parasitism and Resistance 372

Penetration — Parasitism — Resistance — Summ ary .

18. Physiological Variation and Inheritance of Physiological Char-
acters 400

Physiological Variation — Inheritance of Physiological Characters — Summary.

Suggested Laboratory Exercises 419

Index 441

CHAPTER 1
INTRODUCTION

The primary role of the fungi in nature has been fittingly described in
the prophetic statement of B. O. Dodge (1939) :

. . . the fungi are not degenerate organisms which are on their way out in a
scheme of evolution, and so of little economic importance and scientific interest.
The fungi, on the contrary, are progressive, ever changing and evolving rapidly
in their own way so that they are capable of becoming readily adapted to every
condition of life. We may rest assured that as green plants and animals disappear
one by one from the face of the globe, some of the fungi will always be present
to dispose of the last remains.

The most important role of the fungi in the economy of nature is to
act as scavengers in disposing of dead and fallen vegetation. In this way
the biologically essential elements are released for reuse, and the balance
of nature is maintained. However, these are not the only functions of the
fungi which are of interest and importance to man. Since the beginning
of agriculture fungi have been used to prepare bread and other foods,
as well as fermented beverages. Some fungi cause diseases of plants
and animals. Knowledge of their role as the causal agents of plant dis-
eases long antedated the recognition of bacterial diseases. While yeasts
have long been used to produce alcohol, the vast potentialities of other
species for the industrial production of organic acids and antibiotics have
been recognized more recently. An understanding of life processes of
the fungi is essential whether one wishes to control the fungi which cause
disease, to employ them in industry, or to use them in the laboratory to
unlock the secrets of nature.

The domain of physiology is the study of functions or life processes.
Fungus physiology is the study of living fungi, their functions and ac-
tivities, how they affect their environment and how the environment
affects them. Like other branches of science, fungus physiology has
four phases of development: (1) the discovery and verification of facts,
which are the foundation of any science, (2) the organization of these
facts into a systematic and coherent body of knowledge, (3) the dissemina-
tion of newly discovered facts, and (4) use of the newly discovered facts
and others already known to formulate principles. Facts are the basis of
science, but facts alone are sterile unless they are seen in relation to

2 PHYSIOLOGY OF THE FUNGI

previous knowledge. Organization and interpretation of facts are equally
as important as the experimentation which reveals them.

The fungi as a group are highly responsive to their environment and
are thus excellent test organisms for inquiring into the secrets of nature.
Nature always answers correctly the questions we ask, and, in this sense,
no experiment is a failure, although we may fail to ask the question we
intended, or we may misunderstand the answer given. Infinite care is
required to frame a question so that a definite answer may be obtained.
By observing fungi in nature we are limited to questions asked by nature.
Commonly, the environmental and nutritional factors are so complex that
the influence of a single variable cannot be evaluated. By controlling the
conditions under which a fungus is placed in the laboratory it is possible
to ask questions of great precision. Indeed, the number and scope of
the questions which we may ask fungi are limited only by the present-day
techniques and the curiosity of the investigator.

Since most of our knowledge of the physiology of the fungi has been
gained from laboratory investigations, the experimental approach will
be emphasized in the discussions which follow. However, this choice is
not meant to minimize the importance of and need for critical observa-
tions in nature. By emphasizing the results of careful laboratory re-
search, we are better able in the following chapters to present the facts
necessary for an understanding of the vital principles of fungus physi-
ology, and also to show that these principles, theories, and hypotheses
are founded upon experimental evidence.

FUNGUS PHYSIOLOGY IN RELATION TO OTHER SCIENCES

Physiology is that branch of science which deals with the life processes or
the activities of organisms. The activities of the whole organism or of
any of its parts may be hmited by its form or structure. Both the
activity and the form of an individual are determined to a great extent
by its genetic constitution and are modified by the environment to which
the organism is exposed. Physiology, therefore, is not an independent
subject. An understanding of physiological principles is based, in part,
upon facts and theories from many other fields of science, such as chem-
istry, physics, anatomy, cytology, bacteriology, and genetics.

Many of the physiological principles which have been established for
one group of organisms apply equally well to other groups. The vita-
mins essential to the normal growth of the fungi are the same as those
required by man, animals, and the higher plants. The general functions
of these vitamins appear to be the same in all organisms. The differ-
ence in the vitamin requirements seems to lie in the different abilities of
these groups of organisms (or individuals within the group) to synthesize
these necessary compounds. As Schopfer (1943) has pointed out, the

INTRODUCTION 3

vitamin problem is common to many branches of science. Many other
problems investigated in fungus physiology are likewise common to other
related fields of study.

In a similar way, a better understanding of certain related fields is
gained by knowledge of fungus physiology. The plant pathologist com-
monly finds it necessary to study the living parasitic fungus apart from
its host and must know something of the cultural methods and the spe-
cific nutritional requirements of the fungus at hand. The mycologist
and plant pathologist are faced with numerous unsolved problems which
must be investigated by physiological methods. One of the most chal-
lenging problems is the cultivation of certain fungi now classed as obligate
parasites on synthetic media of known composition. Until this is accom-
plished, the nutritional requirements of these fungi cannot be fully deter-
mined. Such knowledge would without doubt lead to a better under-
standing of parasitism and resistance.

The taxonomic mycologist uses morphological characters almost exclu-
sively in his identification and classification studies, while the bacteriol-
ogist, being unable to use distinct morphological features to any great
extent, emphasizes the physiological characters in classifying bacteria.
Much more information is needed before it can be determined whether
any physiological characters are sufficiently valuable and uniform to be
used to supplement morphological characters in taxonomy of fungi. It
seems logical that such physiological differences between groups of fungi
do exist, and that the main problem lies in the discovery and recognition
of these characters and their application to taxonomy. On the other
hand, caution must be observed, for nutritional and environmental con-
ditions are known to affect, to a certain extent, some morphological
characters used in classification.

The geneticist and the biochemist may find the fungi interesting and
suitable subjects for the study of their respective problems, while the
bacteriologist finds many points of similarity between the physiology of
the bacteria and that of the fungi. Industry has used many species of
fungi to its ovnx advantage for many decades. Yeasts were used long
before the physiology of the fungi became an organized study, but the
search for superior strains of yeast continues. The widespread use of
antibiotics has brought under laboratory study many species of fungi
which would otherwise have been ignored. This has created many new
problems of nutrition, especially with regard to large-scale cultivation of
these fungi.

Thus, knowledge of the life activities of the fungi is important and useful
in many related fields of science, just as some knowledge of these related
fields is essential to an understanding of the fimgi. The study of fungup
physiology is justified as a separate field in which the basic or fundamental

4 PHYSIOLOGY OF THE FUNGI

principles are the aim, or as a study closely integrated with the fields of
science concerned with more practical problems. Often, the most
\aluablc results are obtained when research is not restricted by the
boundaries of practical application.

AIMS

This book is a discussion of living fungi, of their life processes and the
factors which influence them. It is written primarily for the student who
is acquainted with the structure of fungi and who is beginning the study
of their activities. From the discussions which follow the student should
gain a knowledge and understanding of the basic principles of fungus
physiology. To this end a considerable amount of factual material con-
cerning the behavior of specific fungi under specific conditions is cited.
The secondary aim of this book is to present a limited number of selected
references which may be of use to the student or investigator who wishes
more detailed information. Where possible, review articles have been
included. Complete documentation is impossible because of the tre-
mendous volume of literature. However, becoming familiar with the
literature is an essential part of a student's education.

SCOPE

As a text this book must cover many phases of the subject. One of the
first problems to be considered is the choice and preparation of suitable
media for growth and sporulation of the fungi under study. Since there
is no universal medium suitable for all fungi, a wise choice of media for
the purpose at hand is of fundamental importance in any investigation.

Before a fungus can be studied in any great detail in the laboratory, it
is necessary to determine the conditions which affect growth. Growth is
a complex phenomenon, and some discussion of the phenomenon itself
and the ways of measuring growth is necessary for the understanding of
these conditions. Nutritional factors, such as source of nitrogen, source
of carbon, the presence of essential elements and vitamins, and the pH of
the substrate, afTect growth in interrelated ways. Each of these factors
and its importance in growth and other activities of fungi are discussed at
some length.

The life processes of the fungi involve numerous chemical transforma-
tions. Living organisms make and use special organic catalysts, enzymes,
which control these reactions. The actions of the enzymes in the living
organism are coordinated and interrelated. A knowledge of the princi-
ples of enzyme action is essential to the study of fungus physiology.

The fungi are able to make a far greater contribution to the production
of food and many other valuable products than they do at present. Both

INTRODUCTION 5

the useful metabolic products, such as alcohols, organic acids, and anti-
biotics, and the harmful products (toxins) are discussed at some length.

Certain fungi cause diseases of plants and animals. The action of
fimgicides used to control these pathogens will be discussed from a theo-
retical viewpoint, since there is an enormous amount of literature dealing
with the practical application of fungicides. Too little attention has
been devoted to the mechanism of fungicidal action.

The production of spores, which is of fundamental importance to the
fungus in the perpetuation of the species, affords many interesting prob-
lems in fungus physiology. Environmental and nutritional factors play
important roles in determining whether a fungus will spoiiilate under a
given set of conditions. These factors are discussed in some detail.

The latter portion of the text emphasizes the activities of fungi in
nature. These topics include the discharge, dissemination, and germina-
tion of spores and the physiological aspects of parasitism, variation, and
inheritance. The physiology of parasitism and resistance is of special
interest to plant pathologists and medical mycologists. Most of the
examples are taken from the field of plant pathology. Perhaps the dis-
cussion of these problems will stimulate the interest and curiosity of the
student. A better understanding of parasitism will surely lead to a wiser
choice of control methods for certain fungi.

No study of fungus physiology is complete without experimental work
in the laboratory. The judgment necessary to evaluate one's own work
is founded upon experience. Suggested laboratory exercises and demon-
strations, with brief instructions, are given at the end of the text. These
are selected to illustrate important principles, many of which can be
illustrated clearly only by direct observation of the varied reactions of
fungi to their environment.

HISTORICAL DEVELOPMENT

The development of fungus physiology is far from complete. While
some of the main outlines are clearly visible, much remains to be done.
Although space and time do not permit a complete review of the history
of this science, it is important to realize that its development was the
work of many minds and hands. The influence of the early investigators
continues, not only in their published work but also in the students they
trained.

Some of the outstanding leaders in the development of fungus physi-
ology are Avorthy of special mention. Their names and their contribu-
tions are encountered frequently bj'" all students of this subject. Brief
mention of some of these men and their fields of interest and investigation
is made below.

6 PHYSIOLOGY OF THE FUNGI

Louis Pasteur (1822-1895), France. Pasteur was a chemist who, as a
result of his interest in microorganisms which cause disease and fermenta-
tions, became a biologist. No other scientist has opened up so many
fields of fruitful study. Early in his career he discovered that fungi are
able to discriminate between the optical isomers of tartaric acid. His
student Raulin devised the first synthetic medium for the cultivation of
fungi and published the first thorough study of the nutritional require-
ments of a fungus. Pasteur discovered that some organisms are inhibited
by free oxygen and that some fungi change both their morphology and
physiology when cultivated anaerobically. Pasteur's complete works
have been collected and edited (1933-1939) by his grandson, Professor
Pasteur Vallery-Radot. Dubos (1950) has published an evaluation of
Pasteur's work.

Heinrich Anton de Bary (1831-1888), Germany. His principal contribu-
tions to mycology dealt with life histories and parasitism of fungi. His
interests were primarily with biological adaptations and were more physi-
ological than taxonomic. De Bary's influence as a teacher attracted
many students who later were responsible for much of the development of
plant pathology and mycology. Among his writings was " Morphologic
und Physiologic der Pilze" (first edition 1866, second edition, English
translation, 1887), which may be considered as the first book containing
discussions of the physiology of the fungi.

Oscar Brefeld (1839-1925), Germany. We owe a great debt to this
patient investigator, who developed methods of ensuring sterile media
and apparatus for pure culture work. He was equally insistent with
regard to the purity of his cultures. His chief interest in mycology was
the study of life histories and development of fungi. This meant to him
observation of a fungus from ''Spore zu Spore." He was the first to use
the single-spore technique. Besides his occasional papers, he published
his monumental work (1872-1912) in 15 parts. This beautifully illus-
trated work is still of great value.

Georg Klehs (1857-1918), Germany. His important contributions to
the study of fungus physiology concerned problems related to sporulation.
In 1900 he summarized his conclusions in four statements or laws (Chap.
14). No better generalizations on this subject have appeared in the 50
years which have elapsed since they were published. For an evaluation
of the significance of Klebs' work, see Kauffman (1929).

A. H. Reginald Buller (1874-1944), England and Canada. Many of his
studies involved the activities of fungi in relation to structure. His chief
interests lay in production of fruit bodies and spores, in spore discharge
and dissemination, and in the effects of the environment on these activi-
ties. His keen observations are recorded in detail in seven volumes,
"Researches on Fungi." These volumes are written in an interesting,

INTRODiCTION 7

readable style and should be frequently consulted by all students of
mycology.

Leon H. Leonian (1888-1945), United States. Trained as a mycologist
under Kauffman, he was always interested in discovering the potentiali-
ties of living fungi. His principal contributions were made in the studj'-
of fungus nutrition with emphasis on the factors which are required by
fungi for growth and reproduction. For a bibliography of his papers see
Orton (1946).

The number of living investigators who have made and are continuing
to make important contributions to fungus physiology is far too great to
list here, and for this reason they have been omitted. An idea of the
scope of their interests and activities may be gained from the references in
the following chapters.

REFERENCES

Bkefeld, O.: Botanische Untersuchungen iiber Schimmelpilze, Hefte 1-4, 1872-
1881. Title changed to Botanische Untersuchungen iiber Hefenpilze Fortset-
zung der Schimmelpilze for Heft 5, 1883; thereafter Untersuchungen aus dem
Gesamtgebiet der Mykologie, Hefte 6-15, 1884-1912. Hefte 1-8, Arthur Felix,
Leipzig. Hefte 9-15, Heinrich Schoningh, Muenster.

BuLLER, A. H. R.: Researches on Fungi, Longmans, Roberts and Green, London.

Vol. I, 1909; Vol. II, 1922; Vol. Ill, 1924; Vol. IV, 1931; Vol. V, 1933; Vol. VI,

1934; Vol. VII, The University of Toronto Press, Toronto, 1950.

*De Bary, a.: Comparative Morphology and Biology of the Fungi, Mycetozoa and

Bacteria (trans. H. E. F. Garnsey), Oxford University Press, New York, 1887.

Dodge, B. O. : Some problems in the genetics of the fungi. Science 90 : 379-385, 1939.
*DuBos, R. J.: Louis Pasteur, Free Lance of Science, Little, Bro^Ti & Company,
Boston, 1950.

Kauffman, C. H.: Klebs' theory of the control of developmental processes in
organisms, and its application to fungi, Proc. Intern. Congr. Plant Sci. 2 : 1603-
1611, 1929.

Klebs, G.: Zur Physiologic der Fortpflanzung einiger Pilze. III. Allgemeine
Betrachtungen. Jahrb. wiss. Botan. 35 : 80-203, 1900.

Orton, C. R.: Leon Hatchig Leonian. Phytopathology 36: 241-244, 1946.

Pasteur, L.: Oeuvres de Pasteur, reunies par Pastevur Vallery-Radot, 7 vols.,
Masson et Cie, Paris, 1933-1939.

ScHOPFER, W. H.: Plants and Vitamins, Chronica Botanica Co., Waltham, 1943.

CHAPTER 2
CULTURE MEDIA

Before discussing the nutrition of the fungi in detail, it will be helpful to
consider the basic problems involved. For many purposes a knowledge
of the nutrition of the fungi is necessary for culturing them in the labora-
tory or in industry. Like all living organisms the fungi must obtain from
their environment the materials needed for the synthesis of protoplasm
and other cellular constituents. Directly or indirectly, the fungi as well
as animals and most bacteria are dependent upon green plants for "food"
and energy.

Not all natui'al substrates are equally suitable for all fungi. In nature,
the saprophytes are more widely distributed than the parasites, which are
usually restricted to the range of their hosts. Many of the substances
upon which the fungi grow in nature are chemically complex, and some,
such as cellulose, starch, and proteins are insoluble or are only colloidally
soluble. Before such compounds can be utilized, they must be changed
into low-molecular-weight compounds which are soluble in water. This
"digestion" is accomplished by means of enzymes which are excreted by
the fungi. This is analogous to digestion in animals, which is also an
enzymatic process. The complete utilization of a natural substrate is
frequently due to the combined action of a succession of microorganisms.
More than one organism may act at the same time, and often this simul-
taneous action is more effective than that of a single organism.

One may ask. Do the fungi simply incorporate within their own proto-
plasm the suitable elements and compounds found in the medium, or do
they transform the compounds of the medium before building their own
structures? Apparently the fungi do both. The essential elements such
as potassium and magnesium are taken up as ions, although these ele-
ments may be in the state of chemical combination in the substrate and
also in the fungus cells. Certain organic compounds, such as the vita-
mins, are undoubtedly absorbed as such from the medium by vitamin-
deficient fungi; otherwise, these fungi would derive no benefit from them.
The same statement is true for other necessary compounds which the
various fungi are unable to synthesize.

By far the greater part of the compounds utilized by the fungi are
modified or changed either before or after they are taken into the cells.
Outside the fungus cells, these changes are largely in the direction of
simplifying the molecular structure of compounds used. Within the
fungus cells some of the metabolite molecules are oxidized to carbon

CULTURE MEDIA 9

dioxide and water or to intermediate products. By this process the
fungus obtains the chemical energy which it requires for the processes of
synthesis.

KINDS OF MEDIA

No one knows when man began to cultivate fungi, but certainly it was
many thousands of years ago. This cultivation was no doubt uninten-
tional at first and was later developed into an art, in connection with the
preparation of foodstuffs and beverages. The use of leaven (yeast)
extends back to the beginning of agriculture. The yeast culture was
preserved in a piece of dough which in turn was added to the next batch,
much as buckwheat batter is prepared today. In the Orient, species of
Mucor and Aspergillus have been used from the dawn of civilization in
preparing food from rice and soybeans. Brewers used yeast many cen-
turies before it was learned that yeast is a living organism. On the other
hand, the science of growing fungi in pure culture is fairly recent.

Natural media. It was quite natural that, when mycologists and
others began to cultivate fungi in the laboratory, they should turn to
natural materials as media. A natural medium is one which is composed
entirely of complex natural materials of unknown composition. Among
the natural substances so used are the following: plant parts, malt, yeast,
peptone, manure, bread, wort, fruit, and vegetables. Many of these
substances are used in the form of extracts, infusions, or decoctions. The
very diversity of these natural media is strong testimony to the fact that
different species have different nutritional requirements. Brefeld (1881)
was among the first to grow fungi in pure culture, and many of his tech-
niques are in use today. Since his interest in cultivating the fungi was
largely for the purpose of observing their development, it was necessary
for him to select suitable media. He found two natural media to be of
great utility: a decoction prepared from dried plums or raisins and a
manure extract. This latter medium he considered "als Universal-
nahrlosung flir Pilzculturen." This medium is still used in some labora-
tories. Natural media have many advantages. They are cheap and
easy to prepare. In many instances it is necessary only to add water to
the base material and autoclave. More important yet is the fact that
many fungi grow well upon a wide variety of natural media.

Certain of the more fastidious fungi have never been cultivated in the
laboratory. These obligate parasites live only upon or ^^^thin the living
tissues of their hosts. Puccinia graminis tritici lives only on wheat, some
species of grasses, and some species of barberry. These host plants when
killed will no longer support growth of this fungus. However, many
species, which in the past were considered to be obligate parasites, have
since been cultured on nonliving media.

10 PHYSIOLOGY OF THE FVSGI

Semisynthetic media. A semisynthetic medium is one Avhich is com-
posed in part of natural materials of unknown composition. Such media
are made by adding compounds of known composition to one or more
natural materials. The widely used potato-glucose (dextrose) medium is
an example of this type. The addition of agar to an otherwise synthetic
medium introduces a natural material of unknown composition. Media
Avhich contain agar cannot be classed strictly as synthetic media. Semi-
synthetic media may be used for many types of physiological investigations.

The composition of a given natural or semisynthetic medium is not
constant. Potato-glucose medium may vary greatly in composition
depending upon whether or not the potatoes were peeled and upon the
variety and age of the potatoes used. Neuberger and Sanger (1942)
found a twofold difference in the amide nitrogen (asparagine and gluta-
mine) among varieties. In this laboratory we have found that the
amount of potato pulp Avhich is allowed to enter this medium exerts a
marked influence upon growth and reproduction of certain fungi. These
differences, which may seem minor, are great enough to make comparisons
between work done in different laboratories difficult.

Synthetic media. As the term is used in this book, a synthetic medium
is one of known composition and concentration. It does not mean that
every compound used is a product of the chemist's art. Some of the
constituents, such as the sugars, may be of natural origin. The important
condition is that the compounds used be pure, and this is difficult to
attain in practice. "Chemically pure" compounds are usually far from
being pure, as a glance at the labels will show. The ideal of using pure
compounds is seldom realized, but the closer it is approached, the more we
shall learn about the nutrition of the fungi.

Natural media and most semisynthetic media are of limited usefulness
in studying nutrition of the fungi. The chief value of synthetic media is
for nutritional studies. However, growth and reproduction are fre-
quently poorer on a synthetic medium than on one containing some
natural material. For example, Aspergillus niger grew well in a synthetic
medium composed of sucrose, ammonium nitrate, magnesium sulfate, and
dipotassium hydrogen phosphate (Steinberg, 1939). In addition to
these major constituents, iron, zinc, copper, manganese, molybdenum,
and gallium salts were present. Extraordinary care was taken in prepar-
ing the medium. The concentration of every constituent was so balanced
that a decrease in concentration of any constituent resulted in diminished
growth. Growth and sporulation were excellent upon this medium.
When 20 mg. per liter of either peptone or yeast extract was added, the
rate of growth was greatly increased and the time required for sporulation
was decreased. The small amount of yeast extract or peptone used
could have added only an insignificant amount of material from which

CULTURE MEDIA ii

the fungus could synthesize protoplasm or derive energy. Steinberg's
synthetic medium was adequate but not optimum for most rapid growth
and sporulation. We may suppose that the yeast extract and peptone
contained compounds the synthesis of which constituted a limiting effect
upon the rate of growth and sporulation.

Synthetic media may be simple or complex but must contain the essen-
tial elements in utilizable form. Brefeld (1881) gave the following direc-
tions for preparing a synthetic medium: Add cigar ashes dissolved in
nitric or citric acid to a solution containing a soluble carbohydrate, such
as glucose, and an ammonium salt. The amount of ashes was not speci-
fied. The first vsynthetic medium was devised by Raulin (1869).

Table 1. Composition of the First Synthetic Medium for CuLTrvATiNG Fungi

(Raulin, Ann. sci. nat., Ser. V, 11, 1869.)

Ammonium nitrate 4.0 g.

Ammonium phosphate 0.6 g

Magnesium carbonate 0.4 g

Potassium carbonate 0.6 g

Ammonium sulfate 0 . 25 g

Zinc sulfate 0 . 07 g,

Iron sulfate 0 . 07 g

Potassium silicate 0 . 07 g,

Sucrose 70 g,

Tartaric acid 4 g

Water 1,500 ml.

However, not enough information is given in Table 1 for the duplication
of this medium. Which ammonium phosphate, (NH4)H2P04 or (NH4)2-
HPO4, was used by Raulin in the original work? Which zinc sulfate,
ZnS04-7H20 or ZnS04-H20, was used? Was the iron sulfate FeS04,
FeS04-7H20, or re2(S04) 3? Did he use D-tartaric, L-tartaric, DL-tartaric,
or weso-tartaric acid? These questions are asked for the purpose of
emphasizing the need for exactness in reporting the composition of media
used in experimental work. These uncertainties creep into the literature
through ignorance or carelessness, or both. Nor are these ambiguities to
be found only in the older literature, for they are present in papers pub-
lished only yesterday. Either the specific name or the formula, or both,
should be stated. If it is stated that dipotassium phosphate, K2HPO4,
was used, the reader is certain of the identity of the compound. Potas-
sium phosphate may designate at least five distinct chemical compounds.

NATURAL VERSUS SYNTHETIC MEDIA

In addition to the fact already noted that the composition of natural
media is unknown, natural and synthetic media differ in two further
respects. Natural media are more complex; i.e., they contain more

12 PHYSIOLOGY OF THE FUNGI

chemical compound.s than synthetic media. They also contain com
pounds ordinarily not present in synthetic media.

Specific metabolites. Only certain chemical compounds are utilized
by fungi, but not all fungi are able to utilize the same compounds. Any
compound utilized by a fungus is called a metabolite. Some fungi are
unable to synthesize certain essential metabolites and are said to be
"deficient" for the specific metabolites they are unable to synthesize.
In order to cultivate such deficient fungi, these metabolites must be pres-
ent in the medium. Natural media usually contain these metabolites.
If a fungus grows upon a natural medium and fails to grow upon a variety
of simple synthetic media, it may be suspected that specific metabolites
are involved in its nutrition.

The following example will illustrate the role of specific metabolites in
fungus nutrition. Fellows (1936) investigated the ability of Ophiobolus
grayninis to utilize different nitrogen compounds for growth. A sucrose-
mineral salts solution was used as the basal medium to which various
nitrogen sources were added. Only complex nitrogen sources such as egg
albumen, peptone, casein, and nucleic acid allowed growth. Under the
conditions used no growth resulted when ammonium compounds, nitrates,
nitrites, and amino acids were tested. 0. graminis in the presence of egg
albumen utilized glucose, maltose, lactose, fructose, xylose, starch, and
dextrin, in addition to sucrose. From these experimental results it was
concluded that 0. graminis requires a complex source of nitrogen for
growth.

Later, White (1941) found that this fungus requires two specific
metabolites, thiamine and biotin. When these vitamins were added to
synthetic media containing simple nitrogen sources (sodium nitrate,
ammonium nitrate, asparagine, or glycine), good growth was obtained.
Thus, it appears obvious that 0. graminis does not require a complex
nitrogen source, but that it is unable to synthesize two specific chemical
compounds. These papers illustrate the fact that fungus physiology is a
young and developing science. Much of the early work needs reevalua-
tion in the light of recent discoveries. A student should strive to develop
a critical attitude toward the work of others, but he should be no less
critical with regard to his own work. The evaluation of experimental
results depends upon the conditions under which the work was done, and
among these conditions the medium used is of first importance.

Complexity of media. It is a common experience to find that a trace of
some crude natural product stimulates the rate of growth and sporulation
of a fungus. This stimulation frequently occurs with fungi which grow
well on synthetic media and which are not deficient for vitamins or amino
acids. It appears that the complexity of natural media offers a clue to
understanding this stimulatory effect. If a fungus is grown upon a simple

CULTURE MEDIA 13

synthetic medium which has only one source of carbon and one source of
nitrogen, it must synthesize many complex chemical compounds from
constituents present in the medium. It may be suspected that these
biochemical syntheses are slowed up under these conditions. When a
mixture of many carbon and nitrogen sources is present, the fungus may
function more efficiently, because the biochemical syntheses are easier
since some of the intermediates are furnished. These speculations
receive some support from evidence to be presented in Chaps. 6 and 7_

CHOICE AND PREPARATION OF MEDIA

Considerable care is needed in the selection of a suitable medium. A
medium may be excellent for growth and unsuitable for reproduction or
the production of an antibiotic. The method of preparation may influ-
ence the composition of a medium in unsuspected ways.

Choice of media. In selecting a medium the purpose for which it is to
be used should be kept in view. For many purposes a natural medium is
the one of choice. This is especially true for routine maintenance of
cultures, for isolations, and for preliminary investigations. The composi-
tion of natural media may be varied by choosing different substrates.
Frequentl}^, a combination of natural products may be used to advantage,
e.g., malt and yeast extracts. In addition, these natural substrates may
be fortified with one or more pure chemical compounds. The constitu-
ents of natural media are fixed by the substances used, but the amounts
used may be changed at will.

More judgment enters into the selection of synthetic media. The
essentials of a synthetic medium may be stated as follows : sources of car-
bon and nitrogen in utilizable forms; phosphate and sulfate ions; the
metallic ions potassium, magnesium, iron, zinc, manganese, and others
which are usually present as impurities in the chemicals used. These are
the essential elements and will be considered at length in later chapters.
Most fungi utilize glucose, so this sugar is frequently used as the carbon
source. More fungi utilize nitrogen in organic combinations than in
inorganic compounds. The question of specificity enters into the choice
of the carbon and nitrogen sources, and this can be determined only by
experiment. In order to cultivate deficient fungi on synthetic media, the
specific metabolites for which the fungi are deficient must be added.
Since synthetic media are used to study nutrition, the development of a
suitable synthetic medium for a specific fungus may require considerable
investigation. In our laboratory we commonly first use a glucose-casein
hydrolysate medium containing the essential inorganic elements. This
medium has been very useful in vitamin studies. Its composition is
given in Chap. 10.

14 PHYSIOLOGY OF THE FUNGI

Solid versus liquid media. Both solid and liquid media are used in
cultivating fungi. Media solidified with agar, or semisolid substrates
such as corn meal, offer many advantages in that the culture vessels can
be freely handled without disturbing the fungus. This feature is particu-
larly valuable when one wishes to follow the development of a fungus.
Microscopic examination is facilitated, and contaminants are more easily
detected. Single-spore isolations can be made more easily from solid
media. Agar media are used to maintain stock cultures and are recom-
mended for many preliminary experiments.

Frau Hesse (Kitchens and Leikind, 1939) introduced the use of agar
into microbiological procedures in 1881. Agar, which is obtained from
various marine red algae, is a complex polysaccharide sulfate ester
(Pigman and Goepp, 1948). It forms colloidal solutions at elevated
temperatures and sets to a gel at temperatures around 45°C. On acid
hydrolysis both D-galactose and its enantiomorph, L-galactose, as well
as sulfuric acid, are formed. Agar must exist in the form of a salt
(Ca, Mg, Na, K, etc.) to form a gel. Agar introduces physiologically
active elements into media. It may contain significant amounts of
zinc (Leonian and Lilly, 1940) and other micro essential elements.
Mulder (1940) found that magnesium could be efficiently removed from
agar by repeated soakings in 10 per cent sodium chloride solution, followed
by washing with distilled w^ater until the filtrate was free from chloride
ion. Agar also contains growth factors such as thiamine (Day, 1942)
(see Fig. 1). Many fungi make some growth on water agar, which indi-
cates that agar or the ''impurities" contained in it are utilized by fungi.
Robbins (1939) found that leaching agar with 5 per cent aqueous pyridine
removed many of the physiologically active compounds.

Liquid media should be used for precise investigations where it is
desired to control as many variables as possible. The composition of the
medium may be controlled and the amounts used measured accurately.
Cultures may be aerated by shaking or by blowing sterile air through the
media. Weighing the mycelium is facilitated. When it is desired to
study the metabolic by-products of fungus metabolism (except gaseous
products), it is almost necessary to use liquid media. Isolation of
by-products is less complicated when liquid media are used. Studies of
various metabolite deficiencies and many microbiological assays (Chap.
10) almost always require the use of liquid media. The choice between
the use of solid or liquid media should be made on the basis of the known
advantages and disadvantages of both and with regard to the purpose of
the problem under investigation.

Designating media. It is common to find references to a medium by
the name of the investigator who first used it. These names have served
as convenient abbreviations and commemorate the pioneers in the art of

CULTURE MEDIA

cultivating fungi. Some of these names are Blakeslee, Uschinsky, Coons,
Czapek, Leonian, Sabouraud, Richard, Thaxter, Shear, Raulin. From a
historical standpoint this practice has much to recommend it. However,
this usage has many disadvantages. These distinguished names give no
clue to the composition of these media. The original formulas have in
many instances been changed. Some of these modifications have received

A B

Fig. 1. Growth of Phycomyces blakesleeanus on vitamin-free liquid medium solidified
with two different brands of agar. Growth in A indicates relatively high content of
thiamine of this agar. The trace of growth in B shows that this agar is relatively
free of thiamine.

hyphenated names: e.g., Czapek-Dox. Frequently the originator of a
medium modified it from time to time. This introduces a further uncer-
tainty as to its composition. In our opinion the use of personal names to
designate media should be abandoned. It is much more helpful to
designate media by descriptive titles than by names which tell nothing of
the composition. The carbon and nitrogen sources are important con-
stituents of every medium. Thus, sucrose-nitrate medium, glucose-
asparagine medium, or malt extract-yeast extract medium are preferred
to Czapek's medium, Schopfer's medium, or Leonian's medium. These
descriptive terms afford valuable information that personal names do not.
Even when the reader is familiar with the composition of a named medium,

16 PHYSIOLOGY OF THE FUNGI

there is a tendency to fail to associate experimental results with the
composition.

Effect of autoclaving. Media are commonly and effectively sterilized
by autoclaving. It should be noted, however, that such high tempera-
tures may cause destruction or alteration of some constituents in the
media. These changes are not serious for many uses; at least media pre-
pared in this way are satisfactory. Sugars are among the substances
most easily altered by autoclaving. The extent of decomposition depends
upon the specific sugar used, the other constituents of the medium, and
the time of autoclaving. It is desirable to adopt a uniform schedule for
autoclaving media. An increase in the amount of caramelization occurs
as the time of heating is increased. Maillard (1912) showed that a brown
color results when reducing sugars (glucose, fi-uctose, etc.) are autoclaved
with amino acids. Hill and Patton (1947) have shown that growth of
Streptococcus faecalis is reduced when tryptophane is autoclaved with
sugars. Margolin (1942) found that no one method of sterilization
resulted in best growth for all of the 14 species tested. Phythophthora
erythroseptica made three times the amount of growth on glucose sterilized
by filtration as when the entire medium was autoclaved. Syncephalastrum
racemosum, however, made more growth on autoclaved than on sterile-
filtered glucose (Table 2). The organisms most sensitive to heated
glucose appear to be various species of Cytophaga, which failed to grow
on glucose which had been heated to 50°C. (Stanier, 1942). These
organisms utilized glucose which had been sterilized by filtration. Phos-
phates, a universal constituent of media, are active in converting glucose
into ketoses and other products (Englis and Hanahan, 1945) during
autoclaving.

Complex sugars and polysaccharides undergo some hydrolysis during
autoclaving. The amount of hydrolysis is dependent upon the carbo-
hydrate, the time and temperature of autoclaving, and the pH of the
medium. Sucrose, when autoclaved in acidic media, may undergo suffi-
cient hydrolysis to support some growth of species unable to utilize
sucrose. This possibility must be guarded against in experiments on the
availability of complex sugars.

Other substances used in media may be destroyed during autoclaving.
To minimize or avoid such effects, heat-sensitive substances may be auto-
claved separately, or they may be sterilized using special bacteriological
filters. The Berkefeld and Chamberland filters are less used than
formerly, while at present Seitz and fritted-glass filters are widely used.
Fritted-glass filters are best for most purposes, inasmuch as the asbestos
pad used in the Seitz filter may adsorb active compounds. All methods
of sterilization w^hich depend upon filtration are slow and can be used only
with liquid media. Various volatile chemical sterilization agents such as

CULTURE MEDIA

alcohol and acetone have been used. Hansen and Snyder (1947) have
recommended the use of propylene oxide for the sterilization of plant
parts used for culture media. Frequently a seemingly insignificant
change in the method of preparing a medium may result in significant
changes in the composition of the medium, which in turn may be reflected
in the behavior of the organisms grown upon it. Even the volume of
medium in culture vessels affects the amount of decomposition during
autoclaving. Cotton plugs may introduce hnt into the medium. Less
refined grades of cotton release a volatile substance which affects the

Table 2. The Effect of Different Methods of Sterilizing Glucose upon

THE Growth of Sex Fungi, at 25°C.

Growth reported as milligrams of dry mycelium.

The entire medium, containing a mixture of amino acids, was autoclaved in the
control experiment. In the other experiments the glucose was sterilized by either
Seitz filtration or treatment with acetone and added aseptically to the remainder of
the sterile medium. (Margolin, thesis. West Virginia University, 1942.)

Species

Days of
incuba-
tion

Control,

entire

medium

autoclaved

Glucose

sterilized by

filtration

Glucose
sterilized by

treating
with acetone

Phycomyces blakesleeanus

Rhizopus suinus

130
122
103

142

140

123

241

88
147

132
115

80
192

85
103

Syncephalastrum racemosxun

Phytophthora erythroseptica

Diplodia macrospora

Phytophthora cadonim

germination of some spores {Phycomyces blakesleeanus, Robbins and
Schmitt, 1945). Paper or aluminium caps may be used to replace cotton
plugs. Residual soap films on improperly rinsed glassware may cause
trouble in some cases.

Preparation of media. Directions for the preparation of specific media
are given at the end of the text in the section Suggested Laboratory
Exercises. Additional details concerning various media are to be found
in Riker and Riker (1936) and Rawlins (1933).

WAYS OF EXPRESSING CONCENTRATION

Concentrations are frequently expressed in the literature as percentages.
Unless the basis for calculating these values is given, percentage is an
ambiguous way of reporting concentration. Buchanan and Fulmer
(1928) have pointed out that there are six ways of calculating the percent-
age composition of a solution. A 10 per cent sulfuric acid solution may
represent six different concentrations. For any precise work it is best to

18 PHYSIOLOGY OF THE FUNGI

avoid the use of percentages, but for routine work, where the composition
of media is of less importance, the use of percentages may be allowed.
Before the same medium can be prepared repeatedly, it is necessary to
know what constituents are used and the amount of each.

Two general methods are used for reporting the composition of media.
Either the weights of the constituents and the volume of water used are
given, or the weights of the constituents are given and the medium made
up to a definite volume. The first method is in common use; its sim-
plicity conceals its disadvantages. The volume of a medium prepared by
this method is never the same as the volume of water used. It is neces-
sary to measure the volume of the medium after preparation in order to
calculate the amount of any constituent in an alicjuot.

The method of choice in accurate work is to weigh the constituents and
make the medium up to a given volume. The amovmt of any constituent
in any volume of medium may then be calculated. If a liter of medium
contains 25 g. of sucrose, and 25-ml lots are dispensed, each lot contains
^/iooo X 25, or 0.625 g. of sucrose.

Direct units. The units of volume most used are the liter (1.) and the
milliliter (ml.). A cubic centimeter (cc.) is nearly, but not exactly,
equivalent to a milliliter. Its use should be discouraged. The formulas
for media are usually given on the basis of a liter. This practice is to be
encouraged, as the liter is a convenient volume in preparing media. The
weights of solid constituents should be reported as grams (g.) or decimal
divisions thereof. The most commonly used decimal fractions of the
gram are the milligram (mg.), the microgram (jug), and the millimicrogram
(m/ig), each of which is one-thousandth of the preceding weight. Since
it is easy to make mistakes in reading small decimals, it is recommended
that no decimals smaller than 0.1 be used. The use of 12 mg. is preferable
to 0.012 g., although both mean exactly the same. It is easier to read
5 jug than 0.000005 g. One milligram of a substance in a Uter of solution
equals one part per million (p. p.m.). Each milliliter of such a solution
wiU contain 1 fxg of the substance. Similarly, a microgram of a substance
in a liter of solution is present as one part per billion. The microgram
has also been called the gamma (7), but this usage should be abandoned
inasmuch as gamma is not a regular prefix used in the metric system.
The necessity of using such small units of weight arises from the physio-
logical activity of certain compounds and elements. For example, a
concentration of 1 mg. of biotin in a liter of medium is a relatively enor-
mous concentration.

Derived units. Derived units must be used in comparing the effect of
compounds which have different molecular weights. Among these
derived units the mole is the most useful. A mole is the molecular weight
of a chemical compoimd expressed in grams. A mole of glucose is 180 g.,

\^ K^ ±^ J. \^ X\/X-J 1.WX JUt X-^X iX.

while a mole of sucrose is 342 g. A liter of solution containing one mole
of a compound is said to be one molar (il/). Equimolar solutions contain
the same number of molecules. In problems in physiology, such as
osmotic pressure, which have to do with the numbers of molecules it is
necessary to use this way of expressing concentration. If it is desired to
compare the effect of the osmotic pressure due to glucose and sucrose, the
concentration must be expressed in terms of molar strengths, for the
osmotic pressure is a function of the number of molecules of solute in a
solution. If it is the purpose to compare the effect of glucose and sucrose
on the amount of growth of a fungus, this method of expressing concen-
trations should not be used. Media of equal molarity with respect to
sucrose and glucose do not contain the same amount of carbon. The
first contains twice as much carbon as the second. Just as a milligram is
one-thousandth of a gram, a millimole is one-thousandth of a mole. The
meaning of micromole and millimicromole should be obvious.

If the weight of a compound is given in grams, this datum may be con-
verted into moles. If a medium contains 50 g. of glucose per liter, the
glucose concentration may be expressed as 50/180 or 5/18ilf. Con-
versely, if the concentration of sucrose in a medium is stated to be 0.15ilf,
the weight of sucrose is 0.15 X 342 or 51.3 g. per liter. These conversions
imply that the molecular weight is known or can be calculated. In pre-
paratory work compounds are weighed on a balance as grams, not as
moles, and unless the interpretation of the results demands conversion to
moles, it is better to record the weights than to convert these data to
derived units. The mole and molar solutions are particularly useful in
dealing with non-ionizing compounds.

Another derived unit, the equivalent, is frequently used to express the
concentration of ionized compounds. An equivalent is the atomic weight
of an ion expressed in grams divided by the valence of the ion. If an ion
is composed of more than one atom, the ion weight is computed by adding
together the atomic weights. It is important to remember that, if an
element has more than one valence, the equivalent weight depends upon
the valence. An equivalent of ferrous (Fe++) ion is 55.8/2 or 27.9 g.,
while an equivalent of ferric (Fe+++) ion is 55.8/3 or 18.6 g. A normal
solution (A^) is one which contains one equivalent in a liter of solution.
In dealing with small amounts it is convenient to use milliequivalents or
microequivalents.

In preparing a series of media for the purpose of comparing the growth
of a fungus on different nitrogen sources, the nitrogen content of the media
should be equal. If urea, CO(NH2)2, and aspartic acid, HOOC — CH2 —
CH(NH2) — COOH, are used, it is obvious that different weights of these
nitrogen sources must be used if the media are to contain equal amounts of
nitrogen. Whenever media are modified by replacing one compound by

PHYSIOLOGY OF THE FUNGI

another, it should be done in such a way that the same amount of the
essential element is present in all the media. If this is not done, the basis
upon which the replacement was made should be stated. If 25 g. of
glucose, C6H12OC is replaced by 25 g. of sucrose, C12H22O11, it should be
realized that the carbon contents of the two media are different. It is
frequently difficult or impossible to find out from some papers in the
literature how substitutions in the media were made.

Table 3. A Compakison of Two Synthetic Media upon the Basis of Amounts

OF Essential Elements and Compounds Present in One Liter

Both media were made with double-distilled water.

Glucose-asparagine *

Sucrose-ammonium nitrate f

Element or
compound

Unit of
meas.

Source

Unit of
meas.

Source

4.0

0.427

0.049

0.065

0.287

0.228

Mg.

0.2
0.2
0.1

100

D-Glucose, 10 g.
L-Asparagine, 2 g.
MgS04-7H.,0, 0.5 g.
MgS04-7H.20, 0.5 g.
KH2PO4, 1.0 g.
KH,P04, 1.0 g.

As sulfate
As sulfate
As sulfate

21.4
0.720
0.025
0.032
0.125
0.062

Mg.

0.3

0.075

0.02

Sucrose, 50 g.

NH4NO3, 2.06 g.

MgS04-7HoO, 0.25 g.

MgS04-7H,0, 0.25 g.

K2HPO4, 0.35 g.

As chloride

As chloride
As chloride

As chloride

Thiamine

hydrochloride

Biotin

* Medium 5, Suggested Laboratory Exercises,
t Steinberg, 1941.

Finally, it should be noted that the common practice of using one com-
pound as the source of two essential elements does not permit perfect
freedom in adjusting the composition of a medium. If magnesium sulfate
heptahydrate is used to supply both magnesium and sulfur, it is obvious
that the ratio Mg/S is fixed. If it is desired to vary the amounts of
magnesium and sulfur independently, it is necessary to use different com-
pounds of magnesium and sulfur; e.g., magnesium chloride and sodium
sulfate. This practice introduces other elements into the medium.

CULTURE MEDIA 21

COMPARISON OF MEDIA

Media differ only in constituents and amounts used. It is desirable to
be able to compare media in some uniform way. To do this, it is neces-
sary to know not only the amounts of the elements present, but also the
compounds in which these elements occur. A comparison of two syn-
thetic media is given in Table 3.

From Table 3 it will be noted that these media contain the same essen-
tial elements. Copper, molybdenum, and gallium do not appear in the
composition of the glucose-asparagine medium, but it should not be con-
cluded that these elements were not present, since only c.p. chemicals
were used to prepare this medium. Stout and Arnon (1939) note that a
distinction must be made between ordinary chemical purity and biological
purity. This will be considered in detail in Chap. 5. The two features
which make these media quite distinct are the different sources of carbon
and nitrogen used and the addition of two vitamins to the glucose-
asparagine medium. The latter medium is suitable for the growth of
more species of fungi than is the sucrose-ammonium nitrate medium.

SUMMARY

Fungi secure food and energy from the substrates upon which they live
in nature. In order to culture fungi in the laboratory, it is necessary to
furnish in the medium those essential elements and compounds they
require for the synthesis of their cell constituents and for the operation of
their life processes. The synthetic abilities of fungi differ. Some fungi
are unable to s5Tithesize certain key compoimds that they require and
must obtain them from the medium upon which they grow. All the fungi
require much the same essential elements but differ widely in their ability
to utilize compounds in which these elements occur. There is no uni-
versal natural substrate or artificial medium upon which all fungi will

grow.

On the basis of composition there are three general types of media:
natural media, which are composed entirely of natural products; semi-
synthetic media, which are composed in part of natural substances; and
synthetic media, which are of kno^\^^ composition. Natural media are
most useful for routine work, while synthetic media and, to a limited
extent, semisynthetic media are used to investigate the nutritional
requirements of the fungi. Media differ only with respect to constituents
and concentrations.

The compounds and the amounts used in preparing a medium must be
specified exactly. Media should be designated by naming the carbon
and nitrogen sources used, e.g., glucose-asparagine medium. The use

22 PHYSIOLOGY OF THE FUNGI

of proper names to designate the composition of a medium should be
avoided.

The selection of a suitable medium depends upon the fungus under
study and the purpose of the experiment. Not all media are equally
suitable for all fungi, nor is one medium suitable for a complete physio-
logical study of one fungus.

REFERENCES

Brefeld, O.: Botanische Untersuchungen liber Schimmelpilze, Heft, IV Verlag

Arthur Felix, Leipzig, 1881.
Buchanan, R. E., and E. I. Fulmer: Physiology and Biochemistry of Bacteria,

Vol. I, The Williams & Wilkins Company, Baltimore, 1928.
Day, D.: Thiamin content of agar. Bull. Torrey Botan. Club 69: 11-20, 1942.
Englis, D. T., and D. Hanahan: Changes in autoclaved glucose, Jour. Am. Chevi.

Soc. 67 : 51-54, 1945.
Fellows, H.: Nitrogen utilization by Ophiobolus graminis, Jour. Agr. Research 53:

765-769, 1936.
*Hansen, H. N., and W. C. Snyder: Gaseous sterilization of biological materials for

use as culture media, Phytopathology 37: 369-371, 1947.
Hill, E. G., and A. R. Patton: The Maillard reaction in microbiological assay.

Science 105 : 481-482, 1947.
HiTCHENS, A. P., and M. C. Leikind: The introduction of agar-agar into bacteri-
ology. Jour. Bad. 37: 485-493, 1939.
Leonian, L. H., and V. G. Lilly: Studies on the nutrition of fungi. IV. Factors

influencing the growth of some thiamin-requiring fungi. Am. Jour. Botany 27:

18-26, 1940.
Maillard, L. C: Action des acides amines sur les sucres; formation des melanoldines

par voie methodique, Conipt. rend. acad. sci. 154: 66-68, 1912.
Margolin, A. S.: The effect of various carbohydrates upon the growth of some

fungi, thesis. West Virginia University, 1942.
Mulder, E. G. : On the use of micro-organisms in measuring a deficiency of copper,

magnesium and molybdenum in soils, Antonie van Leeuwenhoek 6 : 99-109,

1939-1940.
Neuberger, a., and F. Sanger: The nitrogen of the potato, Biochem. Jour. 36:

662-671, 1942.
Pigman, W. W., and R. M. Goepp, Jr.: Chemistry of the Carbohydrates, Academic

Press, Inc., New York, 1948.
Raulin, J.: Etudes chimiques sur la v^g6tation, Ann. sci. nat., Ser. V, 11: 93-229,

1869.
Rawlins, T. E.: Phytopathological and Botanical Research Methods, John Wiley

& Sons, Inc., New York, 1933.
RiKER, A. J., and R. S. Riker: Introduction to Research on Plant Diseases, John S.

Swift Co., St. Louis, 1936.
*RoBBiNS, W. J.: Growth substances in agar. Am. Jour. Botany 26: 772-778, 1939.
Robbins, W. J., and M. B. Schmitt: Effect of cotton on the germination of Phyco-

myces spores, Bull. Torrey Botan. Club 72 : 76-85, 1945.
Stanier, R. Y. : The Cytophaga group: a contribution to the biology of myxobacteria,

Bact. Revs. 6 : 143-196, 1942.
Steinberg, R. A.: Relation of carbon nutrition to trace-element and accessory

requirements of Aspergillus niger, Jour. Agr. Research 59: 749-763, 1939.

CULTURE MEDIA 23

Steinberg, R. A.: Sulfur and trace element nutrition of Aspergillus niger, Jour.

Agr. Research 63: 109-127, 1941.
Stout, P. R., and D. I. Arnon: Experimental methods for the study of the role of

copper, manganese, and zinc in the nutrition of higher plants, Am. Jour. Botany

26: 144-149, 1939.
*■ White, N. H.: Physiological studies of the fungus Ophiobolus graminis Sacc, Jour.

Council Sci. Ind. Research 14: 137-146, 1941.

CHAPTER 3
GROWTH

Growth may be considered either as an increase in cell number or as an
increase in mass. Usually both these processes are concurrent in the
phenomenon called growth. To a limited degree, fungus cells may
divide and form new cells without an increase in mass. A spore may
germinate in distilled water and give rise to a germ tube, but in the
absence of nutrients this process soon stops. A few cell divisions exhaust
the reserve material originally present in the spore, and growth soon
ceases unless these new cells obtain nutrients from the external environ-
ment. Under certain conditions fungus cells may increase their store of
reserve materials, and thus their mass, without an increase in cell number,
but this process is also limited. Growth, excluding the limited meanings
given above, involves an increase in both the number and the mass of cells.

This definition of growth neither ''explains" the processes involved nor
indicates their complexity. Rahn (1932) has expressed doubt that we
will ever fully understand the process of growing. A yeast cell which
buds and produces a daughter cell illustrates one of the striking features
of growth : growth involves duplication. From a dozen or so simple chemi-
cal substances present in the medium the parent cell synthesizes at least
a portion of the protoplasm of the daughter cell. The daughter cell has
the same genetic constitution as the parent cell, and thus a duplication of
genes is a feature of cell multiplication. The compounds which comprise
protoplasm, enzymes, genes, and other substances are extraordinarily
complex. Our meager knowledge concerning the chemical architecture
of these substances only confirms this view. In the synthesis of such
compounds we may assume that the chemical reactions which produce
them are perfectly timed and coordinated, for no series of uncorrelated
reactions could produce such compounds.

The growth processes of the filamentous fungi are still more complex
than those of yeast, because of greater differentiation in structure. In
those species of fungi which produce aerial mycelium these parts are
nourished through the mycelium in contact with the medium. This
involves translocation of nutrients over considerable distances. This is
especially true of sporangiophores and aerial fruit bodies. The develop-
ment of fruiting structures and spores is growth, in that the formation of
new cells is involved. The formation of fruit bodies in many species

GROWTH 25

takes place at the expense of reserve materials and protoplasm formed by
and stored in the vegetative mycelium.

PHASES OF GROWTH

Growth in the fungi, as in other organisms, follows a definite pattern.
The way this development takes place depends upon the species and the
environmental and nutritional conditions. In the present discussion, it
will be assumed that the external conditions are favorable and that growth
takes place in a limited volume of medium.

Unicellular organisms. The bacteriologists have long been interested
in the mathematical analysis of the phenomenon of growth. The student
is referred to Buchanan and Fulmer (1928) and to Rahn (1932, 1939) for
further information on this subject. Among the fungi, the yeasts have
somewhat the same type of development as the bacteria. Since bacteria
multiply by fission and the yeasts (except Schizosaccharomyces) by
budding, we cannot expect the growth pattern of yeasts to fit exactly the
same formulas which have been developed for bacteria. But, in a general
way, yeasts follow closely the phases of growth shown by bacteria.
These phases of growth are as follows: (1) Stationary phase. When cells
are inoculated into a medium, there is a period of time following inocu-
lation when there appears to be no change in number. The stationary
phase may be long or short depending upon the age and vigor of the
inoculum, the medium, and other factors. (2) Phase of accelerated growth.
Not until cell division is established and new protoplasm is being formed
from the constituents of the medium may growth be considered as begun.
This phase is characterized by an increase in the rate of cell division, i.e.,
the generation time is decreasing. (3) Exponential or logarithmic phase.
This phase is clearly defined for bacteria and approached by yeasts. It
is characterized by a constant generation time. If the logarithms of the
cell numbers are plotted against time, the curve is a straight line. (4)
Phase of declining acceleration. As the nutrients become exhausted, or
as toxic by-products accumulate, the average generation time increases.
A combination of these and other factors results in a lessened rate of
growth. If fresh medium were continuously supplied and toxic by-prod-
ucts removed, it is possible that this phase would never be attained. (5)
Maximum stationary phase. This marks the attainment of maximum
weight, or numbers of living cells. It is quite likely that the death of old
cells is balanced by new growth. The duration of this phase is dependent
upon the organism and upon the composition of the medium at this time.
(6) Phase of decline or autolysis. Sooner or later, following attainment of
maximum development, autolysis sets in. As the cells die, the cellular
enzymes begin to digest the various cell constituents. Only the more
resistant portions of the cell remain. Microscopic examination at this

26 PHYSIOLOGY OF THE FUNGI

time reveals that many cells are devoid of protoplasm. It is quite possi-
ble that some of the materials released by autolysis are used by the
remaining living cells.

Filamentous fungi. With exception of the third phase of growth dis-
cussed above, the filamentous fungi follow the same order of development
as the yeasts. The most obvious difference between the filamentous
fungi and unicellular organisms is the failure to attain an exponential rate
of growth. Usually, the exponential phase is replaced by a more or less
linear phase of growth. Emerson (1950) found a straight-line relation
between the cube root of the weight of mycelium produced by Neurospora
crassa grown in nonagitated liquid medium and the time of incubation.
This relation held for three surface-volume ratios. A comparison of the
linear, logarithmic, and cube-root growth curves indicates that this fungus
has a cube-root phase of growth during the interval when the linear graph
is concave upward. Growth in the filamentous fungi is limited to the
tips of the hyphae. The influence of neighboring cells which compete for
nutrients is a much more important factor in the growth of filamentous
fungi than in submerged unicellular organisms. In unagitaged cultures a
portion of the mycelium is usually aerial at some stage of growth. The
aerial mycelium derives its nutrients from the submerged cells, which
involves the transport of these substances over some distance.

RATE OF GROWTH

To study growth, it is necessary to consider both the rate and amount of
production of cells formed during incubation. The average rate of
growth is obtained by measuring the amount of growth at two intervals of
incubation and dividing the difference by the time interval. If the
weight of a fungus colony increased from 50 to 98 mg. between the fourth
and sixth days of incubation, the average rate of growth is 24 mg. per day,
or 1 mg. per hr. In experimental work, measurements of growth should
be made sufficiently often during the period of incubation so that a
smooth graph (growth curve) can be plotted from the data. The inter-
vals between measurements of growth may be as short as 1 day for a
rapidly growing fungus and as long as a week for species which grow
slowly. The rate of growth at any time may be determined by finding
the slope (tangent) of the curve. The growth rates of fungi differ, as is
illustrated in Fig. 2.

Since growth is a process which takes place in time, it can be studied
only by making many growth measurements during the period of incuba-
tion. Such a study is not complete until the phase of autolysis is attained.
Much of the information in the literature is incomplete because growth
was measured only at one time. Many of the potentialities of the fungi
can be discovered only by prolonged observation.

GROWTH

WAYS OF MEASURING GROWTH

The discussion of phases of growth presupposes methods of measuring
growth. In choosing a method of measuring growth, or any other physio-
logical process, the accuracy and type of information desired must be kept
in mind. For some purposes the simplest methods are satisfactory; for
others the most accurate methods should be chosen.

Visual inspection. The simplest way to measure growth is by inspec-
tion and comparison. The value of this method lies in the speed with
which growth measurements are made. Elaborate equipment is not

400

300

• 200

too

4 6 8 10 12 14

Doys of incubation

Fig. 2. Growth of four fungi under the same conditions, in 25 ml. of liquid glucose-
casein hydrolysate medium at 25°C.

needed, as test tubes and Petri dishes are satisfactory culture vessels.
This method has the further advantage that the same cultures may be
kept under observation. It is frequently the method of choice for pre-
liminary experiments, for the very appearance of the mycelium is a clue
to the amount of growth. Growth under varying conditions may be
compared if some condition is used as a standard for comparison (see
Suggested Laboratory Exercises). It is obvious that a great deal of sub-
jective judgment enters into this method of estimating growth, but it is
veiy useful where fine distinctions are not required.

Linear growth. A second widely used method of measuring growth
consists in growing fungi in Petri dishes and measuring either the diameter
or the area of the colony. This is a useful method in some instances but

PHYSIOLOGY OF THE FUNGI

almost useless in others. At least these measurements can be made in an
objective way. In this method, the diameter, radius, or area of a colony
is used to express the amount of growth, while the daily increase repre-
sents the rate of growth. It is obvious that this method neglects the
thickness of the colony. Worley (1939) has proposed to take the thick-
ness of the mycelium into account when growth is measured by this
method. Such measurements are difficult and neglect the mycelium
buried in the agar. The rate of linear growth of some fungi has little
relation to the composition of the medium. The rapid extension of
mycelium on water-agar medium may serve as a familiar example.

It has been frequently assumed that fungi grow at a constant rate when
maintained under constant environmental conditions. This assumption
is not necessarily true, for the growth of Aspergillus rugulosiis and many
other fungi is self-limited under cultural conditions. Two factors may
contribute to cause nonuniform rates of growth: (1) the change in con-
centration of nutrients due to diffusion and utilization; (2) the excretion
of inhibitory metabolic products into the medium.

The same fungus may have a constant rate of growth at one tempera-
ture and not at another. The rate of growth is frequently not constant
when fungi are cultured at temperatures higher than optimum. Fawcett
(1921) found the rate of growth of Phytiacystis citrophthora, Phytophthora
terrestris, Phoviopsis citri, and Diplodia natalensis to decrease with time
when these fungi were cultivated above the optimum temperature. Some
of Fawcett's data which illustrate this phenomenon are given in Table 4.

Table 4. The Effect of Temperature upon the Rate of Growth

OF Three Fungi
The daily increase in the average radius of the colonies is given in milhmeters,
(From the data of Fawcett, Univ. Calif. {Berkeley) Pubs. Agr. Sci. 4, 1921.)

Phytiacystis

Phytophthora

Phomopsis

Days of
incubation

citrophthora

terrestris

citri

23.5°C.

31.0°C.

30.0°C.

35.5°C.

27.5°C.

32.0°C.

5.4

6.3

5.5

4.8

4.6

0.9

10.0

5.5

13.8

4.2

8.0

0.3

10.2

3.5

13.3

2.6

8.0

0.2

10.5

1.5

13.2

2.5

8.5

10.5

0.5

10.9

8.5

If the rate of growth under a given condition does not change with
time, this method is useful and simple. It permits observation of the
same culture for the duration of the experiment. Ryan et al. (1943) have
proposed the use of an ingenious growth tube in which linear growth can

GROWTH 29

be measured with ease and accuracy. This growth tube is illustrated in
Fig. 3.

These authors (Ryan et al, 1943) found the rate of linear growth of
Ncurospora sitophila in such a growth tube to be constant for 200 hr.
The growth-tube method has been used to study the effect of temperature,
pH, vitamin content, and other variables upon Neurospora. These
special tubes have another advantage over Petri dishes in that cultures
are well protected from contamination. The same culture may be
exposed to a variety of environmental conditions such as hght and tem-
perature. These tubes have the disadvantage that it is more difficult to
remove mycelium or fruit bodies for examination. In addition, aeration
may be poor and become a limiting factor for some fungi.

Fig. 3. Growth tube patterned after those described by Ryan, Beadle, and Tatum
{Am. Jour. Botany 30: 784-799, 1943) for measuring linear growth.

Dry weight. By weighing the mycelium and spores produced, an
accurate and objective measure of growth is obtained. For precise work
it is the method of choice. Where any significant weight of spores is pro-
duced, either Gooch or Alundum crucibles may be used to collect both
mycelium and spores. For most purposes the mycelium may be filtered
from the culture medium by use of a finely woven cloth and then trans-
ferred to weighing bottles or small aluminum cups. The excess medium
should be removed by washing and pressing the mycelium, which is then
dried to constant weight at 80 to 100°C. After the mycelium is dry, it is
weighed on an analytical balance. It is usually sufficient to record the
weight to the nearest milligram.

Some fungi make better growth and sporulate more readily on agar
than in liquid medium. It is desirable to have an objective measure of
growth of agar cultures. Fries (1943) and Day and Hervey (1946) have
obtained the dry weight of cultures grown on agar. This technique
should be more widely used. The mycelium is freed from agar by briefly
autoclaving the cultures, filtering off the mycelial mats, and washing with

:36

PHYSIOLOGY OF THE FUNGI

hot water. Frequently the mat can be removed from the melted agar
with a pair of forceps instead of by filtering. Autoclaving removes some
soluble constituents from the mycelium, but if a uniform procedure is
adopted, the results are comparable.

Measuring yeast growth. The growth of yeasts may be measured by
four methods. (1) Yeast cells may be counted in an aliquot of the
medium by the use of a hemocytometer or other counting chamber. The
method is tedious. (2) The volume of yeast cells in a given volume of
medium may be measured in special graduated centrifuge tubes. Yeast

80r

3.1

6.2

0.8 1 .6

iig. fhiomine per culture

Fig. 4. Direct comparison between diameters and dry weights of the same 10-day-
old cultures of Ceratostomella fimbriata in the presence of varying amounts of thiamine.
Cultures were grown in Petri dishes on 25 ml. of glucose-casein hydro lysate agar
at 25°C.

cells are large and easily separated from the medium by centrifuging.
This method is less tedious than counting. (3) Turbidity may be used to
measure the amount of yeast growth. Accurate determinations by this
method require the use of a photoelectric photometer. This method is
rapid and sufficiently accurate for many purposes. Lindegren and Raut
(1947) have cultivated yeasts in colorimeter tubes and have followed the
rate and amount of growth for as long as desired. (4) Yeast cells may be
filtered under vacuum, washed, dried, and weighed. Selas porcelain
crucibles with fritted bottoms are suitable. This method is accurate but
somewhat time-consuming.

Comparison of methods. It should be clearly recognized that one
method of measuring growth may not agree with another. This is illus-

GROWTH 31

trated by Fig. 4, where two methods of measuring the amount of growth
of Ceratostomella fimbriata were used. This figure demonstrates that the
diameter of a colony may be a very poor measure of the amount of growth.
Fries (1943) grew Ophiostoma {Ceratostomella) ulmi on agar medium and
measured the radii of the colonies and also w^eighed the mycelium after
removing the agar. After 5 days the average radius of cultures without
pyridoxine was 16.3 mm., while the average radius of cultures receiving
pyridoxine was 12.3 mm; the weights of mycelium produced under these
two conditions were 5.2 and 18.1 mg., respectively. It is clear from these
examples that different methods of measuring growth do not always give
comparable results. Before valid conclusions can be reached, it is neces-
sary to use valid methods of measuring the quantities involved.

METHODS OF PRESENTING RESULTS

The data obtained in a well-planned and carefully executed experiment
have value in themselves, but more frequently data are a means to an end.
Experimental data form the basis upon which conclusions are reached and
serve as a guide to further investigation. A conclusion is sound only if
the data are sound. To be of greatest value, data must be presented in an
understandable manner. Extensive data may be presented either as
tables or graphs; each method has certain advantages.

Tables. The utility and conciseness of tables make them desirable for
many purposes. Tables are especially suitable in comparing the amount
of growth (or any other function under study) of a number of fungi under
standard conditions or under a number of conditions. They give the
reader the same basic and fundamental information available to the
original investigator. The utility of such information can be appreciated
only when one attempts to assess the reports in the literature.

Derived data, such as ratios or percentages, may be needed for the pur-
poses of interpretation and study, and as such they are entirely proper.
However, the original data from which the derived data were calculated
should always be published. The original data frequently have values
which are not perceived or considered by the original investigator.
Derived data as such afford no clue as to the original magnitudes.
Without the original data no comparison can be made with other experi-
ments, whether in the same or other laboratories. The usefulness of
many publications is severely limited because the author presented only
ratios or percentages instead of the original data. If a datum represents
an average value, the number of determinations upon which it is based
should be stated. It is desirable to indicate the range of variation among
replicates, or the standard deviation should be given if the number of
observations is large.

32 PHYSIOLOGY OF THE FUNGI

Graphs. The significance of data is frequently best appreciated when
presented in graphical form. A graph reminds one that growth is a con-
tinuous function in time, whereas a table may suggest a discontinuous
process. Growth curves are especially suited to illustrate the rate and
amount of growth as a function of time. In Fig. 2 the growth curves of
four fungi illustrate differences among species. Growth curves are
equally applicable to the study of a single species under different condi-
tions. The points representing the data should be given, so that the
reader may see how closely the curve fits the data.

Three-dimensional graphs may be used to represent the relations among
three variables. Three-dimensional graphs take the form of a surface.
Rahn (1939) has given concise directions for constructing such graphs and
models. Schopfer (1943) has used such graphs to represent the growth of
Phycomyces hlakesleeanus with respect to the amount of thiamine and
asparagine in the medium as a function of time of incubation (Fig. 33).

Another way of showing the relations among the variables involves the
use of a triangular graph. Such a presentation is effective if one desires,
for example, to show the effect of the concentrations of three constituents
of a medium upon growth. For examples of the use of triangular graphs
see Haenseler (1921) and Pratt and Hok (1946).

Photographs. The presentation of experimental results is frequently
improved by the judicious use of photographs. Photographs are
particularly useful in comparing the behavior of fungi under different
experimental conditions. The behavior of different species under
identical conditions may be effectively compared by the use of photo-
graphs. Well-labeled photographs also make excellent permanent
records of certain types of experimental results.

FACTORS AFFECTING GROWTH

All the separate factors comprising the internal and external environ-
ment may affect either the rate or the amount of growth, or both. Among
the internal factors are the genetic constitution and the internal modifica-
tions due to age and to the previous external environment. While more
is known about the external factors which affect growth than about the
internal factors, it should always be remembered that the external envi-
ronment acts by modifying the internal environment.

Internal factors. One species differs from another, and even one isolate
of a species may differ from another in genetic composition. Many
mutations have been produced in the laboratory by the action of X rays,
ultraviolet rays, and certain chemicals (see Chaps. 10 and 18). These
mutants of a single species produced in the laboratory differ from the
parent type in one or more biochemical or morphological characteristics
and thus correspond to the different isolates of a species found in nature.

GROWTH 33

There is no reason to suppose that mutants produced in the laboratory-
differ fundamentally from those isolated in nature.

The potentiahties of a fungus are limited by its genetic constitution.
The realization of these potentialities may be denied or favored by the
external environment, and only as the environment is suitable do these
inherent factors find expression. Diversity, rather than uniformity, in
behavior among species and isolates is the rule.

Only a small amount of inoculum is used in most studies. It is impor-
tant to learn if the age, history, or kind of inoculum has any effect on the
subsequent development of the fungus. All these factors may influence
the rate and amount of growth and other functions of the fungi. Young
and vigorously growing inoculum is most suitable, since old cells as a
general rule are slow to start growth. Apparently one of the first func-
tions a cell loses is the power of division. From this standpoint such cells
are "dead," although they may be still capable of performing many vital
functions, such as respiration. Difficulty is frequently experienced in
making subcultures from old cultures. Certain species are difficult to
maintain in culture unless they are frequently subcultured. In general,
these species do not readily form resting cells. Among these are various
species of Pythium and Phytophthora, test-tube cultures of Choanephora
cucurhitarum, and others.

In experimental work of the highest precision neither the temperature
nor the medium upon which the inoculum is grown may be neglected.
Zikes (1919) investigated the generation time of six strains of yeast and
found that the storage temperature of the inoculum affected the time
required for cell division. These original cultures were grown at 8°C. and
25°C., and subcultures were incubated over a range of temperatures.
When the inoculum which was grown and stored at 8°C. was subcultured
at low temperatures, the generation time was less than that of the culture
grown and stored at 25°C. At temperatures above 25°C. the generation
time of the high-temperature yeast was less than that of the low-tempera-
ture yeast. In some way, yeast cells cultured over long periods of time
at a certain temperature become adapted to this temperature, and when
such cells are transferred to other temperatures, the influence of the
original temperature of incubation persists for a time. It is evident that
some change in the internal environment has occurred.

Comparable studies on the filamentous fungi are rare. From Fawcett's
data on the rate of linear growth of four citrus pathogens it appears that
the same phenomenon takes place with some filamentous fungi. Fawcett
grew the inoculum at 20°C., and on subculturing at 7.5°C. the linear rate
of growth increased with time, as is shown in Table 5.

Many fungi have latent abilities to synthesize various essential metab-
olites. In the virtual absence of these compounds in the medium and

PHYSIOLOGY OF THE FUNGI

after a shorter or longer period of incubation, a fungus may begin to
synthesize these essential metabolites, and growth then takes place in a
normal way. This is especially true of the yeasts with respect to vitamins.
Many fungi lose their pathogenicity Avhen cultured for a long time on
laboratory medium. Host passage frequently restores pathogenicity.
The indiscriminate use of inoculum from a variety of substrates and of
different ages may introduce unexpected variation in experimental work
and should be guarded against.

Table 5. Daily Increase (in Millimeters) in Diameter of Colonies

OF Four Fungi
Inoculum grown at 20°C.; subcultures incubated at 7.5°C. (From the data of
Fawcett, Univ. Calij. {Berkeley) Pubs. Agr. Sci. i, 1921.)

Species

Phythiacystis citrophthora
Phytophthora terrestris . . .

Phomopsis citri

Diplodia natalensis

1st
day

0.04
0.02
0.01
0.05

2d
day

0.4
0.14
0.16
1.9

3d
day

0.6
0.21
0.83
2.1

4th
day

0.8
0.7
0.9

5th
day

1.2
0.8
1.0

External factors. Among the external factors which influence the
growth of fungi, temperature plays an extremely important role. Tem-
perature affects almost every function of the fungi. For each fungus
there is a temperature below which it will not grow, the minimum tem-
perature. Likewise there is a temperature above which growth ceases,
the maximum temperature. These two temperatures indicate the tem-
perature range of an organism. A few fungi are capable of growing below
0°C., but for most species the minimum temperature is 0 to 5°C. The
maximum temperature varies from 27°C. ior Phacidium infestans (Pehrson,
1948) and Sclerotinia cameUiae (Barnett and Lilly, 1948) to 45 or 50°C. for
Aspergillus fumigatus (Thom and Raper, 1945). The maximum tempera-

Table 6. Cardinal Temperatures for Various

Fungi

Species

Minimum,

Optimum,
°C.

Maximum,
°C.

Citation

Neurospora sitophila

Ceratostomella pilifera

C. ips

-3

12.0
0.5

36
25-30

18-21

31.5

25-30

36.1

Ryan et al, 1943
Lindgren, 1942
Lindgren, 1942

Phacidium infestans

Phytophthora infestans

P. terrestris

Pehrson, 1948
Crosier, 1933
Fawcett, 1921

Various yeasts

Zikes, 1919

GROWTH 35

ture is sometimes an important factor limiting the attack of plant
pathogens.

The cardinal temperatures of a few fungi are given in Table 6. A more
extensive compilation is given by Wolf and Wolf (1947). The character-
istic effect of different temperatures on the rate of growth of two fungi is
shown in Fig. 5. Further examples may be found in the work of Lindgren
(1942).

Most reports on the effect of light on the fungi have been concerned
with reproduction rather than vegetative growth. However, Elfving
(1890) found strong diffuse daylight to depress the growth of Penicillium
glaucum and a species of Briarea. The amount of inhibition was least
when the culture medium contained complex nutrients such as peptone.
Greater inhibition resulted when the media contained glucose, mannitol,
and malic acid. Scattered observations indicate that the depressing
effect of strong light may be rather common. In the old literature some
mention is made of the favorable effect of light on red yeasts. The
sporangiophores of Phycomyces hlakesleeanus attain a greater length in
darkness than in intense light. The role of light in the sporulation of
some fungi is discussed in Chap. 14.

Conclusive evidence that light affects the amount of growth of Karlingia
(Rhizophijlctis) rosea, one of the lower Chytridiales, was presented by
Haskins and Weston (1950). This fungus when grown in liquid glucose-
nitrate medium produced twice the amount of dry weight of cells when
cultured in light than when the cultures were kept in total darkness.
With the exception of the factor of illumination, the experimental con-
ditions were the same. Approximately twice as much glucose was
utilized by cultures exposed to light as those kept in darkness. On the
other hand, when K. rosea was grown in a liquid cellobiose-nitrate
medium, more growth resulted in total darkness than in light. The
explanation for this behavior of K. rosea is not known.

The moisture requirements of fungi differ. Most species in nature live
on substrates which are not saturated with water. The low moisture
content of a substrate is often a factor which limits the growth of fungi.
Particularly is this true of the species which live on wood or in soil. As a
general rule, wood which contains less than 20 per cent moisture is immune
to fungus decay. A difference of a few per cent in the moisture content
may determine whether a species will be able to grow or not. Lindgren
(1942) has reported that Ceratostomella pilifera, a wood-staining fungus,
does not grow in pine wood having a moisture content of 23 per cent but
develops in wood containing 24.5 per cent moisture. The maximum rate
of penetration was attained on wood having a moisture content of 29 per
cent or more. Jute sacking is subject to fungus attack only if the mois-
ture content exceeds 17 per cent.

PHYSIOLOGY OF THE FUNGI

In physiological studies dealing with high concentrations of nutrients,
it is important to distinguish between osmosis and osmotic pressure.
Osmosis is the transfer of water through a membrane permeable to water

160

120

^80

i"^"^ ^^^

Degrees centigrade

10 20 30

Temperature in degrees centigrade

Fig. 5. A, the effect of temperature on the dry weight of mycelium produced by
Glomerella cingulata after 5 days in 25 ml. of liquid glucose-asparagine medium.
(Drawn from the data of I. G. Bennett, 1951.) B, the effect of temperature on the
rate of linear growth of Neurospora crassa. (Courtesy of Ryan, Beadle, and Tatum,
Am. Jour. Botany 30 : 785, 1943.)

but not to the solute molecules. In simple systems water passes from a
dilute to a more concentrated solution. Osmotic pressure is the force
necessary to restrain the movement of water from a dilute to a concen-
trated solution through a semipermeable membrane. The osmotic pres-
sure which a solution is capable of developing is a function of the number

GROWTH

of ions and molecules of solute contained in a unit \'olume of solution. A
mole of a non-ionized compound in 1,000 g. of water at 0°C. has an
osmotic pressure of 22.4 atm. if separated from pure water by a semiper-
meable membrane. For a fuller discussion of osmosis and osmotic pres-
sure the student is referred to Gortner (1949), Seifriz (1936), and Meyer
and Anderson (1948).

If concentration were the sole factor which determines whether growth
is possible, all solutions having the same osmotic pressure would be
equally inhibitory. Table 7 indicates that this is not true.

Table 7. Highest Osmotic Pressures (Atmospheres) op Solutions of Four
Compounds in Which Various Fungi Grew
(Hawkins, Jour. Agr. Research 7, 1916.)

Species

Glucose*

Sucrose

Potassium
nitrate

Calcium
nitrate

Plenodomus destruens

Diplodia tubericola

Rhizopus nigricans

Botrytis cinerea ....

58.3
63.2
63.2
63.2
63.2

47.4
42.1
42.1
47.4
47.4

54.5
58.8
27.5
54.5
54.5

33.6
33.6

15.9
27.7

Ceratoslomella fimhriata

19.5

* Limiting concentrations not used.

These data and others show that the limiting osmotic pressure depends
upon the fungus and the compounds used. It is difficult to evaluate the
effects of osmotic pressure upon the fungi, for the cell membrane is per-
meable to other compounds in addition to water. Calculations of osmotic
pressure are made by assuming that an indifferent semipermeable mem-
brane separates solutions of different concentrations. The effect of
osmotic pressure upon the fungi cannot be considered as a simple physio-
chemical process. However, the ability of many fungi to grow in solu-
tions having high osmotic pressures is advantageous. Parasitic fungi
characteristically have a higher osmotic pressure than the cell sap of the
plants they parasitize (Thatcher, 1939). For further references to the
effect of osmotic pressure on fungi, see Kroemer and Krumbholz (1931).

Another process involved in the entrance of water into fungus cells is
imbibition. Gortner (1949) has defined imbibition as the process whereby
colloidal substances such as protoplasm take up water, and imbibition
pressure as the pressure against which a colloid will imbibe liquid.
Raciborski (1905) grew a species of Torula in saturated lithium chloride
(1,000 atm.) and Aspergillus glaucus in a saturated sodium chloride
solution.

Aside from osmotic effects, the concentration of the medium has a great
effect on the rate and amount of growth of fungi. The concentration of

PHYSIOLOGY OF THE FUNGI

nutrients which is most favorable for growth may be poor in other
respects, e.g., for reproduction. The concentration may be varied in two
ways: (1) by dihiting the entire medium, whereby the ratios among the
constituents remain unchanged, and (2) by varying the concentration of
one constituent. These methods are not equivalent and yield different
results.

When an entire medium is diluted, it might be expected that the de-
crease in amount of mycelium produced would be directly proportional
to the amount of dilution. Such is not always the case. When Chae-
tomium convolutum was grown in full-strength medium and in medium
diluted to one-fourth and one-sixteenth full strength, the maximum
weights of mycelium produced were 220, 75, and 22 mg., respectively
(Lilly and Barnett, 1949). C convolutum grew most efficiently in the
most dilute medium. This principle appears to be generally valid and is
also illustrated by Ceratostomella fimbriata (Table 57) .

Table 8.

The Effect of Different Volumes of Medium upon the Rate and
Maximum Amount of Growth of Sordaria fimicola
Dry weight of mycelium in milligrams.

Days of

Ml. medium per 250-ml. Erlenmeyer flask

incubation

6.25

12.5

25.0

50.0

129

113

166

160

100

156

238

107

168

269

When the concentration of one constituent in the medium is changed,
over a certain range, the amount of growth will be proportional to the
concentration. Above a certain concentration there will be no further
increase in the amount of growth. This is due to the limiting concentra-
tion of some other constituent in the medium. This is the principle upon
which fungi are used in vitamin and other assays (Chap. 10).

The maximum weight of mycelium which is obtained from a given vol-
ume of medium depends upon the type and size of the culture vessels used.
The rate of growth is also affected. These results appear to be due
mainly to differences in aeration, and perhaps to a lesser degree to diffu-
sion. The effect of depth of medium on rate and amount of growth in non-
agitated cultures may be demonstrated by using a constant volume of
medium in different-sized flasks, or by varying the volume of medium in
flasks of the same size. Data illustrating this latter condition are pre-
sented in Table 8. The slow initial rate of growth when the mycelium is

GROWTH 39

entirely submerged is due to lack of an adequate supply of oxygen. The
efficiency of Sordaria fimicola in converting the constituents of the
medium into mycelium decreased as the depth of the medium increased.
This fungus was less than half as efficient when grown in 50 ml. of medium
as when grown in 6.25 ml.

EFFECT OF EXTERNAL FACTORS ON MORPHOLOGY

While the study of morphology, as such, is not within the province of
physiology, there is a close connection between these two aspects of
mycology. Form and function are the two ways in which the poten-
tialities of organisms come to expression. The morphology of a fungus
may be modified by environmental factors to such a degree as to be
unrecognizable. These changes in morphology may be microscopic as
well as grossly visible.

Pasteur (1879) noted that species of Mucor, when grown submerged in
liquid and in the absence of air, assumed a yeast-like form. Not only did
they resemble yeasts, but under these conditions they fermented sugar to
alcohol. Under aerobic conditions no detectable amounts of alcohol were
formed. Reproductions of Pasteur's drawings have been published by
Foster (1949).

When yeasts are cultured in liquid media and allowed to age undis-
turbed, a film or membrane frequently covers the surface of the liquid.
Film formation frequently starts as a ring of cells on the wall of the flask
at the air-liquid interface. The morphology of the yeast cells in such
films is unusual in that the cells are joined together in filaments. The
supply of oxygen must play an important role in the formation of fila-
ments. The temperature range within which film formation occurs varies
with the species of yeast and is usually considerably less than the tempera-
ture range for growth. Most species of yeasts forms films only between
6 and 30°C., although Zikes (1919) found Monilia Candida and Mycoderma
cerevisiae to form films at 37°C. The early literature on this subject has
been summarized by La Far (1911).

Nickerson and Van Rij (1949) have reviewed the mechanisms of fila-
ment formation in yeast and conclude that the processes of cell elongation
and cell division are controlled by different enzyme systems. Appar-
ently, the sulfhydryl enzymes which regulate the process of cell division
may be inhibited without greatly interfering with cell elongation. Among
the agents which inhibit cell division are cobalt, iodoacetate, and peni-
cillin. The effect of penicillin on Saccharomyces cerevisiae is shown in
Fig. 6. Camphor and other narcotizing agents produce somewhat the
same changes in morphology of yeast cells (Levan, 1947).

Many pathogenic fungi which cause disease in man are dimorphic.
These fungi are usually yeast-like in the host but frequently form myce-

PHYSIOLOGY OF THE FUNGI

Fig. 6. Saccharomyces cerevisine, camera lucida drawings of cells from agar cultures.
A, culture treated with penicillin; B, culture treated with penicillin plus cysteine.
(Courtesy of Nickerson and Van Rij, Biochim. et Biophijs. Acta 3: 461-475, 1949.
Published by permission of Elsevier Book Company, Inc.)

\ . \*'

i/^/\ ""

A B

Fig. 7. The effect of hydrogen-ion concentration on the morphology of cells of
Sordaria fimicola. A, rounded swollen cells produced in glucose-casein hydrolysate
medium at initial pH 3.6. B, normal mycelium from the same culture a few days
after a drop of NaOH was added.

Hum in culture. Blastomyces dermatitidis and B. hrasiliensis exhibit
thermal dimorphi.sm (Nickerson and Edwards, 1949). When these fungi
are cultured on certain media at 37°C., they are yeast-like, while at lower
temperatures of incubation they form mycelium. This change in mor-
phology is accompanied by changes in the rate of respiration and type of

GROWTH

Fig. 8. The effect of environment on the morphology of fruit bodies of Forties
applanatus. A, normal fruit body developed in nature; B, C, malformed fruit bodies
of the same (?) fungus developed under water in abandoned coal mines. The "nodes "
in B are believed to be caused by different water levels.

42 PHYSIOLOGY OF THE FUNGI

metabolism. Chemical agents may favor or prevent similar morphologi-
cal changes. Trichophyton ruhrum produces two metabolic products of
unknown constitution which inhibit the transformation of Candida
albicans to the mycelial form (Jillson and Nickerson, 1948). The addi-
tion of excessive amounts of inositol to the culture medium causes
Ophiostoma (Ceratostomella) muUiannulatum to grow almost entirely in
the form of conidia (Fries, 1949). The morphology of the vegetative
mycelium and sporangia of various species of Phytophthora was found to
depend upon the medium used (Leonian, 1925).

The form of mycelial growth of many species, when grown on agar
media, is an aid in identification. The colony form may be altered
beyond recognition when cultures are grown in agitated liquid medium.
In general, spherical colonies or balls form in agitated medium. Burk-
holder and Sinnott (1945) investigated colony form of a large number of
species when subjected to agitation.

The acidity of the medium affects the size and shape of the vegetative
cells of some fungi. In a medium so acid as to allow only very slow
growth the cells often become swollen or nearly spherical in shape, much
like chlamydospores, but the wall remains thin (Fig. 7). This may be
accompanied by excessive branching.

Unusual environmental conditions often affect the morphology of both
vegetative and reproductive structures. The environment which exists
in coal mines is unnaturally uniform with respect to temperature, mois-
ture,'and absence of light. Basidiomycetes growing on old mine timbers
either fail to fruit or produce odd-shaped sterile fruit bodies (Fig. 8).

SUMMARY

Normal growth results in an increase in cell number and mass. Limited
growth may result from either of these two processes alone. Growth is a
phenomenon which requires time for its various manifestations. Growth
follows a pattern which differs from species to species, but the general
sequence of phases is much the same for all fungi. Growth studies are
based upon measuring both the amount and the rate of growth. The rate
and amount of growth are controlled by the internal and external environ-
ment. The potentialities of a fungus are limited by its genetic constitu-
tion, but the expression of these potentialities is controlled by external
factors such as temperature, light, composition, and concentration of the
medium. Even the size and shape of the culture vessels used affect the
rate and amount of growth.

The amount of growth can be estimated by visual comparison or meas-
ured by determining the diameter of a colony or by harvesting the myce-
lium and weighing it after drying to constant weight. The amount of
yeast growth may be measured by counting the numbers of cells produced,

GROWTH 43

by centrifuging and measuring the volume of cells, by turbidity, or by
weighing. The most direct way of measuring growth of either yeast or
filamentous fungi is by weighing the crop produced. The various meth-
ods of measuring growth are not strictly comparable.

The morphology of a fungus may be changed by environmental factors
so that it becomes unrecognizable. The processes of cell elongation and
cell division are controlled by different enzyme systems. In some
instances it has been possible to inhibit cell division without interrupting
cell elongation. Frequently a change in physiology accompanies a
change in morphology.

REFERENCES

*Barnett, H. L., and V. G. Lilly: The interrelated effects of vitamins, temperature,
and pH upon vegetative growth of Sclerotinia camelliae, At7i. Jour. Botany 35:
297-302, 1948.

Bennett, I. G.: Thesis, West Virginia University, 1951.

Buchanan, R. E., and E. I. Fulmer: Physiology and Biochemistry of Bacteria,
Vol. I, The Williams & Wilkins Company, Baltimore, 1928.

Burkholder, p. R., and E. W. Sinnott: Morphogenesis of fungus colonies in sub-
merged shaken culture. Am. Jour. Botany 32: 424-431, 1945.

Crosier, W.: Studies in the biology of Phytophthora infestans (Mont.) de Bary,
Cornell Univ. Agr. Expt. Sta. Mem. 155, 1933.

Day, D., and A. Hervey: Phycomyces in the assay of thiamine in agar. Plant
Physiol. 21 : 233-236, 1946.

Elfving, F.: Studien iiber die Einwirkung des Lichtes auf die Pilze, Helsingfors
Central-Druckerei, Helsingfors, 1890.

Emerson, S.: The growth phase in Neurospora corresponding to the logarithmic
phase in unicellular organisms, Jour. Bad. 60: 221-223, 1950.
*Fawcett, H. S. : The temperature relations of growth in certain parasitic fungi,
Univ. Calif. (Berkeley) Pubs. Agr. Sci. 4: 183-232, 1921.

Foster, J. W.: Chemical Activities of Fungi, Academic Press, Inc., New York, 1949.

Fries, N.: Die Einwirkung von Adermin, Aneurin und Biotin auf das Wachstum
einiger Ascomyceten. Symbolae Botan. Upsalienses 7(2): 1-73, 1943.

Fries, N.: Ophiostoma multiannulatum (Hedge, and Davids) as a test object for the
determination of pyridoxin and various nucleotide constituents. Arkiv fiir
Botanik 1: 271-287, 1949.

Gortner, R. a.: Outlines of Biochemistry, 3d ed., John Wiley & Sons, Inc., New
York, 1949.

Haenseler, C. M.: The effect of salt proportions and concentrations on the growth
of Aspergillus niger, Am. Jour. Botany 8: 147-163, 1921.
*Haskins, R. H., and W. H. Weston, Jr.: Studies in the lower Chytridiales. I. Fac-
tors affecting pigmentation, growth, and metabolism of a strain of Karlingia
(Rhizophlyctis) rosea, Am. Jour. Botany 37 : 739-750, 1950.

Hawkins, L. A.: Growth of parasitic fungi in concentrated solutions, Jour. Agr.
Research!: 255-260, 1916.

JiLLSON, O. F., and W. J. Nickerson: Mutual antagonism between pathogenic
fungi. Inhibition of dimorphism in Candida albicans, Mycologia 40: 369-385,
1948.

Kroemer, K., and G. Krumbholz: Untersuchung iiber Osmophile Sprosspilze.
I. Mitteilung, Beitrage zur Kenntniss der Garungsvorgange und der Garungs-
erreger der Trockenbeerenauslesen, Arch. Mikrobiol. 2: 352-410, 1931.

44 PHYSIOLOGY OF THE FUNGI

La Far, F.: Tochnical Mycology. Vol. II, Eumycetic Fermentation (trans.

C. T. C. Salter), Chas. Griffin & Co., Ltd., London, 1911.
Leonian, L. H.: Physiological studies on the genus Phytoyhthora, Am. Jour. Botany

12:444-498, 1925.
Lev AN, A.: Studies on the camphor reaction of yeast, Heredilas 33: 457-514, 1947.
Lilly, V. G., and H. L. Barnett: The influence of concentrations of nutrients,

thiamin, and biotin upon growth and formation of perithecia and ascospores by

Chaetomium convolutum, Mycologia 41 : 186-196, 1949.
LiNDEGREN, C. C, and C. Raut: The effect of the medium on apparent vitamin-
synthesizing deficiencies of microorganisms. A direct relationship between

pantothenate concentration and the time required to induce the production of

pantothenate-synthesizing "mutants" in yeasts, Ann. Missouri Botan. Garden

34 : 75-90, 1947.
LiNDGREN, R. M. : Temperature, moisture, and penetration studies of wood-staining

Ceratostomellae in relation to their control, U.S. Dept. Agr. Tech. Bull. 807,

1942.
Meyer, B. S., and D. B. Anderson: Plant Physiology, D. Van Nostrand Company,

Inc., New York, 1948.
NiCKERSON, W. J., and G. A. Edwards: Studies on the physiological bases of

morphogenesis. I. The respiratory metabolism of dimorphic pathogenic fungi,

Jour. Gen. Physiol. 33: 41-55, 1949.
NiCKERSON, W. J., and N. J. W. van Rij: The effect of sulfhydryl compounds,

penicillin, and cobalt on the cell division mechanism of yeasts, Biochim. et

Biophys. Acta 3: 461-475, 1949.
*Pasteur, L.: Studies on Fermentation. The Diseases of Beer, Their Causes, and

the Means of Preventing Them (trans, from Etudes sur la biere by F. Faulkner

and D. C. Robb.), Macmillan & Co., Ltd., London, 1879.
Pehrson, S. O.: Studies of the growth physiology of Phacidium infestans Karst.,

Physiologia Plantarum. 1 : 38-56, 1948.
Pratt, R., and K. A. Hok: Influence of the proportions of KH2PO4, MgS04, and

NaNOs in the nutrient solution on the production of penicillin in submerged

cultures, Am. Jour. Botany 33: 149-156, 1946.
Raciborski, M.: Ueber die obere Grenze des osmotischen Druckes der lebenden

Zelle, Bull, internat. acad. sci. Cracovie, CI. sci. math, et nat. 7: 461-471, 1905.
Rahn, 0.: Physiology of Bacteria, The Blakiston Company, Philadelphia, 1932.
Rahn, O.: Mathematics in Bacteriology, Burgess Publishing Co., Minneapolis, 1939.
Ryan, F. J., G. W. Beadle, and E. L. Tatum: The tube method of measuring the

growth rate of Neurospora, Atn. Jour. Botany 30: 784r-799, 1943.
ScHOPFER, W. H.: Plants and Vitamins, Chronica Botanica Co., Waltham, 1943.
Seifriz, W.: Protoplasm, McGraw-Hill Book Company, Inc., New York, 1936.
*Thatcher, F. S.: Osmotic and permeability relations in the nutrition of fungus

parasites. Am. Jour. Botany 26: 449-458, 1939.
Thom, C, and K. B. Raper: A Manual of the Aspergilli, The Williams & Wilkins

Company, Baltimore, 1945.
Wolf, F. A., and F. T. Wolf: The Fungi, Vol. II, John Wiley & Sons, Inc., New

York, 1947.
WoRLEY, C. L.: Interpretation of comparative growths of fungal colonies on different

solid substrata. Plant Physiol. 14 : 589-593, 1939.
ZiKEs, H.: Ueber den Einfluss der Temperature auf verschiedene Funktionen der

Hefe, Cent. Bakt., Abt. II, 49: 353-373, 1919.

CHAPTER 4
ENZYMES AND ENZYME ACTION

The fungi, in common with other hving organisms, possess tools or
reagents far more specific, more deHcate, and more powerful than those
available in the laboratory. The most complex natural substances such
as proteins, polysaccharides, and Hpoids are degraded into simpler com-
pounds which are soluble in water. Fungi also synthesize similar com-
plex compounds from relatively simple molecules. These transformations
are carried out under such mild conditions of temperature and pressure
and in such low concentrations of acid and alkali that it is certain the
means used are of a peculiar kind. For in the absence of these special
agents formed by the living organisms, these reactions do not take place
or do so at a very slow rate. These organic catalysts produced by living
organisms are called enzymes. The life processes of organisms are con-
trolled and directed by a complicated and interrelated series of enzymes
or enzyme systems (Dixon, 1949).

Some enzymes formed by fungi are excreted and normally perform their
functions outside the cells that produce them. These are termed exo-
enzymes (extracellular enzymes), such as cellulase, amylase, and pectinase.
Exoenzymes perform the functions of digestion; i.e., the degradation of
complex food materials into low-molecular-weight compounds which are
able to enter the cell. After entering the cell, these metabolites are acted
upon by the enzymes within the cell. These enzymes are called endo-
enzymes (intracellular enzymes).

Naturally enough, exoenzymes were recognized and studied first. In
the early literature, these exoenzymes were called unorganized ferments
because of their solubility. In contrast to these unorganized ferments it
was recognized that other ferments (enzymes) occurred in an insoluble
organized form. These were called organized ferments. Pasteur (1875)
still spoke of yeast as "ferment alcoohque ordinaire du vin." Thus, the
name organized ferment took on a dual meaning, that of a living organism
and the various chemical reactions caused by these organisms. In 1878
Kiihne suggested that the word enzyme be used to replace the terms
organized and unorganized ferments. Enzyme is derived from the Greek
phrase, en zyme, which means in yeast or leaven. For excellent sum-
maries of the historical development of the relation between fermentation

46 PHYSIOLOGY OF THE FUNGI

and the action of microorganisms, see Stephenson (1939) and Harden
(1932).

It was not until late in the nineteenth century that Buchner (1897)
succeeded in releasing certain enzymes from yeast cells and demonstrating
that the endoenzyme(s) in yeast causing fermentation was also active
entirely apart from the living yeast cells. While yeast juice prepared
according to the method of Buchner contained a variety of enzymes, it
contained fermentative enzymes never before obtained apart from the
living cell. These enzymes cleaved sugar into alcohol and carbon dioxide.
This was truly a monumental step in the science of enzymes, for it
afforded a way of studying "life" processes apart from the terrible com-
plexity of the living organism. The study of isolated enzyme systems
has led to important advances in our knowledge and understanding of
life processes; yet the student should be reminded that life is more com-
plex than its parts. Leibowitz and Hestrin (1945) say:

... it has become clear that the risk involved in translating results from lifeless
to living systems is a two-way one: not only may mechanisms which operate
in vivo be absent in vitro; mechanisms may be present in vitro and yet not neces-
sarily function in vivo. In fermentative physiology, as in biology generally,
selective and restrictive activity by the living organism must always be taken into
account.

The rate of many chemical reactions is changed by the presence of
traces of substances which do not appear to enter into permanent chemi-
cal combination with the reactants and which appear unchanged when
the reaction has come to equilibrium. Substances which alter the rates
of chemical reactions are called catalysts, and the process catalysis.
Enzymes are catalysts of a very special kind, and many of them catalyze
but a single reaction. For example, lactose reacts with water to form
glucose and galactose. Unless a catalyst is present, this reaction occurs
at a very slow rate. Even at 100°C. a long time is required for an appreci-
able amount of lactose to react with water. If, however, some acid is
added to the lactose solution, the rate of the reaction is greatly increased,
varying in degree with the amount and kind of acid used. This same
reaction is catalyzed w^hen the enzyme, lactase, produced by some yeasts
and certain other fungi, is added to a solution of lactose. In general,
enzymes are specific catalysts. There is no stoichometric relation
between the amount of catalyst (acid or enzyme) and the amount of sub-
strate decomposed. Within limits, the amount of substrate decomposed
per unit of time is dependent upon the amount of catalyst present.

For a given set of conditions there is a position of equilibrium where the
rate of reaction of the reactants is equal and opposite to the rate of com-
bination of the products. The position of equilibrium is not changed by

ENZYMES 47

the presence of a catalyst. The same catalyst will effect synthesis as well
as decomposition; the position of equilibrium as well as the relative con-
centrations of reactants and products determines which reaction pre-
dominates. It is possible to choose conditions, in some instances, so the
equilibrium conditions favor synthesis. Bourquelot (1915) demonstrated
a-methylglucoside was readily formed from methyl alcohol and glucose in
the presence of yeast juice.

CLASSIFICATION OF ENZYMES

It is more important to classify enzymes upon the basis of function
rather than the site of action (endo- and exoenzymes). Many enzymes
catalyze reactions in which water is either a product (synthesis) or a
reactant (degradation). These enzymes are called hydrolases. These
reactions usually involve only moderate energy changes. Another class
of enzymes, usually intracellular, catalyze oxidation and reduction reac-
tions and reactions involving the scission (or formation) of carbon-to-car-
bon linkages. These enzymes are known as desmolyzing enzymes and
include oxidases, dehydrogenases, and desmolases. Energy changes
involved in these reactions are usually large. For more detailed classifi-
cations of enzymes see Gortner (1949) and Sumner and Somers (1947).

Since an enzyme acts upon a restricted number of compounds, it is
convenient to name enzymes with reference to the substrate acted upon.
In general, enzymes are named either by adding the suffix -ase to the
name of the substrate or by replacing the final syllable of the name of the
substrate by this suffix. The following examples give the substrate fol-
lowed by the name of the enzyme: maltose, maltase; lactose, lactase;
cellulose, cellulase; starch (amylum), amylase; protein, proteinase; pectin,
pectinase. The suffix -ase is also used to designate classes of enzymes.
Thus, esterases are members of that group of enzymes which catalyze the
hydrolysis and synthesis of esters; oxidases are enzymes which activate
oxygen, and dehydrogenases are enzymes which activate the hydrogen of
various metabolites. An enzyme may have several names. The enzyme
which catalyzes the hydrolysis of sucrose is known also as saccharase and
invertase. Amylase is also called diastase.

Hydrolases. The hydrolases catalyze a wide variety of reactions in
which water is either a reactant or a product. Hydrolysis is generally
thought of as a process whereby complex molecules react with water to
form simpler substances. Many hydrolases are exoenzymes which func-
tion by preparing the substrate for assimilation. Among these the follow-
ing should be noted : cellulase, amylase, pectinase, various disaccharidases,
proteinases, and peptidases. Others are endoenzymes (the same enzymes
in some instances), which catalyze the same or similar reactions within the
cells. It would be expected that the process of synthesis within the cells

PHYSIOLOGY OF THE FUXGI

would be of much more common occurrence than outside the cells. In the
medium the process of degradation may be expected to go more or less to
completion, since the soluble products of the reaction are assimilated by
the organism and hence equilibrium is not reached. Within the cell,
however, the reverse may be true. Here, the products of hydrolysis may
accumulate, a situation which would tend to favor the reverse reaction, or
synthesis. Therefore, synthesis within the cell would be expected to
occur when a plentiful supply of simple metabolite molecules continue to
reach the cell. When few, if any, metabolite molecules are entering the
cell, the hydrolysis of reserve materials would take place. These prod-
ucts of hydrolysis within the cell are then used in other metabolic processes
until the store of reserve material is exhausted. Some of these functions
are illustrated in scheme I.

Scheme I. General Scheme of Starch Utilization
Outside the cell

Starch

->- Maltose

*- Glucose

amylase ^^■^^ ^-^^ maltase

Fungus cells

many enzymes

Carbon dioxide,
alcohol and other
products of anaerobic
respiration

Within the cell

Glucose -<-

many enzymes

Carbon dioxide,
water and other
products of aerobic
respiration

Glycogen, or
other storage
products

Esterases. These enzymes catalyze the hydrolysis of esters, an acid
and an alcohol being formed. The most important natural esters are the
fats, which are the glycerol esters of the long-chain fatty acids. Enzymes
which catalyze the hydrolysis of fats are called lipases. Both exo- and

ENZYMES 49

endolipases are known. Many fungi store fat as reserve material, and
presumably the first step in utilization is hydrolysis.

Phosphatases are classified as esterases because of the fact that they
catalyze the hydrolysis of esters of phosphoric acid. Phosphorus is an
essential element which enters into many metabolic processes and is a
constituent of many physiologically important compounds. Many
coenzymes are esters of pyrophosphoric acid (thiamine pyrophosphate,
and diphosphopyridine nucleotide, DPN), while triphosphoric acid is a
constituent of triphosphopyridine nucleotide, TPN. The synthetic
capacity of the phosphatases has been rarely demonstrated. Other
enzymes, phosphorylases, are apparently the catalytic agents active in
forming many phosphate esters. In many instances the substrates from
which these esters are formed are different from the products of phos-
phatase hydrolysis.

Carbohydrases. The enzymes which catalyze the hydrolysis of com-
plex carbohydrates, or polysaccharides, are called carbohydrases. These
enzymes appear to be highly specific ; thus each of the common disaccha-
rides requires a different enzyme for hydrolysis. Sucrase is found in
many fungi, including the common strains of Saccharomyces cerevisiae,
although it is apparently absent in Schizosaccharomyces octosporus. The
enzyme which hydrolyzes maltose to glucose is called maltase. Maltase
is very widely distributed among the fungi. The enzyme which catalyzes
the hydrolysis of lactose to glucose and galactose is called lactase. While
this enzyme is less widely distributed among the fungi than sucrase and
maltase, it is produced by many species.

While it is doubtless correct to assume that the more complex and in-
soluble carbohydrates must be hiydrolyzed before utilization, this assump-
tion may, in some instances, be false with regard to the disaccharides. It
is possible that some fungi may employ a phosphorylative degradation of
the disaccharides rather than hydrolysis. For a critical review of carbo-
hydrate utilization without preliminary hydrolysis, see Hestrin (1948).

In addition to the water-soluble polysaccharides there is a wide variety
of water-insoluble high-molecular-weight carbohydrates which are utilized
by many fungi as carbon sources. Only two of these complex polysaccha-
rides will be considered here. The empirical formula for cellulose is
(C6Hio05)„. On complete hydrolysis by acids, glucose is the only prod-
uct. Less complete hydrolysis produces a disaccharide known as cello-
biose. The majority of fungi, according to Norman and Fuller (1942),
are able to attack cellulose. The early work is reviewed by Thaysen and
Bunker (1927). With respect to the fungi which attack cellulose, a great
deal of variation in cellulolytic ability is found (see White et al., 1948).
The enzyme which catalyzes the hydrolysis of cellulose is called cellulase.

While starch has the same empirical formula as cellulose, it is more

50 PHYSIOLOGY OF THE FUNGI

easily hydrolyzed. Glucose is likewise the end product of hydrolysis.
The enzyme (or enzymes) which catalyzes the hydrolysis of starch is
called amylase. In general, the end product of enzymatic hydrolysis of
starch is maltose and glucose. The various intermediate degradation
products are called dextrins.

Starch appears to be composed of two main types of compounds:
amylose (20 to 25 per cent) and amylopectin. Amylose appears to con-
sist of long, unbranched molecules containing some 300 glucose residues,
whereas amylopectin has a branched structure. There are two types of
amylase: jS-amylase, which hydrolyzes off two glucose residues at a time
to form maltose, and oi-amylase, which attacks the 1,4-glucosidic linkages
in such a way as to produce starch fragments (dextrins) as the primary
products. The dextrins are further hydrolyzed to form maltose and some
glucose. The primary function of a-amylase is thus liquefaction; that of
the /3-amylase is saccharification. The Aspergillus amylases are of the
alpha type. The student is referred to the excellent reviews of Hopkins
(1946) and Myrback (1948) for critical summaries of amylase activity.
Amylase is widely distributed among the fungi but is not universal.

Pectinase. The pectins are colloidal carbohydrate-like compounds
found in fruits and in the middle lamellae of plants. Many fungi produce
pectinase, which catalyzes the hydrolysis of pectin. When the pectin is
hydrolyzed, the cells fall apart. Harter and Weimer (1921) tested the
ability of nine species of Rhizoyus to produce pectinase in culture but were
unable to correlate the pathenogenicity of these species with the amount
of pectinase secreted. In fact, some of the pathogenic species {R.
nigricans and R. autocarpi) secreted less pectinase than did two non-
pathogenic species {R. chinensis and R. microsporus) .

Pectins were formerly believed to yield a considerable variety of hydro-
lytic products, including acetic acid, galactose, and arabinose in addition
to methyl alcohol and D-galacturonic acid. More recent work indicates
that pectins are methylated polymers of D-galacturonic acid (Schneider
and Bock, 1937). The chemistry and physiology of the pectins have been
reviewed by Bonner (1936).

Proteinases and peptidases. These enzymes, also called proteolytic
enzymes, catalyze the hydrolysis (and synthesis) of proteins and peptides.
These enzymes have been separated into two groups upon the basis of
ability to attack native protein. Those enzymes which act upon intact
proteins are called proteinases, while those which attack peptides are
called peptidases. It seems that the fundamental difference between
these two classes of enzymes lies in the point of attack. The proteinases
attack the protein molecule in such a way as to produce various peptides
as well as amino acids, while the peptidases act only on the ends of the
peptide chains. This is analogous to the action of the two amylases.

ENZYMES 51

The proteolytic enzymes are a very complex group of hydrolases. In
view of the complexity of protein structure this is not unexpected. The
question of specificity of the proteolytic enzymes has been considered by
Bergmann (1942), who emphasizes that the specificity of a given enzyme
for a certain substrate may be modified by the presence of a second sub-
strate. Johnson and Berger (1942) have reviewed the enzymatic proper-
ties of the peptidases, including those produced by the fungi.

Oxidases, hydrogenases, and desmolases. One of the central prob-
lems in metabolic processes is how and by what means oxidation of
metabolites to carbon dioxide and water is brought about. Some
organisms (bacteria) are inhibited or killed by free oxygen (anaerobes).
Others may live either in the presence or absence of free oxygen (faculta-
tive anaerobes), while others require free oxygen (aerobes) to carry on
their metabohc processes and to maintain life. Thus, one organism may
degrade a substrate only partially, and these intermediate oxidation
products become substrates for other organisms. In the end complete
oxidation takes place. In other instances an organism may first carry
out a partial degradation and complete it later. Thus, yeast produces
alcohol by fermentation. In the presence of oxygen, alcohol is utilized
for the synthesis of cellular constituents and as a source of energy. Many
fungi possess two ways of obtaining energy by the degradation of metabo-
lites: an anaerobic (fermentative) and an aerobic (oxidative) pathway.
Both may function in the same organism at the same time, although
external conditions may favor one process at the expense of the other, or a
substance may inhibit one without affecting the other.

Biological oxidations are carried out in two ways: by the removal of
hydrogen from, or by the addition of oxygen to, substrates. The name
of Wieland is associated with the process of dehydrogenation, and that of
Warburg with the second process.

The theory of Wieland stressed the importance of the enzyme systems
which activated hydrogen or removed hydrogen from substrate molecules,
while Warburg's theory focused attention upon the enzyme systems
which activated oxygen and which carried oxygen to the substrates.
These two theories might seem irreconcilable, but today they are con-
sidered as mutually complementary. Both types of enzymatic oxidation
are known for the same organism. For further discussion of this problem
the student is referred to Elvehjem and Wilson (1944) and Meyerhof et al.
(1942) . For a classification of the respiratory enzymes see Gortner (1949)
and Sumner and Somers (1947). For the electronic mechanism involved
in biological oxidation-reduction see Michaelis (1946).

Some representative dehydrogenases and oxidases are aerobic dehj^dro-
genases (xanthine oxidase, and uricase); anaerobic dehydrogenases,
(succinic dehydrogenase, glucose dehydrogenase, triose phosphate dehy-

52 PHYSIOLOGY OF THE FUNGI

drogenase) ; oxidases (cytochrome oxidase, tyrosinase, polyphenol oxi-
dase). Succinic acid dehydrogenase oxidizes succinic acid to fumaric
acid by the removal of two hydrogens ; but this reaction takes place only
in the presence of another system (cytochromes) which "carries" the
hydrogen to an oxidizing enzyme, which converts the hydrogen to water
and regenerates the cytochrome system so that it can transport more
hydrogen. In the cell, succinic acid dehydrogenase is said to be cyto-
chrome-linked. In the laboratory, hydrogen carriers other than cyto-
chrome may be used. Various other dehydrogenases are linked to the
cytochrome system.

Another oxidase, tyrosinase, is found in many fungi. It is well estab-
lished that copper is an essential constituent of this enzyme system
(Kubowitz, 1937) and may be removed by dialyzing the enzyme against
cyanide solutions. The activity which is lost by this treatment is restored
by cupric ion, Cu++, but other divalent metals do not replace copper.
Various reagents which react with copper, such as cyanide, diethyl
dithiocarbamate, salicylaldoxine, and carbon monoxide, inhibit the action
of tyrosinase. Among the fungi which produce tyrosinase are the follow-
ing species (Nelson and Dawson, 1944) : Boletus luridis, Russula foetens,
R. niger, Lactarius piperatus, and PsalUota campestris. It is probable that
the darkening and coloration of the fruit bodies of these fungi depend upon
the activity of tyrosinase.

Pyruvic acid, CHs — CO — COOH, is a key compound in carbohydrate
utilization, and perhaps in other metabolic processes as well. The
enzyme, carboxylase, catalyzes the decomposition of pyruvic acid in
the following way:

carboxylase

CHs— CO— COOH > CO. + CHs— CHO

Pyruvic acid Carbon dioxide Acetaldehyde

The carbon dioxide formed escapes, while the acetaldehyde formed may
be either oxidized to acetic acid or reduced to ethyl alcohol. The enzyme
which catalyzes the decarboxylation of pyruvic acid to carbon dioxide and
acetaldehyde is abundant in yeast and other fungi. This enzyme con-
sists of three moieties, a specific protein, a magnesium ion, and thiamine
pyrophosphate.

CHEMICAL NATURE OF ENZYMES

In the past there has been a great deal of controversy over the chemical
nature of enzymes. Sumner (1926) was the first to isolate an enzyme
(urease) in pure crystalline condition. Since then a dozen or more
enzymes have been prepared in pure crystalline form. All the enzymes
which have been isolated in pure crystalline condition have proved to be
proteins.

ENZYMES 53

Some enzymes are specific proteins requiring neither coenzymes nor
metals for activity. These enzymes must contain as an integral part of
their structure the specific groups whereby they react with the substrate.
Other enzymes consist of two moieties, a specific protein and a specific
nonprotein compound which can be detached from the protein. In the
process of purifying an enzyme by dialysis the activity may be lost and
later restored by adding to the dialyzed material some boiled juice from
the tissue under investigation. These specific nonprotein compounds are
known as coenzymes. Neither the specific protein nor the coenzyme alone
functions as the enzyme; both are required for activity. The specific
protein is called the apoenzyme, while the combination of apoenzyme and
coenzyme is called the holoenzyme. Still other holoenzymes consist of an
apoenzyme, a coenzyme, and a metallic ion. Coenzymes, being non-
protein in nature, have proved to be more easily isolated and studied than
the specific protein moieties of enzymes. Coenzymes are a varied group
of compounds, some relatively simple in structure and others more com-
plex. The vitamins are known to enter into the structure of some
coenzymes, and it is supposed it is through such coenzyme molecules that
the vitamins exert their specific effects. The same coenzyme may com-
bine with many specific proteins to form different enzymes.

FACTORS AFFECTING ENZYME ACTIVITY

Some of the factors influencing enzyme activity affect the intact organ-
ism as well as isolated enzyme systems. While the situation within the
intact organism is more complex, a knowledge of the behavior of isolated
systems will be useful in interpreting the behavior of living fungi. The
factors which will be discussed are temperature, hydrogen-ion concentra-
tion (pH), chemical reagents (activators and inhibitors), and radiation.

Temperature. The rate of many reactions is approximately doubled
for each 10°C. increase in temperature. The rate of reactions catalyzed
by enzymes also increases with temperature. This increase is not main-
tained indefinitely, for enzymes are destroyed by temperatures of less
than 100°C. Although there are some reports in the literature of the rate
of enzymatic reactions being increased as much as fivefold by a 10°C.
increase in temperature, for most enzymatic reactions the increase in rate
is less than twofold. This increase between two temperatures 10°C.
apart is called the temperature coefficient, or Qio. Since the increase in
rate is not exactly constant, it is desirable to specify the temperatures
involved; e.g., Q20-30.

A reaction with a Qio of 2 proceeds sixteen times faster at 40°C. than
at 0°C. Or, the transformation of a given amount of substrate which
requires 16 hr. at 0°C. will occur within 1 hr. at 40°C. Figure 9 shows the
theoretical effect of temperature upon the amount of substrate trans-

PHYSIOLOGY OF THE FUNGI

formed when Qio is 2, 3, and 4. It was assumed that one unit of substrate
was transformed per unit of time at 0°C. For a reaction with a Qio of 2
an increase in temperature from 28 to 30°C. causes as great an increase in
the amount of substrate transformed as does the increase from 0 to 10°C.
A small increase in temperature in the range 25 to 35°C. has a greater
effect on the rate of reaction than a much greater increase in temperature
in the lower temperature range.

70
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Temperature in degrees centjgrode

I' ;g. 9. The theoretical effect of temperature on the rate of enzymatic reactions for
different assumed values of Qio.

Enzymes are inactivated by heat. The inactivation may be reversible
or irreversible depending upon the enzyme involved, the duration of heat-
ing, and other factors. The temperature at which the increased rate of
reaction is balanced by destruction of an isolated enzyme is the so-called
optimum temperature (Bayliss, 1925).

The life processes of a fungus are mediated by a large number of en-
zymes, which differ in their sensitivity to heat. Fungi cease to grow or
reproduce at temperatures lower than that required to kill them. It may
be assumed that the enzymes most sensitive to heat are gradually inac-
tivated as the temperature increases. This situation in the li^'ing fungus
is different from that of an isolated system in that the enzyme is in its
natural surroundings and the fungus is able to synthesize or repair the
vital enzymes in question. At some temperature we may suppose that
the rate of synthesis or repair of the enzyme system is exceeded by the
rate of inactivation. When this temperature is reached, or exceeded, the
activity of these enzyme systems decrease. This decreased activity is

ENZYMES 55

reflected in a lowered rate of growth or may be seen in other behavior of
the fungus. With further increases in temperature, the enzyme systems
become less and less operative. So long as the temperature does not
exceed the point which produces irreversible inactivation, lowering the
temperature will enable the fungus to resume growth or other activity.
The temperature of inactivation is not fixed unless the length of exposure
is also considered.

The effect of temperature upon growth is shown in Figs. 5 and 39.
The portions of the curves in the optimum temperature range represent a
balance between inactivation and increased rate of reaction. Above
optimum temperature, the rate of growth falls off abruptly. In a general
way the rate of growth parallels that expected of enzymatic processes.

Hydrogen-ion concentration. Long ago it was recognized that strong
acids and alkalies were destructive to enzymes. A second effect was also
recognized: some enzj^mes exhibited maximum activity only in the pres-
ence of weakly acidic or alkaline solutions (see Chap. 8 for a discussion
of pH). The effect of pH on the activity of urease is shown in Fig. 25.
It should be noted that the pH optimum is dependent upon the concen-
tration of urea.

Haldane (1930) compiled the pH optima of 105 enzymes and found
that the range extended from pH 2 to 10. However, all but nine of these
enzymes had pH optima between 4 and 8. Most fungi grow between
these limits. The effect of the pH of the medium upon the pH of the cell
contents is unknown in most instances. Biinning (1936) has reported
that the internal pH of the cells of Aspergillus niger is influenced by the
pH of the medium. The activities of the exoenzymes are affected by the
pH of the medium.

Chemical reagents. Some enzymes are inactive or nearly so until they
have been treated with certain reagents. A group of the plant proteinases
which includes papain and bromelin are activated by hydrogen sulfide and
hydrogen cyanide (inhibitors for many enzymes), glutathione, and other
thiol compounds. These various activators do not act by removing
heavy metals (inactivators for many enzymes) but by reducing the disul-
fide linkage, — S — S — , to thiol (sulf hydril) , — SH. Neutral salts
activate some enzymes (emulsin, pancreatic amylase). The mode of
activation by neutral salts is unknown. Many of the metallic ions
(Mg++, Ca++, Fe++, Cu++, Mn++) are required for enzyme activity, but
it seems better to consider them as essential parts of some enzymes rather
than activators.

Inhibitors are substances which reduce or destroy enzyme activity.
Inhibition may be reversible or irreversible. A few enzyme inhibitors are
cyanides, monoiodoacetate, fluoride, and the hea\^ metals (lead, copper,
mercury, silver, etc.). An inhibitor is active against certain enzymes and

PHYSIOLOGY OF THE FUNGI

not others. There appears to be a close relation between the chemical
constitution of the prosthetic group of the enzyme and the inhibitors
which inactivate it. We may postulate that inactivation results from a
chemical reaction between the inhibitor and the prosthetic group of an
enzyme.

One characteristic of an oxidase is inhibition by cyanide and hydrogen
sulfide. This points to some common moiety in these enzymes which is

/Vo cyanide

0.95 X 10'^ M NaCN

o — '-' — o-
2.5xlO'^M NaCN

U P — o — o- — o — o,

l2.4xlO'^MNaCN

50 75 100

Oxygen tension (mm Hg)

Fig. 10. The effect of cyanide on yeast respiration. (Courtesy of Winzler, Jour.
Cellular Comp. Physiol. 21: 238, 1943. Published by permission of Wistar Institute
of Anatomy and Biology.)

able to react with cyanide. The oxidases are metalloproteins, and in
view of the property of cyanides of reacting with metals to form complexes,
it would appear likely that cyanide reacts with the metal to form inactive
little-ionized compounds. The typical properties of ferrous and ferric
ions are masked by cyanide. Tyrosinase, a copper-containing enzyme, is
inactivated by cyanide. Hydrogen sulfide acts on many of the same
enzymes which are inhibited by cyanide ; the action may be assumed to be
due to the formation of insoluble metal compounds rather than the forma-
tion of non-ionized complexes.

Winzler (1943) studied the effect of different concentrations of cyanide
upon the respiration of yeast maintained under different oxygen tensions.
The effect of cyanide on yeast respiration is shown in Fig. 10. It may be

ENZYMES 57

noted that tlie percentages of inhibition of respiration (oxygen uptake)
depend upon two conditions, the amount of oxygen available and the
concentration of cyanide present. We may assume that the cyanide
inhibited one or more respiratory enzymes and that, as the concentration
of cyanide increased, more and more of these enzymes were inactivated.
When the oxygen tension was reduced, these effects were increased.

While it is kno^^^l that salts of the heavy metals may denature proteins,
and this explanation has been advanced to account for enzyme inactiva-
tion by them, recent opinion inclines to the \'iew that the heavy metals
inactivate enzymes either by combining with — SH groups, or, under
alkaline conditions, by oxidizing thiol sulfur to disulfide. Mercuric ions,
especially, may combine with specific metabolites which contain — SH
groups (glutathione, thioamino acids), as found by Fildes (1940). Cer-
tain metals may inactivate enzymes by replacing the normal metal, ren-
dering the enzyme inoperative. It is noteworthy that many enzymes
which are inactivated by heavy metals may be either "protected" or
restored to activity by the addition of thiol compounds. We may assume
for the purpose of illustration that, when a heavy metal combines with an
enzyme, an inactive complex or compound is formed as shown in scheme
II. Two factors would influence the effectiveness of thiol compounds in
preventing or reversing enzyme inactivation, the relative affinity of the
enzyme — SH groups and the thiol compound for mercury, and the rela-
tive concentration of enzyme and thiol compound.

Scheme II. A Scheme Illustrating a Possible Mechanism of Inactivation

OF A Sulfhydril Enzyme by Mercuric Ion and Reactr'ation of the Inactive

Enzyme-Mercury Complex by the Addition of a Thiol Compound

Inactivation

Enzyme — S

2(Enzyme— SH) + Hg++^ Hg + 2H+

Enzyme — S
Active enzyme Inactive enzyme complex

Reactivation
Enzyme— S RS

\ \

Hg + 2RSH^ 2 (Enzyme— SH) + Hg

Enzyme— S RS

Active enzyme

Radiation. Many reports are to be found in the literature that radia-
tion affects enzymes adversely (see the review of Schomer, 1936). Radia-
tion may affect not only the enzymes of an organism but also the sub-
strates. Ionizing short-wave radiations may cause the formation of
hydrogen peroxide from water. Barron et al. (1947) were able, by adding

PHYSIOLOGY OF THE FUNGI

glutathione, to reactivate phosphoglyceral dehydrogenase which had been
inactivated by X rays.

Whether radiation is absorbed or not depends upon the chemical con-
stitution of the absorbing molecule and the wave length of the radiation.
The energy thus obtained may disrupt the molecule or may merely
increase its ability to react. These generalizations are not very helpful in
either predicting the effect of light upon living fungi or interpreting the
observed effects of light on growth and reproduction. It is probable that
light acts on various enzyme systems. Light is known to affect one
specific enzyme system (cytochrome-cytochrome oxidase). Warburg
(1926) showed that the respiration of baker's yeast was inhibited to the
extent of 70 per cent in the dark when exposed to carbon monoxide con-
taining 5 per cent oxygen, while respiration was inhibited only 14 per cent
in light. The same effect of light on carbon monoxide inhil)ition of
respiration has been demonstrated with larvae of Tenehris molitor and the

heart of embryo trout.

Ultraviolet radiation and X rays have a
lethal effect on fungi. A small percentagr^
of the spores which survive exposure
to ultraviolet radiation may produce
mutants. It has been noted recently
(Kelner, 1949) that the lethal effect of
ultraviolet radiation upon spores of Strep-
tomyces griseus is overcome to a considera-
ble extent by exposing irradiated spores
to visible light. Whether this is due to

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Enzyme

molecule

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Fig. 11. Diagrammatic illustra-
tion of the mechanism of enzy-
matic hydrolysis. The substrate
molecules are represented by
small circles, the products of
hydrolysis by semicircles. (Cour-
tesy of Van Slyke, Advances in
Enzymol. 2 : 38, 1942. Published
by permission of Interscience
Publishers, Inc.)

reactivation of certain enzyme systems is
not known.

MECHANISM OF ENZYME ACTION

The most generally accepted theory of
enzyme action postulates that the enzyme
and substrate unite to form a molecular
compound or complex (enzyme-substrate
complex). In favorable instances the ex-
istence of such enzyme-substrate complexes has been demonstrated (Stern,
1936) . During this temporary union the substrate molecule is " strained"
or activated so that it undergoes reaction. The products of the reaction
have less affinity for the enzyme surface than the substrate molecules and
hence diffuse away, and other substrate molecules unite temporarly with
the enzyme and the process continues. If the product molecules are
present in excess, they may compete more successfully for the enzyme
surface than the substrate does. During synthesis, when the reactants

ENZYMES

(products) are present in solution in greater than equilibrium concentra-
tions, the reactants combine with the enzyme, unite, and diffuse away.
Figure 11 gives a diagram which is helpful in visualizing these processes.

ADAPTIVE ENZYMES

Some fungi produce certain enzymes only in response to particular
environmental conditions. Such enzymes are called adaptive enzymes.
\\^ether they are produced under all cultural conditions, but in such small
amounts as to be undetectable, or whether they are produced de novo is
questionable. How^ever, this phenomenon is of great importance. Two
types of behavior may be noted when a fungus is placed upon unsuitable
medium for the first time. Either the fungus may die, owing to lack of
ability to synthesize the enzymes to cope with the new environment; or

240

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• -Anaerobic adaptation

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24
Hours
Fig. 12. Rate of adaptation of a strain of Saccharomyces carlsbergensis to galactose
under aerobic and anaerobic conditions. (Courtesy of Spiegelman, Jour. Cellular
Comp. Physiol. 25: 128, 1945. Published by permission of Wistar Institute of
Anatomy and Biology.)

after a time it may synthesize the necessary enzymes, and the fungus is
then able to grow and function under the new surroundings. Wlrether or
not the fungus is able to synthesize "new" enzymes depends upon its
genetic constitution. The biochemical and physiological responses of an
organism may change when it is placed on a different kind of medium.
These changes ordinarily are called forth by deficiencies in the medium.
The substrate upon which the inoculum grew- may be very important in
governing the various responses of the organism.

Spiegelman (1945) has shown that the adaptation of yeasts to galactose
is affected by aerobic and anaerobic conditions. Adaptation is more
rapid in air than in nitrogen, and some strains of yeast are unable to adapt
to galactose in the absence of oxygen. Figure 12 shows that only some
30 min. is required for Saccharomyces carlshergensis to begin to utilize
galactose under aerobic conditions, while about 20 hr. are required under
anaerobic conditions.

The effect of composition of the medium on the readaptation of panto-

()0 PHYSIOLOGY OF THE FUNGI

thenate-dependent strains of yeast to the synthesis of pantothenate has
been studied in some detail (Lindegren and Rant, 1947; Lindegren, 1949).
Changes to pantothenate independence occurred by an adaptation, which
was transmitted vegetatively, and by a gene mutation. The adaptation
occurred only in the media of low pantothenate content, while the muta-
tions were apparently not affected by the concentration of pantothenate.
Leonian and Lilly (1943) studied the induced ability of eight strains of
Saccharoniyces cerevisiae to synthesize various vitamins for which they
were normally deficient. This was accomplished by long "training" in
media "free" from various vitamins. The ability of various yeast strains
to synthesize a given vitamin varied. These yeasts which had been
trained "reverted" to their deficient status when cultured for 6 months on
media containing vitamins and yeast extract.

ENERGY AND ENERGY UTILIZATION BY FUNGI

Fungi need energy, as well as certain elements and chemical compounds,
for life, growth, and reproduction. Since the life processes of the fungi
are controlled by interlocking systems of enzymes, the utilization of
energy is also an enzymatic process. The chemical reactions which
accompany or underlie life processes may be divided into those which
yield energy (exergonic) and those which require energy (endergonic)
(Coryell, 1940). The oxidation reactions whereby such substrate mole-
cules as glucose are converted into carbon dioxide and alcohol or carbon
dioxide and water yield energy, while the reactions involved in the syn-
thesis of protoplasm and reserve materials require energy. Let us con-
sider an analogy first. When water falls from a higher to a lower level,
there is a decrease in energy content, and this decrease in energy content is
the same whether the water has passed through a turbine or not. The
water that passes over a spillway does no useful work, while the water that
turns a turbine makes part of the energy available (as mechanical or
electrical power) for doing useful work. The energy given up by the
falling water is the same in both cases, but only where the proper mecha-
nism is available is any useful work obtained.

A similar situation occurs when a fungus oxidizes glucose to water and
carbon dioxide. If energy-requiring synthetic reactions are coupled with
the degradation reactions, a portion of the available energy becomes useful
to the fungus. The remainder of the energy liberated appears as heat,
which is unavailable to the fungus for lack of suitable mechanisms to
utilize it.

Winzler and Baumberger (1938) have investigated the liberation of
energy by yeast cells during metabolism. Washed yeast cells were sus-
pended in a phosphate buffer containing glucose but no nitrogen. The
reaction vessel was placed in an adiabatic calorimeter, and the heat

ENZYMES

evolved and the amount of oxygen absorbed and of carbon dioxide evolved
were measured. In the absence of a nitrogen source the synthesis of
protoplasm was avoided. The rate at which heat was evolved was con-
stant until all the glucose was consumed (exogenous respiration), after
which the rate of heat formation decreased (endogenous respiration) (see
Fig. 13).

30 40

Time in minutes
Fig. 13. Heat produced from glucose oxidation by yeast in the absence of a nitrogen
source; 10, 20, and 30 mg. of glucose were added at zero time in curves I, II, and III,
respectively. In all cases, only 26 per cent of the expected amount of heat was
evolved before the endogenous respiration rate was resumed. (Courtesy of Winzler
and Baumberger, Jour. Cellular Comp. Physiol. 12: 199, 1938. Published by per-
mission of Wistar Institute of Anatomy and Biology.)

In this experiment the theoretical amount of heat could be calculated
for the amounts of glucose used. Only 26 per cent of the theoretical heat
was produced before endogenous respiration set in. The volume of oxy-
gen used was equal to the volume of carbon dioxide evolved, i.e.j the R.Q.
was 1. These data may be interpreted as follows: For every molecule of
glucose oxidized to carbon dioxide and water, three molecules were syn-
thesized into a carbohydrate, presumably glycogen. When sodium
acetate was the substrate, about 59 per cent of the theoretical heat was
evolved, but in the presence of dinitrophenol the theoretical amount of
heat was evolved. This inhibitor, therefore, blocked the assimilative
mechanism but not the oxidative processes.

Within recent years it has been discovered that certain phosphate esters
may play a very important role in energy transfer. The student is
referred to the review of Lipmann (1941) for further information on this
subject.

The utilization of energy derived from degradation reactions depends
upon such energy-yielding reactions being coupled with energy-requiring
reactions. Degradation reactions which are not so coupled (blocked)

62 PHYSIOLOGY OF THE FUNGI

waste energy in the form of heat which is not iitihzed by the fungi. The
efficiency of utiHzation depends upon the substrate utihzcd and upon the
nature of the coupled reactions. In any case only a part of the energy
available in the sulDstrate does useful chemical work for the fungus utiliz-
ing it. The application of these ideas with any rigor requires a sound
knowledge of thermodynamics.

SUMMARY

The chemical reactions which underlie the life processes of fungi and
other organisms are initiated by organic catalysts, or enzymes. Enzymes
catalyze synthetic as well as degradation reactions and are mediators of
energy transfer as well.

Enzymes are specific proteins which in some instances require certain
metallic ions or organic coenzymes, or both, before they are active. In
general, an enzyme controls but a single type of reaction. In living
organisms these enzyme-controlled reactions are correlated and integrated
to a high degree.

Among the external factors which modify the action of enzymes the
following are especially important: temperature, hydrogen-ion concentra-
tion, concentration of substrate and products, and inhibitors. The
effects of these factors on isolated enzymes and intact organisms are much
the same.

While the role of enzymes in maintaining life processes in fungi and
other organisms is well established, the application of this information to
living fungi must be made with due caution and the realization that a
living organism is more complex than its parts.

REFERENCES

Barron, E. S. G., S. Dickman, and T. P. Singer: On the inhibition ol enzymes by

ionizing radiations, Fed. Proc. 6 : 236, 1947.
*Bayliss, W. M.: The Nature of Enzyme Action, 2d ed., Longmans, Roberts and

Green, London, 1925.
Bergmann, M.: A classification of proteolytic enzymes. Advances in Enzymol. 2:

49-68, 1942.
Bonner, J.: The chemistry and physiology of the pectins, Botan. Rev. 2: 475-497,

1936.
Bourqxtelot, E.: La Synthese biochimique des d-glucosides d'alcools monovalents.

II. AlcooW-glucosides a, Ann. chim., Ser. IX, 3: 287-337, 1915.
Buchner, E.: Alcoholische Garung ohne Hefezellen, Ber. d. deut. chem. Ges. 30:

117-124, 1897.
BtJNNiNG, E.: Ueber die Farbstoff- und Nitrataufnahme bei Aspergillus niger, Flora

131:87-112, 1936.
Coryell, C. D.: The proposed terms "exergonic" and "endergonic" for thermo-
dynamics. Science 92 : 380, 1940.
DrxoN, M.: Multi-enzyme Systems, Cambridge University Press, New York, 1949.
Elvehjem, C. a., and P. W. Wilson (Editors): Respiratory Enzymes, Burgess

Publishing Co., Minneapolis, 1944.

ENZYMES 63

IiLDES, P.: The mechanism of the anti-bacterial action of mercury, Brit. Jour.

Exptl. Path. 21: G7-73, 19-40.
GoRTNER, R. A.: Outlines of Biochemistry, 3d ed., John Wiley & Sons, Inc., New

York, 1949.
Haldane, J. B. S.: Enzymes, Longmans, Roberts and Green, London, 1930.
Harden, A.: Alcoholic Fermentation, 4th ed., Longmans, Roberts and Green,

London, 1932.
Harter, L. L., and J. L. Weimer: A comparison of the pectinase produced by

different species of Rhizopus, Jour. Agr. Research 22: 371-377, 1921.
Hestrin, S.: The fermentation of disaccharides. I. Reducing disaccharides and

trehalose, Wallerstein Labs. Communs. 11 : 193-206, 1948.
Hopkins, R. H.: The action of the amylases. Advances in Enzymol. 6: 389-414, 1946.
Johnson, ]\L J., and J. Berger: The enzymatic properties of peptidases. Advances

in Enzymol. 2 : 69-92, 1942.
Kelner, a.: Effect of visible light on the recovery of Streptomyces griseus conidia

from ultraviolet irradiation injury, Proc. Natl. Acad. Sci. U.S. 35: 73-79, 1949.
KuBowiTZ, r.: Ueber die chemische Zusammensetzung der Kartoffeloxydase,

Biocnem. Zeit. 292 : 221-229, 1937.
Leibowitz, J., and S. Hestrin: Alcoholic fermentation of the oligosaccharides,

Advances in Enzymol. 5: 87-127, 1945.
Leonian, L. H., and V. G. Lilly: Induced autotrophism in yeast. Jour. Bad. 45:

329-339, 1943.
Lindegren, C. C.: The Yeast Cell, Its Genetics and Cytology, Educational Pub-
lishers, St. Louis, 1949.
Lindegren, C. C, and C. Ralt: A direct relationship between pantothenate con-
centration and the time required to induce the production of pantothenate-

synthesizing ''mutants" in yeasts. Ann. Missouri Botan. Garden 34: 85-93,

1947.
Lipmann, F.: Metabolic generation and utilization of phosphate bond energy,

Advances in Enzymol. 1: 99-162, 1941.
*Meyerhof, O., et al.: Symposium on Respiratory Enzymes, University of Wisconsin

Press, Madison, 1942.
MicHAELis, L. : Fundamentals of oxidation and reduction in Currents in Biochemical

Research (edited by D. E. Green), Interscience Publishers, Inc., New York,

1946.
Myrback, K.: The structure of starch, Wallerstein Labs. Communs. 11: 209-218,

1948.
Nelson, J. M., and C. R. Dawson: Tyrosinase, Advances in Enzymol. 4: 99-152,

1944.
Norman, A. G., and W. H. Fuller: Cellulose decomposition by microorganisms,

Advances in Enzymol. 2 : 239-264, 1942.
Pasteur, L.: Etudes sur la vin, Librairie F. Savy, Paris, 1875.
Schneider, G. G., and H. Bock: Ueber die Konstitution der Pektinstoffe, Ber. d.

deut. chem. Ges. 70: 1617-1630, 1937.
Schomer, H. a. : The effects of radiation on enzymes in Biological Effects of Radi-
ation (edited by B. M. Duggar), McGraw-Hill Book Company, Inc., New York,

1936.
*Spiegelman, S. : The effect of anaerobiosis on adaptation to galactose fermentation

by yeast cells, Jour. Cellular Comp. Physiol. 25: 121-131, 1945.
Stephenson, M.: Bacterial Metabolism, 2d ed., Longmans, Roberts and Green,

London, 1939.
Stern, K. G.: On the mechanism of enzyme action. A study of the decomposition

64 PHYSIOLOGY OF THE FUNGI

of nioiioctliyl hydrogon peroxide by catalase and of an intermediate enzyme-
substrate eompound, Jour. Biol. Chem. 114: 473-494, 1936.

Sumner, J. B.: The isolation and crystallization of the enzyme urease, Jour. Biol.
Chem. 69: 435-441, 1926.
*SuMNER, J. B., and G. F. Somers: Chemistry and Methods of Enzymes, Academic
Press, Inc., New York, 1947.

Thaysen, a. C, and H. J. Bunker: The Microbiology of Cellulose, Hemicelluloses,
Pectins and Gums, Oxford University Press, New York, 1927.
*Van Slyke, D. D.: The kinetics of hydrolytic enzymes and their bearing on methods
for measuring enzyme activity. Advances in Enzymol. 2 : 33-47, 1942.

Warburg, O. : Ueber die Wirkung des Kohlenoxyds auf den Stoffwechsel der Hefe,
Biochetn. Zeit. 177: 471-486, 1926.

White, W. L., R. T. Darby, G. M. Stechert, and K. Sanderson: Assay of cellulo-
lytic activity of molds isolated from fabrics and related items exposed in the
tropics, Mycologia 40: 34-84, 1948.

WiNZLER, R. J. : A comparative study of the effects of cyanide, azide, and carbon
monoxide on the respiration of bakers yeast. Jour. Cellular Conip. Physiol.
21 : 229-252, 1943.
*WiNZLER, R. J., and J. P. Baumberger: The degradation of energy in the metabo-
lism of yeast cells. Jour. Cellular Comp. Physiol. 12: 183-211. 1938.

CHAPTER 5
ESSENTIAL METALLIC ELEMENTS

The fungi need about 17 elements to supply their nutritional require-
ments. These elements are utilized in the form of specific compounds, as
ions, and as free elements. Some of the essential elements are required by
all fungi. Other elements are required only by certain species. In a
general way, the elements required by the fungi are the same ones
required by bacteria, green plants, and animals. There are, however,
striking differences in the essential-element requirements of different
groups of organisms (Table 15). Differences in ability to utilize specific
compounds containing these essential elements are common in the fungi
and bacteria.

BIOLOGICALLY ESSENTIAL ELEMENTS

Before seeking to determine which elements are essential, it is necessary
to define what is meant by the term hiologically essential element. An
essential element is indispensable in that no other element may entirely
replace it. Without these essential elements life is impossible. An ele-
ment needed in extremely small amounts may be just as essential as car-
bon, which comprises almost half the weight of a fungus.

There are some 92 chemical elements (if we exclude the recently
isolated trans uranic elements), most of which are known to exist, or may
exist, as a mixture of isotopes. So far as is known, all the isotopes of an
element (with the possible exception of the isotopes of hydrogen) have the
same chemical and biological properties. Even radioactive isotopes,
before they decay, exhibit the same biological properties as the stable
isotopes. The biological effects of radiation in inducing mutations are
considered briefly in Chapter 18. In spite of the limited number of ele-
ments, the question of essentiality is not settled completely for all.

The problem of determining which elements are essential for the fungi
has been approached from the standpoint of ultimate analysis of mycelium
and spores. If certain elements, such as carbon, potassium, and mag-
nesium, are always found in all samples analyzed, irrespective of the sub-
strates upon which these fungi grew, it may be concluded with a high
degree of probability that these elements are essential for the fungi.
Some of the analytical results of ultimate analyses of mycelium and spores
have been collected by Buchanan and Fulmer (1928) and Foster (1949).

66 PHYSIOLOGY OF THE FUNGI

Organic materials are dried before analysis. On the average about 75 per
cent of the fresh weight of mycelium is water, while spores contain only
about 40 per cent water. It is probable that the water driven off when
fungus cells are dried to constant weight is in part free water and in part
water bound to various colloidal cell constituents.

Ultimate analyses of mycelium and spores always reveal the presence of
carbon and nitrogen. On the average about 45 per cent of dry mycelium
is carbon. This high content of carbon makes it certain that carbon is an
essential element. The percentage of nitrogen found is quite variable.
Phosphorus, potassium, magnesium, calcium, sodium, sulfur, and iron are
found in the ash that remains after burning mycelium and spores. More
refined methods of analysis reveal that fungus ash contains still other
elements. Richards and Troutman (1940) investigated the composition
of yeast ash by spectrographic analysis and found the following elements :
iron, sodium, boron, bismuth, barium, magnesium, manganese, copper,
zinc, tin, lead, tellurium, silver, chromium, potassium, gold, and lan-
thanum. However, the mere presence of an element in fungus cells does
not necessarily mean that it is essential.

Since many of these elements in fungus ash occur in minute traces only,
it is desirable to approach the problem of essentiality in another way.
This is done by omitting from the medium the element in question.
Raulin (1869) was apparently the first to use this method. He found
that the omission of phosphorus, sulfur, magnesium, zinc, or iron from the
basal medium allowed very little growth of Aspergillus niger. These ele-
ments are thus shown to be essential by the two methods of investigation.
In general, the experimental work in which specific elements have been
omitted from the medium is more convincing than the method of ultimate
analysis. This is the preferred method of testing the essentiality of ele-
ments required in small amounts.

Functions of the essential elements. Thatcher (1934) has attempted
to classify the essential elements into groups: structural, functional, and
those utilized in the transfer of energy. This classification has some
validity and may serve to fix attention upon the more salient biological
features of an element. However, most, if not all, of the essential ele-
ments play many roles in the life processes of the fungi. In general, the
nonmetallic elements may be classified as structural elements. This
means that the compounds which make up the structural units such as
the protoplasm are largely composed of the nonmetallic essential ele-
ments: carbon, nitrogen, hydrogen, oxygen, sulfur, and phosphorus. The
functional uses of these elements by the fungi are no less important. The
essential metallic elements may be classified as functional elements, but
this does not mean that these metallic elements have no structural
functions.

ESSENTIAL METALLIC ELEMENTS 67

The elements are in the form of chemical compounds, some of which
are relatively simple, Avhile others are complex. With the exception of
oxygen the essential elements are usually utilized in the form of com-
pounds or ions. An essential element may exist in a chemical compound
and be unavailable. The properties of a chemical compound are deter-
mined by all the atoms that compose it and by the way in which atoms
are joined together in the compound. It is convenient to consider the
essential elements one by one, but this is done only to simplify the
approach to a complex subject. These separate factors must be con-
sidered in relation to the organism as a whole.

A fungus is no more capable of growth on an iron-free medium than on
a carbon- or nitrogen-free medium. Yet, in a balanced medium the ratio
of iron to carbon is in the neighborhood of 1 to 50,000. The essential
metallic elements function in conjunction with enzyme systems (Chap. 4).
This accounts for the small amounts of these elements required. If a
vital enzyme system lacks an essential metal ion, it will not function. It
appears that in processes such as growth a suboptimal amount of an
essential metal will stop growth because the apoenzymes or coenzymes
synthesized will lack the necessary activating metal. The ratios as well
as the amounts of the various essential metallic ions affect certain
metabolic processes other than growth.

The absolute amounts of the essential metallic elements required differ
widely. Raulin (1869) found that Aspergillus niger required 1 g. of
potassium to produce 64 g. of mj^celium, while 1 g. of magnesium sufficed
for the synthesis of 200 g. of mycelium. Recent work of Steinberg (1946)
with .4. niger indicates still higher yields per gram of these two elements.
The yield of mycelium per gram of iron and zinc was in the neighborhood
of 55,000 g.

The list of metallic elements knoTvni to be essential to fungi has in-
creased over the years. The list now includes potassium, magnesium,
iron, zinc, copper, calcium, gallium, manganese, molybdenum, vanadium,
and scandium. Others will probably be added as cultural methods
become more refined and more species are studied. It is unfortunate that
only a few fungi have been investigated thoroughly with respect to mineral
nutrition. In stating that the above elements are essential, the reserva-
tion must be made that they are essential for some fungi under certain
conditions. WTiile it may be assumed that all fungi require the
same essential elements, experimental evidence is lacking for most
species.

For the purpose of discussion the essential metallic elements will be
divided into two groups, macro and micro metallic elements. This
grouping is made solely for convenience and on the basis of the amounts
ordinarily employed in culturing fungi under laboratory conditions.

68 PHYSIOLOGY OF THE FUNGI

THE ESSENTIAL MACRO ELEMENTS

Potassium. This element is essential for all organisms, so far as is
known. There is an immense amount of information on the specific
effects of potassium on green plants and animals, l)ut such data are not
common for the fungi. The quantitative relation between the amount of
potassium in the medium and the weight of mycelium produced by
Aspergillus niger was studied by Steinberg (1946). This work was done
with extraordinary care using a highly purified optimal medium (except
potassium). The optimum amount of potassium w-as 150 mg. per liter.
The relative amounts of mycelium formed increased as the potassium
content of the medium decreased. The fungus produced almost three
times as much mycelium per milligram of potassium when 15 instead of
150 mg. per liter were used. Jarvis and Johnson (1950) have reported
that Penicillium chrysogenum Q176 requires 40 mg. of potassium and 8
mg. of magnesium per liter of medium for optimum growth.

The physiological effects of potassium on fungi have been studied but
little. The enzymes in yeast maceration juice which ferment glucose are
activated by either potassium or ammonium ions (Muntz, 1947). Mol-
liard (1920) noted that a low potassium content of the medium resulted in
increased synthesis of oxalic acid by A. mger. The chemical composition
of A. niger mycelium varies, depending upon the amount of potassium in
the medium (Rippel and Behr, 1934).

The problem of biological substitution arose early in the study of fungus
nutrition. Biological substitution means that one element can replace
another, in whole or in part. The possibility of biological substitution
was investigated by Steinberg (1946) using A. niger as the test fungus.
This investigation was made to determine whether the alkali metals
(lithium, sodium, rubidium, or cesium) could replace potassium, and
whether the alkaline-earth metals (calcium, beryllium, strontium, or
barium) could replace magnesium. Under these conditions sodium and
beryllium gave increased yield of mycelium in media containing sub-
optimal amounts of potassium and magnesium. These effects are
illustrated in Table 9.

Some increases in weight of mycelium were noted under certain con-
ditions with some of the other metallic ions tested, but the effects of
these elements were ascribed to ion antagonism.

Studies of biological substitution require great care and a detailed and
extensive knowledge of the composition of the media and of the behavior
of the fungus under the experimental conditions used.

Magnesium. This element is one of the alkaline-earth group. It is
essential for green plants and animals as well as for fungi and bacteria.
Aspergillus niger has been more carefully investigated with respect to the

ESSENTIAL METALLIC ELEMENTS

effects of magnesium than any other fungus. Within certain limits of
concentration, the amount of growth of A. niger is proportional to the
concentration of magnesium in the medium. This has been demon-
strated by Steinberg (1946), Lavollay and Laborey (1938), and others.
The application of this principle to the microbiological assay of magne-
sium is discussed in Chap. 10. Penicillium glaucum, Botrytis cinerea, and
Alternaria tenuis failed to grow in the absence of magnesium (Rabinovitz-
Sereni, 1933). Excess magnesium was not harmful to these three fungi
until the concentration of magnesium sulfate in the medium reached about
40 per cent. These three species were able to grow in the presence of
traces of magnesium but sporulated only when the concentration of mag-
nesium was increased. Respiration also increased as the magnesium con-
tent of the medium increased. Failure to sporulate unless sufficient
magnesium is available is probably to be expected with many fungi.

Table 9. The Effect of 50 Milligrams of Sodium on the Amount of Mycelium

Produced by Aspergillus niger in an Optimal Medium Containing Twice

THE Normal Amounts of INIicro Elements when the Concentration

OF Potassium Was Varied
(Steinberg, Avi. Jour. Botany 33, 1946.)

Potassium,
mg. per liter

Control, mg.
myceUum

Sodium added,

50 mg. per liter,

mg. mycelium

256.3

401.3

446.1

783.1

641.2

896.7

823.4

1,042.0

955,2

1,089.0

988.0

1,070.0

105

1,065.2

1,093.1

120

1,059.2

1,095.5

135

1,113.9

1,084.9

150

1,145.9

1,146.5

Most of the magnesium in the mycelium of Aspergillus niger can be
extracted by means of dilute acids (Ripple and Behr, 1930), which indi-
cates that this element does not form stable organic compounds. A rela-
tion between the optimum concentrations of magnesium and phosphorus
for A. niger was discovered by Laborey et at. (1941). Some 36 phosphate
ions are required for every ion of magnesium. Many enzyme sj^stems are
activated by magnesium ion, and in view of the role of phosphate in
enzymatic transformations it is not surprising that there should be a close
relation between magnesium and phosphate concentrations. Magnesium
is involved in many of the enzymatic reactions involved in fermentation

70 PHYSIOLOGY OF THE FUNGI

(Sumner and Somers, 1947). It is equally likely that magnesium is
involved in aerobic oxidation of carbohydrate. Low concentrations of
magnesium in the medium led to increased synthesis of riboflavin by A.
niger (Lavollay and Laborey, 1938).

One ion may affect the physiological action of another. This is called
ion antagonism. In nature and in the laboratory fungi come in contact
with compounds of both essential and nonessential elements. Many of
the nonessential elements are toxic, although toxicity is not limited to the
nonessential elements. Copper is an essential element, but it is toxic to
most fungi when the concentration exceeds certain limits (Chap. 12).
The toxic effect of an ion may be overcome by the presence of one or more
other ions in the medium. Gortner (1949) has reviewed this subject from
the standpoint of colloidal chemistry and suggests that the relative
concentrations of various metallic ions may regulate the process of
adsorption.

As an example of ion antagonism Lohrmann (1940) described the toxic
action of mercuric chloride and boric acid on Aspergillus niger, A. flavus,
Mucor pusillus, Penicillium glaucum, Fusarium coeruleum, Cunning-
hamella elegans, Ahsidia cylindrospora, and Rhizopus nigricans. The
inhibition caused by either of these toxic compounds was overcome in
part by increasing the concentration of magnesium sulfate. Similarly,
the toxic effects of high concentrations of magnesium sulfate were over-
come by mercuric chloride. Either mercuric chloride or boric acid in
certain concentrations "stimulated" growth in the nutrient solution used.
This is not evidence that either boron or mercury is an essential element,
but it does show that the nutrient solution used was unbalanced. The
effect of sodium and calcium ions upon growth and respiration of A. niger
depended upon the ratio of these nonessential ions present in the medium.
A sodium-calcium ratio of 19 to 1 gave the highest rate of respiration,
while a ratio of 4 to 1 was most favorable for growth (Gustafson, 1919).
Aluminum inhibits the production of itaconic acid by A. terreus. This
inhibition is overcome by magnesium sulfate (Lockwood and Reeves,
1945). Nickerson (1946) found the inhibitory effects of zinc ion on the
rate of respiration of Epidermophyton floccosum to be reversed by calcium
or magnesium ions.

The phenomenon of antagonism is not confined to ions. Organic
compounds present in media may modify the activity of ions, and organic
compounds may antagonize the physiological activity of other organic
compounds (Chap. 11). All these possibihties exist. Whether a given
ion or compound will be physiologically active depends upon the other
constituents of the medium and the metabolic compounds excreted by the
fungus under study.

ESSENTIAL METALLIC ELEMENTS 71

ESSENTIAL MICRO ELEMENTS

These elements have been called heavy-metal nutrients, trace elements,
micronutrients, and minor elements. The literature on this subject is
extensive and often conflicting. Reviews of this subject are given by
Perlman (1949), Foster (1939), and Steinberg (1939). A collection of
10,000 abstracts on the effects of the micro elements on green plants and
animals has been published by the Chilean Nitrate Educational Bureau
(1948).

In spite of Raulin's (1869) discovery that iron and zinc are essential for
Aspergillus niger, there arose a school of investigators who considered tho
micro elements to be stimulatory rather than essential. This view is no
longer held. There are a number of reasons for this misinterpretation:

(1) The failure to realize that the "chemically pure" compounds used in
preparing media are grossly contaminated from the biological standpoint
and that rigorous purification of media is essential in work of this kind.

(2) Distilled water is often a source of metallic ions unless it has been
redistilled in Pyrex, or preferably quartz, stills. (3) Many kinds of
chemical glassware are sufficiently soluble to furnish the fungi all or a part
of the micro elements required. (4) The inoculum, whether mycelium or
spores, may introduce sufficient micro elements to obscure the need for
these elements. Serial transfer using media free from the element in
question and the use of small inocula minimizes this source of error.

Steinberg (1936) has indicated that the optimum concentration of the
essential micro metallic elements for A. niger ranges from 0.3 mg. of iron
to 0.02 mg. of gallium per liter of medium. Lest the reader conclude that
these concentrations are so small as to be meaningless, it is revealing to
calculate the number of atoms of iron in 0.3 mg. From the atomic weight
of iron and Avagadro's number it may be calculated that there are about
3 X 10^^ atoms in 0.3 mg. of iron. If the number of cells produced by
A. niger under these conditions were known, the number of iron atoms
available for each cell could be calculated. In lieu of this information we
may use data from experiments on the number of yeast cells produced in
a liter of medium. Under favorable conditions there are roughly 500
billion yeast cells produced in a liter of medium (Stark et al., 1941). If
A. niger produces the same number of cells per liter as yeast, there would
be available 6.4 X 10^ atoms of iron per cell.

The prime essential in investigations dealing with the effects of the
essential micro elements is a medium free from the element under study.
This ideal is difficult to attain in practice. Equal care is necessary in the
choice of culture vessels, for it is wasted effort to remove an element from
the medium rigorously and then contaminate it by using glassware which

72 PHYSIOLOGY OF THE FUNGI

furnishes the metal. The culture A^essels should be of quartz for work of
the most exacting kind, although Pyrex or other suitable glassware may
be used. It is desirable in any event to use a few quartz culture vessels as
controls. In part, the long controversy over the effect of zinc on fungi
was due to the liberation of sufficient amounts of this element from certain
kinds of glassware used as culture vessels. Javillier (1914) showed that
the addition of zinc to cultures of A . niger has little effect when Jena glass
culture flasks were used. When quartz vessels were used, the crop
increased from 291 mg. in the control without added zinc to 1,624 mg.
when zinc was added. Steinberg (1919) found essentially the same
results except that zinc deficiency could be demonstrated for A. niger
when Pyrex vessels were used (Table 10) .

Table 10. The Average Weight of Five Cultures of Aspergillus niger Culti-
vated ON the Same Basal Medium in Three Makes of Glassware
(Steinberg, Am. Jour. Botany 6, 1919.)

Make of glassware

Jena

Kavalier Bohemian .
Pyrex

Mg. mycelium

Zinc, 10 mg. per liter

987
943
957

Purification of culture media. Progress in the study of essential micro
elements depends upon methods of removing them from media. As long
as these elements occur in the ingredients of the media, their need may be
unnoticed and unsuspected. In 1919 Steinberg devised a useful method
of reducing the concentration of heavy metals, especially iron and zinc, in
media. In essentials, this method consists in autoclaving the complete
medium with 15 g. per liter of calcium carbonate. The hot solution after
autoclaving is filtered through paper or a fritted-glass filter, or allowed to
cool and the supernatant liquid decanted off. The precipitate must be
removed; otherwise the essential elements will be released by the fungi.
Calcium oxide and magnesium carbonate may replace calcium carbonate
in some applications (Steinberg, 193oa) . The mode of purification appears
to be as follows: During autoclaving, heavy-metal carbonates or their
hydroxides are formed. The excess calcium carbonate serves to adsorb
these insoluble compounds The composition of a medium is somewhat
changed by this treatment, part of the phosphate being removed as cal-
cium phosphate. In practice this is compensated by using an excess of
phosphate. A medium which is treated by this process is essentially
neutral in reaction, which may lead to some changes in the sugar during
autoclaving.

ESSENTIAL METALLIC ELEMENTS 73

Sugars are frequently highly contaminated with metallic compounds.
Steinberg (1937) has reported a sample of glucose to contain the following
elements: lithium, sodium, strontium, calcium, rubidium, potassium,
manganese, aluminum, iron, rhodium, nickel, silver, copper, magnesium,
tin, boron, and silicon. The metallic contamination of non-ionic com-
pounds such as the sugars can be sharply reduced by a variety of mild
procedures. Shu and Johnson (1948) give these details for an aluminum
hydroxide coprecipitation method: To 140 g. of glucose contained in 500
ml. of solution, 1.25 g. of Al2(S04)3'18H20 were added. Dilute ammo-
nium hydroxide was added until the pH rose to 9, and the precipitate of
A1(0H)3 Avith the adsorbed impurities was filtered off. This treatment
was repeated until the desired degree of purification was attained. Non-
ionic substances such as glucose and urea may be purified by treatment
with cation-exchange materials operating on the hydrogen cycle. Perl-
man (1945) used Zeo-karb H (Permutit Corporation) for this purpose.
Various ion-exchange materials are used to purify beet juice in the manu-
facture of sugar. Mulder (1939-1940) found the combination of ammo-
nium sulfide and Norit to be efficient in removing copper from media.
The sulfide ion forms insoluble heavy-metal sulfides while the activated
carbon serves as a "gatherer."

Complex-forming reagents such as diphenylthiocarbazone (dithizone)
(Stout and Arnon, 1939), and 8-hydroxyquinoline (Waring and Werkman,
1943) are useful in removing heavy-metal ions from, or testing the purity
of, salts used in preparing media. These reagents and the metal com-
plexes they form are removed from solutions by extraction with chloro-
form or other organic solvents. The chemistry of complex formation
between organic compounds and ions is treated by Yoe and Sarver (1941).
Others have merely added such complex-forming reagents to the media,
in which the various metallic ions combine with the reagent to form non-
available compounds. The specificity of the reagent, concentrations of
reagent and the metallic ion or ions, the pH of the medium, as well as the
stability of the complex, enter into the success of this type of treatment.
Hickey (1945) found that 2,2'-bipyridine inactivated ferrous iron in media
treated with this reagent. It is better to remove metallic impurities from
the media by extraction than to depend upon complexing compounds to
hold these ions in non-ionic combination.

Certain compounds used in making media such as the amino acids and
hydroxy acids form non-ionized complexes with various metallic ions.
Media containing these types of compounds are difficult to free from
metallic contamination. In addition, some fungi excrete hydroxy acids,
such as citric acid, which may modify the availability of the essential
micro elements.

Media may be freed of essential micro elements by a biological process.

74 PHYSIOLOGY OF THE FUNGI

If a fungus is grown on a medium, it will absorb and utilize the essential
elements present in the medium. The success of this procedure depends
upon having a low initial concentration of the essential micro elements,
which soon become exhausted so that the culture liquid no longer supports
growth. Removal of the mycelium will thus remove the elements which
have been taken up. The culture filtrate may then be used as a medium
relatively free of micro elements. However, fungi excrete various com-
pounds which may affect the results. MacLeod and Snell (1947) have
recently utilized this method in studying the mineral nutrition of some
lactic acid bacteria.

Iron. Raulin's claim that iron was essential for fungi was questioned
at first, but his findings were soon confirmed. So far as is known, iron is
essential for all fungi. It may be noted that, in the absence of another
essential element in the medium, iron alone may cause little or no response.
If the zinc content of a medium is low, the addition of iron to an iron-free
medium will have little effect. This situation is true of any essential
nutrient. Only one element may be studied at a time, but all the other
essential nutrients must be present before the effect of the nutrient under
investigation can be studied. Some results of Steinberg (1919) with
Aspergillus niger on media purified by the calcium carbonate method are
given in Table 11. Neither iron nor zinc alone had much effect on the
growth of A. niger, since both of these elements are essential for this
fungus.

Table 11. The Effect of Iron and Zinc, Singly and in Combination, on the

Amount of Growth of Aspergillus niger

(Steinberg, Am. Jour. Botany 6, 1919.)
Essential Micro Element Mg. Mycelium

Added

Control (none added) 18

Iron 44

Zinc 40

Iron plus Zinc 731

Little interest has been shown in recent years in proving iron to be an
essential element for a large number of fungi. In view of the almost uni-
versal occurrence of a group of iron-containing enzymes (catalase, the
cytochromes, cytochrome oxidase, etc.), the essential role of iron is taken
for granted.

The most obvious effect of suboptimal iron concentrations upon fungi
is decreased growth. This result is probably due to the decreased and
limited amounts of iron-containing enzymes formed under these condi-
tions. It was shown by Yoshimura (1939-1940) that the amount of
catalase produced by Aspergillus oryzae increased as the amount of iron
in the medium increased. Lilly and Leonian (1945) showed that a rela-

ESSENTIAL METALLIC ELEMENTS

tion existed between the amount of iron supplied in the medium and the
ability of Rhizohium trijolii to synthesize certain vitamins. In the pres-
ence of suboptimal concentrations of iron the addition of certain vitamins
replaced iron to a certain degree. A quantitative study of the ^ntamins
synthesized by Torulopsis utilis has shown the iron concentration to be
important (Lewis, 1944). Increased amounts of thiamine, riboflavin,
nicotinic acid, and pyridoxine were synthesized on media low in iron, while
the amounts of biotin, inositol and p-aminobenzoic acid were decreased.

L) 10 15 20

Mg. ferric sulfate per liter

Fig. 14. The effect of iron [Fe2 (804)3] in overcoming the inhibitory action of copper
(CuS04-5H20) on the production of penicilUn by Penicillium chnjsogenum X-1612.
An amount of copper sufficient to inhibit peniciUin production entirely did not affect
the amount of growth. The fungus was cultured submerged in a lactose-starch-
dextrin-ranmonium sulfate medium for 7 days. (Curves drawn from data of Koffler
et al., Jour. Bad. 53 : 120, 1947. Pubhshed by permission of The Williams & Wilkins
Company.)

There has been a great deal of interest in the effects of iron and other
metallic ions on various microbiological processes. Perlman et al. (1946)
have shown that the iron concentration is an important factor in citric
acid fermentation by Aspergillus niger. The optimum iron concentration
for citric acid production varied over tenfold for different strains of
A. niger. The effect of iron on penicillin production has been studied by
Kofl^ler et al. (1947), who concluded that the effect of the ash of corn steep
is due to iron and phosphate. Chromium increased penicillin production
above that obtained with iron and phosphate, presumably by neutralizing
the effect of other ions. Similarly an antagonism was shown to exist
between copper and iron. The antagonistic effect of copper and iron on
the production of penicillin by Penicillium chrysogenum X-1612 is shoA\Ti
in Fig. 14.

76 PHYSIOLOGY OF THE FUNGI

The iron concentration of the medium has been shown to affect the
amount of pigmentation of Torulopsis pulchcrrima (Roberts, 1946).

Zinc. This element is essential for Aspergillus niger (Raulin, 1869;
Steinberg, 1919). Foster (1939) lists Trycliophytoninterdigitale, Rhizopus
nigricans, and Saccharomyces cerevisiae as recjuiring zinc, and Roberg
(1928) found zinc to be essential for A. fumigatus and A. oryzae. Blank
(1941) reported the amount of growth oi Phymatotrichum omnivorum to be
increased by the addition of zinc to a medium treated with calcium car-
bonate, and Perlman (1948) noted that the sclerotia of Sclerotium
delphinii are more highly pigmented in the presence of added zinc.

Zinc ions activate (and inhibit) various enzymes such as enolase and
dipeptidase. Zinc is contained in carbonic anhydrase, an enzyme which
catalyzes the decomposition of carbonic acid to carbon dioxide and water.
In addition to these specific uses the zinc concentration has a decided
effect on a number of physiological or biochemical processes in fungi.
Foster and Waksman (1939) found that the production of fumaric acid
from glucose by Rhizopus nigricans varied according to the amount of zinc
added to the medium. Fumaric acid was produced most efficiently when
the concentration of zinc was low (1.2 mg. per liter). Higher concentra-
tions of zinc resulted in increased growth and decreased production of
fumaric acid. From these results it appears that zinc plays a role in the
utilization of glucose, the completeness of oxidation and assimilation
being favored by relatively high concentrations of zinc. A somewhat
similar effect of zinc on the production of lactic acid by Rhizopus sp. has
been noted (Waksman and Foster, 1938). Zinc was found to cause
increased growth and a decrease in the production of lactic acid, while the
effect of iron is to increase the yield of lactic acid. For a further dis-
cussion of the mechanism of zinc in fungus metabolism, see Foster (1949).

Copper. This element is essential for animals, green plants, and fungi.
From the work of Steinberg (1936) it appears that 0.04 mg. of added
copper per liter of purified medium is sufficient for the maximum growth
of Aspergillus niger. Under these conditions omission of copper decreased
the yield only from 984.8 to 774.3 mg. It is probable that purification of
the medium by the calcium carbonate treatment is not very satisfactory
for this element. The ^veight of metal needed to obtain maximum growth
with A. niger is much less for copper than for iron or zinc. The experi-
mental difficulties increase as the amount of a micro element needed
becomes less. Apparently it is very difficult to prepare a copper-free
medium. Roberg (1931) made use of Bortel's method of adding a trace
of ammonium sulfide to convert heavy metals to sulfides and adsorbing
these impurities with charcoal. This treatment is very efficient in remov-
ing iron and zinc but somewhat less satisfactory for removing copper.
The essential nature of copper for A. flavus and Rhizopus nigricans was

ESSENTIAL METALLIC ELEMENTS

shown by McHargue and Calfee (1931). The full effect of copper was
dependent upon the presence of other essential elements. The coloration
of conidia of A. niger has been shown to depend upon the copper content
of the medium (Javillier, 1939).

Although copper is an essential element, it is a constituent of many
fungicides (Chap. 12). The concentration, therefore, is a very important
consideration in studying the effect of this element. The phenomenon of
ion antagonism must also be considered, for the effect of a given amount
of copper is dependent upon the other constituents of the medium.
Marsh (1945) investigated the antagonistic effects of three salts upon
copper as it affected germination of conidia of Sclerotinia fructicola

Table 12. The Antagonistic Effect of Three Salts on Copper as Shown by

THE Germination of Conidia of Sclerotinia fructicola

(Marsh, Phytopathology 35, 1945.)

Salt concen-
tration

Percentage Germination in

4 X 10-^/ CuS04, plus

0.01% glucose

MgS04

CaCh

KCl

0.0

1.2

0.8

2.9

10-^M

54.0

31.0

3.9

10-^il/

67.0

62.0

2.6

IQ-'M

78.0

83.0

3.9

10-2M

—

59.0

(Table 12). It was shown that the mechanism of the protective action of
these salts was to decrease absorption of copper. There is no reason to
assume that the absorption and utilization of copper from nutrient solu-
tions would not be affected similarly. Thus, the amount of copper added
to a nutrient solution may reflect only imperfectly the amount absorbed
and used by a fungus.

It was noted in Chap. 4 that copper is an essential constituent of certain
enzymes, including tyrosinase, which occurs in many fungi. Nelson and
Dawson (1944) suggest that tyrosinase functions in the respiration chain
as an oxygen shuttle.

Manganese. The classification of this element as essential rests upon
the experimental findings that omission of this element from media
results in decreased yields. The multiplication of examples strengthens
the validity of this conclusion, although most investigators have confined
their attention to a relatively few species. The results of Robbins and
Hervey (1944) with Pythiomorpha gonapodyoides indicate that investiga-
tion of fungi other than Aspergillus niger with regard to micro-element

I'lIYSlOWGY OF THE FUSGl

nutrition may be rewarding. It was unnecessary to resort to elaborate
methods of medium purification to demonstrate that manganese is essen-
tial for P. gonapodyoides. This situation occurred only when reagent
magnesium sulfate of a certain manufacture was used. Substitution of
another brand of magnesium sulfate revealed heavy (biological) con-
tamination by manganese (Fig. 15). The inoculum was found to carry
sufficient manganese and other micro elements to influence the amount of
growth in the first passage. No growth resulted in the third passage in

A B

Fig. 15. Pythiomorpha gonapodyoides growing in a basal solution \Yith no added
mineral supplements. A, medium prepared with Baker's Analyzed magnesium
sulfate. B, medium prepared with Mallinckrodt's magnesium sulfate analytical
reagent. Age, 5 days. Note the extensive white mj'celium in A and the slight
growth in B. (Courtesy of Robbins and Hervey, Bull. Torrey Botan. Club 71: 263,
1944.)

the absence of added manganese. The range of manganese concentra-
tions for optimum growth was narrow and appeared to depend upon the
concentration of other micro elements present, particularly zinc. Stein-
berg (1935) found manganese to be essential for A. niger. McHargue and
Calfee (1931, 1931a) noted that growth of A. flainis, Rhizopus nigricans,
and Saccharomyces cerevisiae increased in the presence of added
manganese. Steinberg (1945) showed that omission of manganese from
a balanced medium resulted in a decrease in yield of A. niger from
1,084.8 to 356.6 mg. No spores formed when manganese was omitted.
It is interesting to note that, as the numbers of spores used for inoculum

ESSENTIAL METALLIC ELEMENTS 79

decreased, A. niger became more sensitive to micro-element deficiencies
in the medium. The favorable effect of adding biotin to the medium
when only a few spores were used as inoculum suggests an intimate con-
nection between micro-element nutrition and the synthesis of this vita-
min. Whether the decreased yield due to small inoculum was due to
other deficiencies or to a decreased rate of growth is not entirely clear, as
all harvests were made after 4 days.

Manganese (Mn++) has been shown to be the natural activator of
yeast arginase. Other enzymes are activated by this element (Sumner
and Somers, 1947). In view of the small amounts of manganese required
by fungi, it may be assumed that manganese functions as a constituent of
various enzymes.

Molybdenum. The study of the role of this element emphasizes the
similarity in certain physiological processes throughout the plant king-
dom. The most striking feature of this essential element is its role in
nitrogen metabolism. The utilization of nitrate nitrogen by green plants
and fungi and the fixation of atmospheric nitrogen by bacteria {Azotohac-
terchroococcum, Clostridium pasteimanum) is dependent upon molybdenum
(Bortels, 1930, 1936).

Our knowledge of the effect of molybdenum on fungi is largely confined
to Aspergillus niger. Steinberg (1936, 1937) found that more molybde-
num was required by A. niger for maximum growth in media containing
nitrate nitrogen than in media with ammonium nitrogen. Steinberg
expressed the opinion that molybdenum is essential for A . niger even when
ammonium nitrogen is available. Additional studies on A. niger and
other organisms (Mulder, 1948) indicated that an increased need for
molybdenum is associated with nitrate utilization. It may be assumed
that the enzymatic reduction of nitrate is carried out by enzymes which
require molybdenum as an activator.

In view of the important role of molj^bdenum in the utilization of
nitrate nitrogen, care should be used in comparing the value of different
nitrates. Unless sufficient molybdenum is present, misinterpretations
may result. Steinberg (1937) found the amount of molybdenum present
as an impurity in various nitrates to vary. One sample of calcium nitrate
contained enough molybdenum to support maximum growth of A. niger.
Perhaps the report of Young and Bennett (1922) that many fungi made
better growth on calcium than on potassium nitrate may be partially
explained on the basis of the molybdenum content of these two salts.
This explanation, of course, must allow for the effect of calcium, which is
now known to be essential for certain fungi.

Calcium. This element was one of the first to be recognized as essen-
tial for green plants and animals. In 1922, Young and Bennett reported
that Rhizocionia solani made no growth in the absence of this element

PHYSIOLOGY OF THE FUNGI

This report apparently attracted little attention since most investigators
working on this problem confined their attention to Aspergillus niger.
The value of using more than one fungus to demonstrate the essential
nature of calcium was strikingly shown by Steinberg (1948, 1950). These
data are given in Table 13.

It is evident from the data in Table 13 that the essential nature of cal-
cium for certain fungi is established. The concentrations of calcium
required for maximum growth varied from 2 to G mg. per liter of medium.
On the other hand, neither A. niger nor Fusariuni oxysporum needs more

Table 13. Effect of the Omission of Calcium from the Medium on the

Growth of Seven Fungi
(Steinberg, Science 107, 1948.)

Fungus

Aspergillus niger

Rhizoctonia solani

Sclerotium I'olfsii

Cercos-pora nicotianae

Fusarium oxysporum var. nicotianae.

Pythium irregulars

Thielaviopsis basicola

Calcium added,

Calcium not added,

mg. mycelium

percentage of yield

1,250.0

100.0

1,215.1

14.3

1,082.3

49.5

1,380.2

90.1*

823.3

100.0

459.0

60.1*

364.2

82.0*

* Asparagine of unknown purity was used as a sovirce of nitrogen.

than spectroscopic traces of calcium, if they require this element at all.
Steinberg is of the opinion that further advances in purity of nutrient
solutions will reveal more uniformity in the essential element require-
ments of organisms.

Lindeberg (1944) has demonstrated a synergistic effect between manga-
nese and calcium upon the growth of various species of Marasmius.

Table 14. The Effect of Increasing Concentrations of Calcium and Manga-
nese, Alone and in Combination, on the Growth of Marasmius epiphyllus
(Lindeberg, Symbolae Botan. Upsalie7isis, 8: 2, 1944.)
(Dry weight mycelium in milligrams.)

Mn, millimoles
per liter

Ca, millimoles per liter

0.005

0.05

0.5

0.0

0.0005

0.005

0.05

0.5

10.1

11.1

10.7

18.2
20.3

19.8
18.0
18.3
20.8

35.8

33.0
38.4
35.7
47.6
48.6

73.5

83.8
78.5
77.0
52.6

ESSENTIAL METALLIC ELEMENTS 81

Within limits, the growth of M. aUiaceus and M. epiphyllus was propor-
tional to the concentration of either of these elements, and the response
to each element was modified by the presence of the other. The data in
Table 14 illustrate this effect.

In addition to the essential micro elements discussed above, there is
some evidence which indicates the essentiality of other metallic elements
for the fungi. Certain of these elements are essential for other organisms.

Gallium. Under certain conditions Steinberg (1938) was able to show
that omission of this element from the medium led to decreased yield and
sporulation of Aspergillus niger. Extraordinary care was needed to
demonstrate gallium deficiency. The chemicals used were spectroscopi-
cally pure with the exception of traces of iron, calcium, and sodium. The
sucrose, after 6-hr. extraction with alcohol, contained only 0.0014 per cent
ash. The water used was triple-distilled, the last distillation being made
in a quartz still. Spectroscopically pure calcium oxide w^as used to purify
the sucrose further. Under these conditions the yield of A. niger
increased from 814 mg. to 1,053 mg. when gallium (0.02 mg. per liter) was
added to the medium. The salts of 76 other chemical elements were
tested, and none was found to replace gallium. In view of the similar
chemical behavior of gallium and aluminum, Steinberg considers it possi-
ble that the biologic activity sometimes attributed to aluminum may in
reality be due to gallium.

Scandium. In the discussion of the role of manganese in nitrogen
metabolism it w^as noted that the amount of manganese required was
determined by the nitrogen source used. In a somewhat similar fashion,
Steinberg (1939) found that scandium appeared to be essential when
glycerol was used as a carbon source for Aspergillus niger. Growth was
poor on this carbon source ; omission of copper or manganese increased the
yield somewhat. Omission of scandium decreased the yield from 269.4 to
107.4 mg. Interestingly enough, scandium appeared to have no effect on
growth when sucrose was used as a source of carbon. Addition of lysine
or proline (20 mg. per liter) to the glycerol medium increased growth and
at the same time prevented the effect of scandium. These results suggest
that the need for certain elements may be shoA\Ti only under certain
nutritional conditions.

Vanadium. Bertrand (1943) reported the presence of this element in
all fungi examined. Amanita muscaria contained from 61 to 156 mg. of
vanadium per kilogram. Bertrand (1941) considers vanadium as an
essential element for Aspergillus niger.

Cobalt. Whether fungi require some, or all, of the other metallic ele-
ments required by other organisms is not kno^vn. Cobalt is required by
animals. Lack of sufficient amounts of this element in the soil causes
severe cobalt deficiency in animals which are pastured on such soils.

PHYSIOLOGY OF THE FUNGI

Recently, a cobalt-containing vitamin (B12) was isolated. This vitamin
is synthesized by Streptomyces griseus (Rickes et al., 1948) and some
bacteria. Whether S. griseus requires cobalt as an essential element for
growth or reproduction is not known. The synthesis of this vitamin is
necessarily dependent upon a supply of cobalt. Some bacteria are known
to be deficient for vitamin B12.

PERIODICITY OF BIOLOGICALLY ESSENTIAL ELEMENTS

Steinberg (1938a), Frey-Wyssling (1935), and others have considered
the problem of biologically essential elements in relation to the structure

Table 15. A Portion of the Periodic Table of Elements Based on Atomic

Number
The biologically essential elements are set in italics. Those elements essential for
fungi are marked with an asterisk.

Group

Mg*

7v'*

Ca*

Sc*

Mn*

Fe*

Co Ni

27 28

Cu*

Z71*

Ga*

Mo*

Rh Pd

45 46

and atomic number of the elements. The biologically essential elements
are in italics in Table 15. It is noteworthy that the essential elements
tend to occur in groups with consecutive atomic numbers. Atomic num-
ber is a fundamental property of atoms and denotes the number of excess
positive charges on the nucleus. Only those elements which have certain
configurations are required by organisms. Why some organisms require
certain elements not required by others is not known.

ESSENTIAL METALLIC ELEMENTS 83

SUMMARY

The role of the essential metallic elements is primarily functional rather
than structural. Presumably these ions usually function in ionizable
combinations, but some compounds containing metals in non-ionizable
compounds have been isolated from fungi. It may be assumed that
many of these metallic ions activate enzyme systems, while others are
integral parts of enzymes and other essential organic compounds. An
element is essential because some of its vital functions cannot be replaced
by any other element. Some functions may be performed by other closely
related elements.

The concentration of an essential element affects many life processes
besides growth, which is the usual criterion of essentiality. The concen-
trations of various essential ions influence the formation of pigments, the
synthesis of vitamins and other products, and the dissimilation of carbo-
hydrates. While the essential elements may be supposed to participate
uniquely in certain life processes, the concentrations of other ions, both
of essential and nonessential elements, modify the action of a given ele-
ment. The phenomenon of ion antagonism no doubt exists among all
ions, and in evaluating the effects of any element it is necessary to con-
sider the other constituents present in the medium. It is probable that
the mechanism involved is one of modified adsorption rather than any
direct chemical reaction in the medium.

The widespread use of Aspergillus niger as a test fungus in micro-
element studies has had the advantage that the work in many laboratories
may be compared. The careful and long-continued studies by Steinberg
are especially valuable. The almost exclusive use of this fungus has also
had its disadvantages. Comparatively little is known about the need of
other species for micro elements. Other fungi may require some of these
elements in amounts which make it comparatively easy to demonstrate
deficiency. The evidence for the essentiality of iron, zinc, copper,
manganese, molybdenum, and calcium is impressive in most instances,
but the need for the elements on the part of all fungi under all cultural
conditions has not been established. In a few instances the evidence is
confined to a single fungus. The micro-element nutrition of a wide range
of species needs further study.

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Blank, L. M.: Response of Phymatotrichum omnivorum to certain trace elements,

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84 PHYSIOLOGY OF THE FUNGI

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ESSENTIAL METALLIC ELEMENTS 85

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Mulder, E. G.: Importance of molybdenum in the nitrogen metabolism of micro-
organisms and higher plants, Plant and Soil 1: 94-119, 1948.
MuNTZ, J. A. : The role of potassium and ammonium ions in alcoholic fermentation,

Jour. Biol. Chem. 171: 653-665, 1947.
Nelson, J. M., and C. R. Dawson: Tyrosinase, Advances in Enzymol. 4: 99-152,

1944.
NiCKERSON, W. J. : Inhibition of fungus respiration : a metabolic bio-assay method,

Science 103 : 484-486, 1946.
Perlman, D. : Some effects of metallic ions on the metabolism of Aerobacter aerogenes,

Jour. Bad. 49: 167-175, 1945.
Perlman, D.: On the nutrition of Sclerotium delphinii, Am. Jour. Botany 35: 360-

363, 1948.
*Perlman, D.: Effects of minor elements on the physiology of fungi, Botan. Rev. 15:

195-220, 1949.
Perlman, D., D. A. Kita, and W. Peterson: Production of citric acid from cane

molasses. Arch. Biocheni. 11: 123-129, 1946.
Rabinovitz-Sereni, D.: Influenza del magnesio suUo sviluppo di alcuni funghi, Dol.

R. Staz. Veg. 13 : 203-226, 1933.
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Richards, O. W., and M. C. Tkoutman: Spectroscopic analysis of the mineral con-
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Rickes, E. L., N. G. Brink, F. R. Koniuszy, T„ R. Wood, and K. Folkers:

Crystalline vitamin B12, Science 107 : 396-397, 1948.
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Aspergillus niger, Arch. Mikrohiol. 5: 561-577, 1934.
RoBBiNS, W. J., and A. Hervey: Response oi Pythiomorpha gonapodyoides to manga-
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86 PHYSIOLOGY OF THE FUNGI

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Arch. Biochem. 1 : 303-310, 1943.
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culture media, Am. Jour. Botany 9: 459-469, 1922.

CHAPTER 6

THE ESSENTIAL NONMETALLIC ELEMENTS OTHER

THAN CARBON

Fungus mycelium and spores are composed mainly of compounds of the
nonmetallic elements. As a rule, more than 95 per cent of the fungus
consists of hydrogen, oxygen, carbon, nitrogen, sulfur, and phosphorus.
The nonmetallic essential elements are both structural and functional.
The cell wall, which is composed mainly of chitin or cellulose, appears to
be the most stable structure of the fungus. Protoplasm is highly labile,
and the constituent compounds of protoplasm are continually undergoing
destruction, repair, and synthesis. The various structural and functional
compounds of organisms are in a state of continual flux (Hevesy, 1947).
The turnover of essential elements in functional compounds is more rapid
than in structural compounds.

The terms utilization, assimilation, and dissimilation are frequently
used in physiology. Utilization is a broad term and implies that an
organism uses or gains some benefit from a specific substance. Fungi
utilize water as a solvent but derive neither energy nor substance from it.
Assimilation is the incorporation of substances or their degradation
products into cellular materials. Assimilation implies synthesis. Dis-
similation is the degradation, or breakdown, of complex compounds into
simpler ones. This term is particularly applied to those processes such as
alcoholic fermentation where intermediate metabolic products accumu-
late in the medium. Frequently dissimilation must precede assimilation
and may be considered as the first phase of utilization.

HYDROGEN

Hydrogen enters into the composition of nearly all organic compounds
of interest to physiology except carbon dioxide. This is true of the
organic nutrients used by fungi as well as of the fungus protoplasm and
other cellular compounds. Elemental hydrogen is not used by fungi.
All the hydrogen utilized by fungi is in chemical combination. Certain
bacteria (hydrogen bacteria), however, are able to obtain energy by
oxidizing hydrogen.

The importance of water for all living organisms is so great that it
seems impossible to conceive of life without water. The formula H2O is
really the formula of steam. In the liquid state these simple molecules

88 PHYSIOLOGY OF THE FUNGI

associate to form polymers. At room temperature water consists mostly
of (H20)3, which is sometimes called trihydroL For a further discussion
of water see Barnes (1937).

The chemistry of life processes is largely confined to reactions which
take place in the presence of water or in solution. In addition to being a
solvent of remarkable powers, water is associated with the colloids which
comprise protoplasm. Gortner (1949) has distinguished between "free"
and "bound" water. Free water is mobile within the cell and serves as a
solvent and for the purpose of translocation of the various products of
metabolism. Bound water is firmly adsorbed by protoplasm, and in this
form water does not freeze. This property of bound water enables cells
to withstand low temperatures. The ability of fungus spores to with-
stand low temperatures may well be due to their having most of their
water content in the bound form.

Water ionizes to form hydrogen (H+) and hydroxyl (0H~) ions.
The effects of these ions on biological processes are so important that they
will be discussed in detail in Chap. 8.

OXYGEN

Apparently none of the fungi are obligate anaerobes. Many are
strictly aerobic, and some are facultatively anaerobic. An aerobic
organism requires uncombined oxygen, while a facultative anaerobe may
use combined oxygen in addition to free oxygen. The amount of oxygen
required for optimum growth varies with the species. It is common to
express the amount of oxygen available in terms of millimeters of mercury.
Approximately 21 per cent of air is oxygen. The amount of oxygen may
be regulated by controlling the air pressure within the culture vessel. If
the barometric pressure is 740 mm. Hg, the partial pressure due to oxygen
is ^Hoo X 740, or 155.4 mm. Hg. If the pressure within a culture vessel
is reduced to 100 mm. Hg, the partial pressure of oxygen amounts to 21
mm. Hg. Tamiya (1942) has reported that Aspergillus onjzae has a
maximum rate of respiration when the partial pressure of oxygen is 500
to 630 mm. Hg. Such partial pressures of oxygen are readily obtained by
using oxygen-nitrogen mixtures. Ternetz (1900) reported the following
effects of reduced oxygen supply on Ascophanus carneus: at 10 mm. Hg the
mycelium grew with difficulty; at 20 mm. Hg growth was good, but no
spores formed; at 40 mm. Hg some fructification occurred; at 120 to 140
mm. Hg growth was somewhat better than at atmospheric pressure.

The ability of certain soil fungi to exist under conditions of low oxygen
supply is important for survival. The amount of oxygen in soil depends
upon the soil type and the amount of water present. Soil saturated with
water contains but a trace of free oxygen. Hollis (1948) found Fusarium
oxysporum to survive under essentially anaerobic conditions for 13 weeks,

ESSENTIAL NONMETALLIC ELEMENTS 89

while F. eumartii perished within 3 weeks when exposed to the same condi-
tions, The mycehum of F. oxysporuni grown under reduced oxygen ten-
sion was abnormal in its morphology. For further information on the
effect of reduced oxygen tension, see Fellows (1928) and Scheffer and
Livingston (1937).

Enormous amounts of sterile air must be supplied to the 10,000- to
15,000-gal. tanks used in the production of penicillin and other antibiotics.
In the laboratory, aeration is provided by shaking machines of the rotat-
ing or reciprocal type. Aeration under these conditions is more uniform
than is possible in stationary cultures, W'here submerged and aerial
hyphae obtain different amounts of oxygen. This was sho\Mi by Tamiya
(1942) who reported that the enzyme systems of submerged mycelium of
Aspergillus oryzae are more easily poisoned by cyanide than are those of
aerial mycelium.

In a broad sense, respiration denotes all the enzymatic processes which
occur in cells involving a release of energy. There are two general ways
in which energy is released by living cells: (1) Cells obtain energy from
chemical reactions in which free oxygen is a reactant. The oxidation of
metabolite molecules by this process is generally called respiration, or
more specifically aerobic respiration. This process is characterized by the
intake of free oxygen and the formation of carbon dioxide. If the com-
pound being oxidized is composed of carbon, hydrogen, and oxygen only,
the products are carbon dioxide, water, and energJ^ (2) Cells also obtain
energy from chemical reactions in which free oxygen is not a reactant.
This process is called anaerobic respiration, or fermentation. Metabolic
processes of this kind are characterized by the production of carbon
dioxide, the incomplete oxidation of substrate molecules, and the release
of a small amount of energy.

The reactions involved in the aerobic respiration of glucose may be
summarized in a single equation:

CsHisOe + 60.-^ 6CO2 + 6H2O + 673,000 cal.

This equation gives no indication of the intermediate stages in this reac-
tion or how the energy is utilized by the organism performing the oxida-
tion. The number and variety of intermediate reactions do not affect
the total amount of energy released. The reactions involved in the
alcoholic fermentation of glucose are summarized in the following equation :

CeHisOs-* 2CH3CH2OH + 2CO2 + 25,000 cal.

This equation, like the preceding one, gives no indication of the inter-
mediate reactions involved. To obtain the same amount of energy, more
of a compound must be fermented than when it is completely oxidized.
Not all of the energy released by either of these processes is available to
the organism (Chap. 4).

90 PHYSIOLOGY OF THE FUNGI

A knowledge of the amounts of oxygen consumed and carbon dioxide
evolved by organisms is the basis of a useful method of study in many
phases of physiology. The principles of such measurements are simple.
In aerobic respiration both the oxygen and carbon dioxide may be meas-
ured. The ratio of the moles, or volumes, of carbon dioxide evolved and
oxygen used is called the respiratory quotient (R.Q.) and is written
CO2/O2. From the respiratory quotient the nature of the substrate
being oxidized may be deduced. A respiratory quotient of 1 is character-
istic of aerobic oxidation of carbohydrate. The complete oxidation of a
fat may be represented as follows:

(C,8H3602)3C3H5 + 81.50-2^ 57CO0 + 55H.2O

The respiratory quotient for this fat is 57/81.5, or 0.7. If fungus cells are
suspended in a buffer in the absence of nutrients, and the respiratory
quotient determined, it is possible to deduce the type of compound within
the cells being used as a source of energy. Oxidation of the stored com-
pounds within the cell is called endogenous respiration. The oxidation of
substrate molecules from the medium is called exogenous respiration.
Since both types of respiration may occur simultanously in the presence
of nutrients, it is necessary, in order to determine exogenous respiration,
to subtract the value for endogenous respiration from that obtained in the
presence of nutrients.

The rate and amount of respiration are determined by instruments
known as respirometers. Various types of respirometers have been used
to investigate different phases of fungus metabolism and nutrition. In
principle a respirometer is a closed vessel of known volume in which
fungus cells are suspended in a buffer or other solution. The carbon
dioxide evolved is absorbed in a concentrated solution of potassium
hydroxide. The change in volume due to the consumption of oxygen is
measured by the use of suitable manometers. At the end of the experi-
ment the amount of carbon dioxide evolved is measured after the potas-
sium hydroxide solution is treated with a mineral acid. Carbon dioxide
alone may be measured by passing a stream of carbon dioxide-free air
through a culture and absorbing the carbon dioxide evolved in barium
hydroxide or other suitable reagent. The results of such experiments are
reported on the basis of the volumes of oxygen used and carbon dioxide
evolved per milligram of dry weight per hour. These values are reported
as Q02 and Qco2 (see Umbreit et at., 1945).

A modern respirometer is illustrated in Fig. 16. The various manipula-
tive details will not be discussed. For an adequate treatment of these
see Umbreit etal. (1945) and Dixon (1943). These methods are extremely
useful in studying a wide range of problems. Hawker (1944) used
manometric techniques in studying the effect of excess thiamine on

ESSENTIAL NONMETALLIC ELEMENTS

glucose utilization by Melanospora destruens and Phycomyces nitens.
The papers of Siu and Mandels (1950) and Mandels and Siu (1950)
should be consulted for details concerning a simple differential manometer.
This manometer is designed to measure the respiration of intact growing
cultures of filamentous fungi. Dorrell (1948) investigated the effect of

Fig. 16. A constant-temperature bath and shaking device for micro respiration
studies. (Courtesy of American Instrument Company.)

dinitrophenol on endogenous and exogenous respiration of Fusarium
graminearum (Gibberella zeae). As usually carried out, respiration experi-
ments last only a few hours. The initial state of the cells or mycelium

Table 16. The Effect of Age of Zygosaccharomyces acidifaciens Cells on the

Amount of Aerobic Respiration
(Nickerson and Carroll, Jour. Cellular Cornp. Physiol. 22, 1943. Published by
permission of the Wistar Institute of Anatomy and Biology.)

Age of cells,
hr.

Qo.*

Glucose substrate

No substrate
(endogenous)

60
35
35.5

16
7.3

7.0

* Q02 equals lil O2 per hr. per mg. dry cells.

92 PHYSIOLOGY OF THE FUNGI

used has a great effect on the results obtained. Nickerson and Carroll
(1943) have indicated that the culture history of the cells used influences
the amount of aerobic respiration. Some of their data for Zygosaccharo-
myces acidifaciens are shown in Table 16.

SULFUR

Not all compounds which contain an essential element are equally
useful. In fact, some compounds are useless because the essential ele-
ment is unavailable. Among the factors which may affect availability is
the state of oxidation of the essential element. This is particularly true
of sulfur, phosphorus, and nitrogen. Among the organic compounds,
structure is enormously important. The situation is further complicated
in that not all fungi utilize the same compounds. Many examples of thi,
will be cited in connection with nitrogen and carbon nutrition. Atten-
tion must be given the sources of the essential elements as well as the uses
fungi make of them.

Sources of sulfur. This element is present in many types of com-
pounds, both inorganic and organic. The state of oxidation of sulfur, as
well as the specific structure of organic sulfur compounds, affects utiliza-
tion. Sulfate sulfur, SO4"", is the most common source of sulfur used in
media. Some fungi, however, require specific organic sources of sulfur.
Steinberg (1936, 1941) has made an exhaustive study of sulfur sources for
Aspergillus niger and reached the general conclusions that inorganic sulfur
compounds containing oxidized sulfur are utilized, while sulfide and
disulfide sulfur are not utiHzed. Of the organic compounds containing
sulfur, the alkyl thioalcohols, sulfides, and disulfides are not used.
Alkyl sulfonates and sulfinates are excellent sources of sulfur. Steinberg
is of the opinion that oxidized sulfur is reduced to suKoxylate before it
enters the normal metabolic channels. An exception to the nonutiliza-
tion of reduced sulfur was noted for compounds which occur as normal
metabolites, such as cysteine, cystine, methionine, and homocystine.
These are assumed to enter normal metabolic channels without pre-
liminary modification. An exception to this statement was noted with
thiamine (thiazole sulfur), but the enormous (physiologically) amounts
used may have upset the metabolic activities of the fungus.

In spite of the general utility of sulfate sulfur in fungus nutrition, many
fungi either utilize organic sulfur contained in natural metabolites to bet-
ter advantage or require these compounds as a source of sulfur. Leonian
and Lilly (1938) reported that the addition of cystine to a synthetic
medium was necessary for the grovv^th of Saprolegnia mixta, Achlya con-
spicua, Isoachlya monilijera, and Aphanomyces camptostylus. Since other
naturally occurring sulfur-containing amino acids were not tested, it
should not be concluded that these species are deficient for cystine.

ESSENTIAL NONMETALLIC ELEMENTS 93

Volkoiisky (1933, 1934) observed that certain of the aquatic Phycocomy-
cetes failed to utiHze sulfate sulfur. These species were Saprolegnia
parasitica, Isoachlya monilifera, Achlya prolifera, A. polya7idra, A. oblon-
gata, A. conspicua, Dichtyuchus monosporus, and Aphanomyces sp. A
total of 26 isolates failed to utilize sulfate sulfur. This investigator
(1933a) designates ability to utilize 6-valent sulfur as euthiotrophy and
inability to utilize sulfate sulfur and ability to utilize reduced sulfur as
parathiotrophy .

Fries (1946) was able to induce mutation in Ophiostoma (Ceratostomella)
rmiltianmilatum by irradiating the ascospores with X rays. Among
these mutants 13 strains were unable to utilize sulfate sulfur. Only five
of these strains regained this ability when cultivated on media containing
sulfate. These parathiotrophic strains of 0. multiannulaium utilized
ammonium sulfide as well as cystine and cysteine as sources of sulfur.
From the fact that these mutants could utilize sulfide sulfur, it is evident
that these strains were not deficient for specific sulfur-containing amino
acids. Bonner (1946) has, however, found induced mutants of Peni-
cillium to be deficient for specific sulfur-containing amino acids. Blasto-
cladia pringsheimii has been reported to require methionine (Cantino,
1949).

Fries (1948) has reported the occurrence of natural mutants of Ophi-
ostoma multiannulatiim which require reduced sulfur, and also mutants
which are unable to synthesize methionine. Of a total of 51,037 single-
conidium cultures, 2 required reduced sulfur and 30 required methionine.

The role of sulfur. The use fungi make of sulfur may be deduced from
the sulfur-containing compounds which are known to occur in mycelium
and spores. Among these are the proteins. In Chap. 4 it was noted that
the activity of many enzymes depends upon the sulfhydryl or thiol group,
■ — SH. On hydrolysis, fungus protein yields the following sulfur-contain-
ing amino acids: cystine, cysteine, and methionine. Sulfur is thus a
structural element. Another sulfur-containing compound is the tripep-
tide, glutathione, which is abundant in yeast. The formula for gluta-
thione is given below:

COOH CH2SH

H2N— CH— CH2— CH2— CONH— CH— CONH— CH2— COOH

This compound is sometimes represented by the symbol GSH. In spite
of intensive investigation the role of this compound is not fully under-
stood. Perhaps one of its functions is to protect sulfhydryl enzymes
from inactivation.

The probable mechanism of the biosynthesis of cystine has been studied
using mutants of Aspergillus nidulans (Hockenhull, 1949). All these
cystine-deficient mutants were able to utilize thiosulfate sulfur, methio-

94 PHYSIOLOGY OF THE FUNGI

nine, and cystine. It was postulated that sulfate sulfur was first reduced
to sulfite and then to sulfoxylate, which was assumed to dimerize to
thiosulfate. The next reaction was believed to be between serine and
thiosulfate to form cysteine S-sulfonate, which is then converted to
cysteine. Cysteine on being oxidized forms cystine.

Two vitamins, thiamine and biotin, contain sulfur. The role of these
compounds will be considered in Chap. 9. In addition to the sulfur-con-
taining amino acids and vitamins there is evidence that other types of
organic sulfur compounds are formed by fungi. Raistrick and Vincent
(1948) found that many strains and species of Aspergillus and Penicillium
converted essentially all of the sulfate sulfur into organic sulfur com-
pounds, but not all of these compounds were found in the fungus proteins.
Penicillium chrysogenum excretes into the medium various unidentified
organic sulfur compounds (Plumlee and Pollard, 1949). The function of
these compounds is unknown.

The reactions whereby a fungus transforms a single source of sulfur into
these various compounds are obscure. When sulfate or other sources
containing oxidized sulfur are utilized, it is necessary for the fungus to
reduce the sulfur to its lowest valence. Schizophyllum commune has been
shown to reduce sulfate to methyl mercaptan, CH3SH (Birkinshaw et al.,
1942). This substance contributes to the characteristic odor of this
fungus.

PHOSPHORUS

Raulin (1869) found phosphorus to be an essential element for Aspergil-
lus niger. Omission of phosphate from his synthetic medium reduced the
yield approximately 50 per cent. Phosphorus is essential for all forms of
life. Phosphorus may be classified as a structural element in the sense
that definite compounds containing this element have been isolated from
fungi. Phosphorus compounds play an important role in the functions of
chemical transformations and energy transfer.

Sources of phosphorus. Apparently phosphorus is utilized only when
it is in the form of phosphate. This element is taken up as phosphate and
functions in this form, mainly in the form of phosphate esters. It will be
recalled that there are several different phosphates. The formulas for the
potassium salts are K3PO4, potassium orthophosphate ; KPO3, potassium
metaphosphate ; and K4P2O7, potassium pyrophosphate. More complex
phosphates than pyrophosphate occur. Orthophosphoric acid may be
neutralized in three steps to produce the following types of salts: KH2PO4,
monopotassium orthophosphate; K2HPO4, dipotassium orthophosphate;
and K3PO4, tripotassium orthophosphate. All these salts furnish utiliz-
able phosphate, but the effects of these three salts on the acidity of the
medium are quite different. In addition to inorganic phosphates, the

ESSENTIAL NONMETALLIC ELEMENTS

organic phosphates (esters) may also be used as sources of this element.
Dox (1911-1912) investigated the assimilation of various phosphorus
compounds by Aspergillus niger with the following results: Ortho-,
meta-, and pyrophosphates supported excellent growth, as did such
organic compounds of phosphorus as phytin, sodium glycerophosphate,
sodium nucleinate, casein, and ovovitellin. Sodium hypophosphite
(NaH2P02-H20) and sodium phosphite (Na2HP03-5H20) were not
utilized and appeared to be toxic.

Smith (1949) studied the phosphorus metabolism of MeruUus lacrymans
and Marasmius chordalis in connection with the utilization of different
carbon sources. In glucose medium M. lacrymans grew better when
supplied w^ith inorganic phosphate, while M. chordalis grew miore rapidly
when supplied with organic phosphorus (adenylic acid). On cellobiose
medium M. lacrymans grew faster when supplied with organic phosphorus.

The role of phosphorus. An idea of the manifold ways in which phos-
phorus enters into fungus metabolism may be gained from the studies of
Mann (1944, 1944a). Aspergillus niger was grown on a glucose-nitrate
medium containing varying amounts of dipotassium orthophosphate.
Some of Mann's data on the effect of two concentrations of phosphate are
given in Table 17.

Table 17. The Effect of Two Concentrations of Orthophosphate upon the
Appearance, Sporulation, and Other Metabolic Functions of Aspergillus niger
(Mann, Biochem. Jour. 38, 1944. Published by permission of the Cambridge
University Press.)

Characteristics of

Grown in presence of

5-day-old cultures

0.02% K2HPO4

0.2% K2HPO4

MyceUum

Thin, white, smooth.

Thick, yellowish. No

Conidiophores present

conidiophores

Dry weight, mg.

460

1,092

Q02 of intact mycellium, lA

6.12

11.4

Total N, xng.

8.1

23.7

Total P, mg.

1.5

12.1

Thiamine, /xg

3.2

19.0

Riboflavin, ng

16.1

78.7

Nicotinic acid, ng

19.4

302.0

Medium

Colorless

YeUow

From Table 17 it may be seen that suboptimal amounts of phosphorus
affect the metabolism of A. niger in many ways besides diminishing
growth. Nitrogen utilization was affected, and the synthesis of three
vitamins (thiamine, riboflavin, and nicotinic acid) was greatly decreased.
The ability of phosphorus-starved mycelium to utilize oxygen was dimin-
ished, as shown by the lower Qo,- Mann also showed that utilization of

96 PHYSIOLOGY OF THE FUNGI

phosphate by A. niger takes place only in the presence of oxygen. The
utilization of phosphorus by yeasts, and presumably by other fungi which
are capable of anaerobic respiration, may take place in the absence of
oxygen. Various respiratory inhibitors such as iodoacetate, azide, and
cyanide inhibited both respiration and phosphorus metabolism. This
points to an intimate connection between carbohydrate and phosphorus
metabolism. By analysis, ortho-, meta-, and pyrophosphates were
found in the mycelium. Since only orthophosphate was supplied in the
medium, it is shown that A. niger is capable of these transformations.

Phosphorus appears to participate in almost every step in the anaerobic
dissimilation of glucose into alcohol by yeast. Some of these steps may
be common to other fungi. It is remarkable that the formation of alcohol
by yeast and lactic acid in muscle should follow almost the same pathways.
Phosphorus is required in the enzymatic transformation of glucose into
alcohol and carbon dioxide (Harden, 1932). Sumner and Somers (1947)
and Tauber (1949) have summarized the enzymatic reactions involved.

Either starch or glycogen may be transformed into glucose-1-phosphate
by enzymatic esterification. The shift of the phosphate radical to the
other end of the glucose molecules leads to glucose-6-phosphate, which
may also be formed by direct esterification of glucose. Glucose-6-phos-
phate is transformed into fructose-6-phosphate and then into fructose-
1,6-diphosphate. Scission of a molecule of fructose-l,6-diphosphate
yields dihydroxyacetone-1-phosphate and D-1-phosphoglyceric aldehyde.
An equally long series of transformations leads to pyruvic acid,
CHs — CO — COOH, Avhich on decarboxylation by the enzyme carboxylase
yields acetaldehyde, which is enzymatically reduced by DPN-H2 to ethyl
alcohol. Cocarboxylase and diphosphopyridine nucleotide (DPN) are
coenzymes, both of which contain phosphorus.

Gould et al. (1942) studied the formation of alcohol by Fusarnim
tricothecioides and found the limited production of alcohol by this species
was due to insufficient synthesis of diphosphopyridine nucleotide. Alco-
hol production was increased 20- to 25-fold by the addition of either yeast
extract or DPN to the medium. The paper of Semeniuk (1943-1944),
which deals with the relation of phosphorus to glucose dissimilation by
Chaetomium funicola, has an extensive bibliography (117 references).
Nord and Mull (1945) have summarized a long series of papers on the
physiology and biochemistry of Fusarium lini and reached the conclusion
that fermentation by this fungus follows a pathway which does not
involve the sugar phosphates. The review of Barron (1943) on the
mechanisms of carbohydrate metabolisms contains much information
about the role of phosphorus (219 references) in carbohydrate metabo-
lism. The role of phosphorus compounds in the transfer of energy was
noted in Chap. 4.

ESSENTIAL NONMETALLIC ELEMENTS 97

Phosphorus enters into the composition of the nucleoproteins, which are
found in the nucleus and cytoplasm of every cell. The nucleoproteins are
conjugated proteins which consist of a protein moiety in combination
with purine or pyrimidine nucleotides (nucleic acids). These nucleotides
are important functional compounds and may be classified according to
their heterocyclic components.

The preliminary hydrolysis of purine and pyrimidine nucleotides
involves the removal of phosphoric acid and the formation of nucleosides.
Nucleosides on hydrolysis yield sugars, purines (adenine, guanine) or
pyrimidines (cytosine, thymine, uracil). The nucleotides are also classi-
fied according to the sugar moiety, i.e., D-ribose or D-desoxyribose.

The nucleoproteins which contain D-ribose are mainly found in the

cytoplasm, while D-desoxyribose characterizes the nucleoproteins of the

nucleus. The Feulgen stain is used by cytologists to detect the presence

of D-desoxyribose nucleic acid. Viruses, chromosomes, and genes consist

largely of nucleoproteins. For a review of the role of nucleoproteins see

Mirsky (1943).

NITROGEN

This essential element is used by fungi for functional as well as struc-
tural purposes. The cell wall of many species, with the exception of the
Oomycetes and yeasts, appears to be composed of chitin (Brian, 1949).
Chitin is a linear polymer, similar to cellulose, of D-glucosamine. The
amino group of glucosamine in chitin is acetylated. This substance
makes up the exoskeleton of insects and Crustacea. It is interesting that
the chitin formed by fungi, insects, and Crustacea appears to be the same
substance. Protein, the basis of protoplasm, is composed of nitrogenous
substances. Purines, pyrimidines, and some of the vitamins are also
nitrogen-containing compounds.

Not all nitrogen sources are equally suitable for all fungi. Fungi may
be specific in the nitrogen sources they utilize. Our information on this
subject, while extensive, is far from complete. The reports in the litera-
ture which indicate that specific fungi are able to grow on a given source
of nitrogen may be accepted with confidence, but the reported negative
results are to be viewed with caution. Failure of a fungus to grow upon a
given nitrogen source may mean only that the medium used did not con-
tain the necessary growth factors, as in the case of Ophioholus graminis
(See Chap. 2).

Classification according to nitrogen sources used. Robbins (1937),
Steinberg (1939, 1950), and others have classified the fungi according to
their ability to utilize different sources of nitrogen. In the main Rob-
bins's classification is as follows: (1) fungi able to utihze atmospheric
nitrogen, nitrate nitrogen, ammonmm nitrogen, and organic nitrogen; (2)
fungi able to utilize nitrate nitrogen, ammonium nitrogen, and organic

PHYSIOLOGY OF THE FUNGI

nitrogen but not able to utilize atmospheric nitrogen; (3) fungi able to
utilize ammonium and organic nitrogen but unable to utilize atmospheric
or nitrate nitrogen; (4) fungi which are able to utilize only organic
nitrogen and unable to utilize atmospheric, nitrate, or ammonium nitro-
gen. Robbins recognized that the experimental conditions might affect
the classification of some fungi. In spite of admitted imperfections the
above classification is very useful in preparing media and in discovering
the causes of failure of some fungi to grow on certain media.

Nitrogen -fixing fungi. It has been shown to the satisfaction of all
competent investigators that various genera of bacteria {Rhizohium,
Azotobacter, Clostridium) contain species which are able to fix nitrogen.

Table 18. Nitrogen Fixation by Phoma betae and Azotobacter vinlandii
(Duggar and Davis, Ann. Missouri Botan Garden 3, 1916.)

Inoculated flasks

Uninoculated flasks

Mg. N fixed
per flask

Organism

IVIg. N per
flask

Ave,

Mg. N per
flask

Ave.

Aspergillus niger
(30 days)

Phoma betae
(89 days)

Azotobacter vinlandii
(28 days)

62.510
62.545
63.140

31.010
31.360

46.515
46.480
46.445

62 . 732

31.185
46 . 480

62.510
62.335
62.300

25.585
25.655

5.810
6.405

62.382

25.620
6.108

0.350

5.565
40.372

No such agreement exists regarding fungi. Much of the early work on
nitrogen fixation by fungi was done without using proper precautions.
However, in several instances the experimental methods appear to be
beyond reproach. Duggar and Davis (1916) cultured Phoma betae and
Aspergillus niger in Kjeldahl flasks and determined the nitrogen content
after growth without removing either the mycelium or medium prior to
digestion. Two types of controls were used. A number of uninoculated
flasks which had been stored under the same conditions as the inoculated
flasks were analyzed for nitrogen at the end of the experiment. A culture
of Azotobacter vinlandii served as a positive control. The data in Table 18
show that A. niger did not fix nitrogen, while P. betae and A. vinlandii did.
However, the nitrogen-fixing power of P. betae was slight compared with
that of A. vinlandii. In addition, the following fungi were tested for
ability to fix nitrogen, with negative results: Macrosporium commune,
Penicillium digitatum, P. expansum, and Glomerella gossypii. For further

ESSENTIAL NONMETALLIC ELEMENTS 99

references to nitrogen fixation by filamentous fungi see Wolf and Wolf
(1947) and Buchanan and Fulmer (1930).

So far as we are aware, only one study of nitrogen fixation by fungi
using modern isotopic techniques (Tove et at., 1949) has been published.
Phoma causarina was grown on a sucrose-salts medium in oxygen and
nitrogen enriched with N^^. Growth was slow and sparse under these
conditions, but some N^^ was fixed. These authors state that the isotopic
method is about 100 times more sensitive than the Kjeldahl procedure
used by other investigators.

While it is probable that only a relatively few fungi are able to fix nitro-
gen, the importance of biological nitrogen fixation is so great that further
investigations with modern techniques are desirable. Long ago Ternetz
(1907) reported that five species of Phoma isolated from roots of Ericaceae
fixed significant amounts of nitrogen. For a discussion of nitrogen fixa-
tion by bacteria see Wilson (1940).

Fungi utilizing nitrate nitrogen. Nitrates occur in the soil and thus are
a ''natural" source of nitrogen. A fungus which utilizes nitrate nitrogen
(NOs") must be able to reduce the nitrogen to the oxidation level of
ammonia. We may assume that failure of a fungus to utilize nitrate
nitrogen is coupled with inability to perform this reduction. According
to Robbins (1937) no instances have been recorded in the literature of an
organism being able to utilize nitrate nitrogen and unable to utihze
ammonium nitrogen. This does not mean that fungi which are able to
utihze nitrate nitrogen will grow at the same rate on ammonium nitrogen,
or that all sources of organic nitrogen will be as favorable as nitrate nitro-
gen. Yeasts as a rule do not utilize nitrate nitrogen.

The following is a partial list of fungi which have been reported or
observed to utilize nitrate nitrogen:

Armillaria rnellea C. velutipes

Ascobolus denudata Cordyceps militaris

A. leveillei Dendrophoma obscurans
Ascochyta pisi Dothidella quercus
Aspergillus spp. Fusarium spp.
Botryotinia convoluta Glomerella cingulata
Botrytis allii Gyrnnoascus setosus

B. cinerea Helminthosporium spp.
Cephalothecium roseum Lambertella corni-maris
Cercospora apii Lentinus tigrinus

C. beticola Macrosporium sarcinaeforme
Chaetomiiim cochlioides Marasmius Julvobidbillosus
C. convolutum Neocosmopara vasinfecta

C. globosum Ophiobolus graminis

Colletotrichum lagenarium 0. miyabeanus

C. lindeniuthianum Penidllium spp.

Collybia tuberosa Phoma apiicola

100 PHYSIOLOGY OF THE FUNGI

P. betae S. sclerotioruni

Pleurage curvicolla Sderotium bataticola

Pyronema confluens Septoria nodorum

Pythiomorpha gonapodyoides Sordaria fimicola

Pythium debaryanum Sphaeroholus stellatus

P. intermedium Sphaeropsis malorum

P. irregular e Trichoderma lignorum

Rhizodonia solani VerticiUium albo-atrum

Sderotinia minor Xylaria mali

Several of the species in the above Hst were reported by Young and
Bennett (1922) and others by Robbins and Kavanagh (1942). Some
reports are found in the papers of various authors, while some of the fungi
have been observed in our laboratory (see Fig. 17 for illustrations).

Fungi which utilize ammonium nitrogen. In the nitrogenous com-
pounds found in fungi the nitrogen is in the same state of oxidation as in
ammonium compounds. The following is a partial list of fungi which
have been reported or observed to require ammonium or organic nitrogen
and to be unable to assimilate nitrate nitrogen:

Absidia coerulea M. putillus

A. cylindrospora M. ramealis

A. diibia M. rotula

A. glauca M. scorodonius

A. orchidis Monilinia frudicola

Basidiobolus ranarum Mortierella rhizogena

Ceratostomella fimbriata Mucor flavus

C. ulmi M. hiemalis

Choanephora cucurbitarum M. nodosus

Cyathus striatiis M. pyriformis

Endothia parasitica M. saturninus

Lenzites trabea M. stolonifer

Marasmius alliaceus M. stridus

M. androsaceus Phycomyces blakesleeanus

M. chordaiis Pleurotus ostreatus

M. epiphyllus Rhizophlydis rosea

M. foetidis Rhizopus nigricans

M. graminum R. oryzae

M. performis Sporodina grandis

M. personatus Zygorrhynchus moelleri

A number of fungi in the above list were listed by Robbins (1937).
The studies on Marasmius are reported by Lindeberg (1944). Others are
reported by various authors, while some have been observed in our
laboratory. Obviously, this list is far from complete, and numerous
common fungi have been omitted from both this and the previous list
because of lack of definite information regarding their ability to utilize
nitrate nitrogen.

ESSENTIAL NONMETALLIC ELEMENTS

101

Fungi which utiUze only organic nitrogen. Certain fungi are unable to
utilize nitrogen except in the form of amino acids, peptides, and mixtures
of these compounds such as peptone. The use of organic nitrogen does
not extend to all organic compounds which contain this element. Many

A B C D

Fig. 17. Growth of two fungi on four media differing in nitrogen source. .4, no
nitrogen added; B, potassium nitrate; C, ammonium tartrate; D, asparagine. Above,
Helminthosporium sativum; below, Ceratostomella fimbriata.

of the early reports claiming utilization of organic nitrogen only by various
species have been found to be in error.

All the early work where peptone was the nitrogen source used is to be
suspected because the need for growth factors was not recognized. The
use of other complex nitrogen sources such as proteins makes interpreta-
tion doubtful for the same reason. However, in the case of amino-acid-
deficient fungi a portion of the nitrogen source must be supplied in the

102 PHYSIOLOGY OF THE FUNGI

form of a particular amino acid. Cantino (1949) found that Blastocladia
pringsheimii is deficient for methionine and perhaps other amino acids.
Presumably other amino acids are used to supply a portion of the metabolic
nitrogen of this species. The same situation may exist in the nitrogen
utilization of amino-acid-deficient mutants of Neurospora. Leonian and
Lilly (1938) reported Coprinus lagopus and Pleurotus corticatus to grow on
a mixture of five amino acids and not on ammonium nitrate as a source of
nitrogen.

Inorganic sources of nitrogen. The nitrates commonly used in prepar-
ing media are potassium nitrate, sodium nitrate, and calcium nitrate.
These salts are equivalent in so far as they supply the same kind of nitro-
gen. They are not equivalent in that different cations are involved.
Calcium ion may precipitate a varying amount of phosphate, depending
upon the concentrations of the two ions and the pH of the medium.

Some fungi utilize nitrite (N02~) nitrogen. Blakeslea trispora makes
some growth on nitrite nitrogen (Leonian and Lilly, 1938). Owing to the
instability of nitrites in acid solution and the destructive effect of nitrous
acid on proteins and amino acids, nitrite nitrogen is little used in making
media. Nitrite is produced by many fungi from nitrate and may accumu-
late in the medium under certain conditions. The toxic effect is related
to the pH of the medium, being greatest at low pH. Wirth and Nord
(1942) attributed the accumulation of pyruvic acid in the nitrate medium
on which Fusarium lini grew to the presence of the nitrite, which inac-
tivated thiamine pyrophosphate (cocarboxylase).

Yeasts utilize nitrate nitrogen poorly as a general rule. Pirschle (1930)
studied the relative value of nitrate and ammonium nitrogen for a yeast
and concluded that poor utilization of nitrate nitrogen was due in part to
the accumulation of nitrite in the medium. This was shown by the yields
of aerated and nonaerated cultures on media containing nitrate and
ammonium nitrogen as well as by analyses of the culture medium for
nitrite. Aeration prevented the accumulation of toxic amounts of nitrite
or its decomposition product nitrogen trioxide. In other experiments
Pirschle showed that nitrite inhibited the growth of yeast on ammonium
nitrogen. By adding sufficient nitrite to a medium containing ammonium
sulfate, growth was depressed below that obtained on potassium nitrate.
How far these conclusions may be applied to other fungi which do not
utilize nitrate nitrogen is not known.

Inorganic and organic ammonium salts are equivalent in that they
furnish inorganic nitrogen; i.e., ammonium ion. The nitrogen of all
ammonium salts is the same, but the physiological effects of the anions
are not. The ammonium salts of strong inorganic acids generally tend to
make a culture medium more strongly acidic than when an ammonium
salt of a weak acid is used. However, the situation is far more compli-

ESSENTIAL NONMETALLIC ELEMENTS

103

cated than this simple theory Yv'ould predict. It should be emphasized
that nitrates and ammonium salts have opposite effects on the acidity of
culture media. Other conditions being equal, as nitrate ions are con-
sumed, the culture medium becomes more alkaline, while as ammonium
ions are utilized, the culture medium becomes more acid.

Before considering the ammonium salts of the organic acids, the use of
ammonium nitrate should be mentioned. Both ions contain nitrogen, a
feature which has led many investigators to use it in media. If a fungus
is able to utilize both kinds of nitrogen, the pH of the medium will be
somewhat stabilized. This salt should not be used if the purpose of an
experiment is to determine whether a fungus can utilize either one or the
other or both forms of nitrogen. Some fungi apparently use nitrate
nitrogen in preference to ammonium nitrogen when both are supplied in
the medium. Fusarium lini appears to be such a fungus (Wirth and
Nord, 1942).

Table 19. The Effect of Various Organic Acids on the Growth of Four
Fungi on Media Containing Ammonium Nitrate
Initial pH 5.5. Figures are milligrams of mycelium produced. (Leonian and LiUy,
Am. Jour. Botany 27, 1940.)

Organic acids,
0.02M

Mucor raman-
nianus

Phythium
ascophallon

Pythiomorpha
gonapodyoides

Phycomyces
blakesleeanus

Control

Acetic

Lactic

77
144
154
142
135
158
174
152

117

156

113

157

180

112

144
121

Succinic

Glutaric

Fumaric

165
144
189

Tartaric

Citric

149
171

M etarrhizium glutinosum {Myrothecium verrucaria) grew well on nitrate
nitrogen alone and poorly on ammonium nitrogen (Brian et al., 1947).
Ammonium nitrogen inhibited growth of this fungus, whether nitrate was
present or not. Growth was equally poor on ammonium nitrate and
ammonium sulfate. Since this fungus grew well on media containing
nitrate as the sole source of nitrogen, these authors have questioned the
common belief that all fungi which are able to utilize nitrate nitrogen can
also utihze ammonium nitrogen. Most fungi appear to utihze ammonium
nitrogen before nitrate nitrogen when both are supplied in the medium,
but this is not universal. Rippel (1931) found the pH of the medium to
determine which form of nitrogen was utilized by Aspergillus niger and
A. oryzae. Additional examples are given by Foster (1949).

The utilization of ammonium and some forms of organic nitrogen may

104

PHYSIOLOGY OF THE FUNGI

be modified by the presence of other compounds in the medium. Among
these the organic acids, especially the four-carbon dicarboxylic acids, play
an important role. This subject has been studied by Leonian and Lilly
(1940), Burkholder and McVeigh (1940), Brian et at. (1947), and Bernhard
and Albrecht (1947). The data in Table 19 illustrate the effect of organic
acids on the amount of growth of four fungi.

Succinic and fumaric acids were most uniform in their effect on nitrogen
assimilation. Figure 18 shows the reciprocal effect of varying amounts of

200

~~^^

^.-^

1 1

150

/ \

^2.5g.Nh

'4NO3/I

100

7. succinic ac

id /I

°(

g. NH4r

2
OO3/I

3.0

2.0

1.0

g. succinic acid/l

Fig. 18. The reciprocal effect of varying amounts of succinic acid (ammonium
nitrate constant) and ammonium nitrate (succinic acid constant) on the growth of
Phycoviyces blakesleeanus. (Drawn from data of Leonian and Lilly, Am. Jour. Botany
27: 22, 1940.)

succinic acid and ammonium nitrogen on the growth of Phycomyces
blakesleeanus, which does not utilize nitrate nitrogen. The amount of
growth, within certain limits, is directly proportional to the amount of
succinic acid in the medium.

Brian et al. (1947) have suggested on the basis of studies on Myro-
thecium verrucaria that a definite antagonism exists between the metabolic
pathways involved in nitrate and ammonium utilization, and in the
presence of ammonium nitrogen the nitrate pathway is blocked. Ammo-
nium nitrogen is poorly utilized unless certain organic acids are present in
the medium. Malic acid has no effect on utilization of nitrate nitrogen.

ESSENTIAL NONMETALLIC ELEMENTS 105

These authors suggest that different pathways of carbohydrate iitihzation
may be followed, depending upon whether nitrate or ammonium nitrogen
is present.

Organic sources of nitrogen. Of the vast number of organic com-
pounds which contain nitrogen the ones of interest in fungus nutrition are
those which occur naturally. A few exceptions will be noted later. In
practice, this means proteins and the products of protein hydrolysis.
The following steps in protein hydrolysis have been recognized : protein -^
metaprotein — > proteoses — > peptones — > peptides — > amino acids. Pep-
tone, which is a complex mixture of peptides and amino acids, is frequently
used as a nitrogen source in media. According to Gortner (1929),
peptones are neither coagulated by heat nor precipitated by saturating a
solution with ammonium sulfate, properties which distinguish peptones
from proteins, metaproteins, and proteoses. Since peptides having some
11 amino-acid residues are precipitated by ammonium sulfate, it may be
deduced that the peptides in peptone have on the average 10 or less
amino-acid residues. Peptone is a useful source of nitrogen when it is
desired to culture a large number of species upon a single medium. A
part of its virtue may be ascribed to its complex nature, for a mixture of
nitrogen sources may be better utilized than a single source. Peptone
also contains most of the water-soluble vitamins (Stokes et al., 1944).

Most of the amino acids which have been isolated from proteins are
listed in Table 20. In addition, the amides of aspartic and glutamic
acids are included. These compounds are found free in many plants and
are thus available to the fungi in nature.

These amino acids are not of equal value in fungus nutrition. The
relative value of 24 amino acids for 14 fungi was tested by Leonian and
Lilly (1938) who found no one amino acid w^as best for all these species.
Steinberg (1942) made an extensive study of growth of Aspergillus niger
on 22 amino acids. Seven were excellent sources of nitrogen for A. niger:
alanine, arginine, aspartic and glutamic acids, glycine, proline, and
hydroxyproline. Steinberg expressed the opinion that the seven amino
acids which supported the most growth of A. niger are those which are
synthesized first (primary amino acids) by this fungus and from which the
other amino acids (secondary amino acids) are normally formed. It is
assumed that the "primary" amino acids enter directly into the metabolic
pathways, while the "secondary" amino acids must undergo preliminary
deamination before use. The primary amino acids are probably not
the same for all fungi. Lilly and Leonian (1942) investigated the effect
of nitrogen source on the growth of 10 strains of Saccharomyces cerevisiae.
The data in Table 21 show clearly that different amino acids vary in
effectiveness, and that different strains of the same organism respond
differently to the same source of nitrogen.

106

PHYSIOLOGY OF THE FUNGI

Table 20. Common Names and Formulas ok Some Alpha-amino Acius Isolated

FROM Proteins and of Some Amides Found in Plants
Monoamine dicarboxylic acids:

Aspartic acid: HOOC— CH2— CHCNHo)— COOH

Glutamic acid: HOOC— CHo— CH.— CH(NH2)— COOH
Amides of monoamino dicarboxylic acids:

Asparagine :
Glutamine:
Basic amino acids:
Argiiiine*:
Lysine*:
Histidine * :

NH2OC— CH2— CH(NH2)— COOH
NH2OC— CH2— CH,— CH(NH2)— COOH

NHo— C(=NH)— NH— CHo— CH2— CH2— CH(NH2)-
NH2— CH2— CH2— CH2— CH2— CHCNH,)— COOH
CH

-COOH

CH===C— CH2— CH(NH2)— COOH
Monoamino monocarboxylic acids:

Glycine:
Alanine :
Valine * :
Leucine*:
Isoleucine*:
Phenylalanine * :
Serine :
Threonine*:

Tryptophane '*

Tryosine :

CHoCNH.)— COOH
CH3— CH(NH2)— COOH
(CH 3) 2— CH— CH (NHo)— COOH
(CH3)2— CH— CH2— CH(NH2)— COOH
CH3— CH2— CH(CH3)— CHCNH.)— COOH
CeHs- CHo— CH (NH2)— COOH
CH2(0H)— CH(NH2)— COOH
CH3— CH(OH)— CH(NH2)— COOH

C— CH2— CH(NH2)— COOH

HO^^^
CH2— CH

CH2— CH(NH2)— COOH

Proline:

CH2 CH— COOH

NH
CHOH

-CH2

Hydroxy proline: CHo CH— COOH

NH
Sulfur-containing amino acids:

Cysteine: CHsCSH)— CHCNHo)- COOH

Cystine: HOOC— CH(NH2)— CH2— S— S— CH2— CHCNHa)- COOH

Methionine*: CH2(SCH3)CH2— CHCNH.)- COOH

* The 10 amino acids reported by Rose (1938) as essential for the nutrition of the white rat.

Physiological specificity extends to the configuration as well as the
composition of the molecule. Optical isomers (enantiomorphs) usually
have different physiological properties. A mixture of amino acids may or
may not be utilized better than a single amino acid. The effect of one
amino acid on the utilization of another varies with the amino acids

ESSENTIAL XON METALLIC ELEMENTS

107

involved and the specific fungus used. Leonian and Lilly (1940) tested
the growth of Phycomyces blakesleeanus upon five single amino acids and
upon a mixture of these five amino acids with the following results : mix-
ture of five amino acids, 214; asparagine, 209; DL-alanine, 151; arginine,
50; aspartic acid, 203; glycine, 201; and glutamic acid, 189 mg., respec-
tively. Arginine is a poor nitrogen source for P. blakesleeanus, but the
presence of arginine in the amino-acid mixture did not depress growth.
More complex relations were found with yeast (Lilly and Leonian, 1942).
Ten strains of yeast were grown upon media containing a mixture of six
amino acids (aspartic and glutamic acids, arginine, asparagine, alanine,
and leucine). Upon this mixture of amino acids two strains grew as well
as or better than upon the best single amino acid (aspartic acid). The

Table 21. Comparison of Various Soi'rces of Nitrogen for Six Strains of

Yeast
Milligrams of dry yeast cells produced in 72 hr. Each culture received 8 mg. of X.
(Lilly and Leonian, Proc. West Va. Acad. Sci. 16, 1942.)

Nitrogen source

Ammonium sulfate

Urea

L- Aspartic acid . . .

L-Aspargine

Glycine

DL-Norleucine

Yeast strain

18.7
33.2
60.7
49.4
3.0
29.3

21.2
31.5
59.9
45.8
1.2
17.6

22.3
32.9
65.6
50.0
2.0
33.0

17.5
27.3
62.0
49.2
2.1
18.4

21.7
32.0
52.4
47.6
1.0
1.2

23.8
35.1
70.6
35.0
1.1
4.2

amount of growth of one strain was 70.6 mg. on aspartic acid alone, while
on the amino-acid mixture only 38.6 mg. was produced. Omission of
asparagine from the mixture increased the yield to 52.0 mg. These
results show that the effects of multiple nitrogen sources upon growth, and
perhaps other functions, are complex.

Organic acids, especially the four-carbon dicarboxylic acids, affect the
utilization of some amino acids much as they do that of ammonium com-
pounds. Phycomyces hlakesleeamis on a medium containing arginine
produced 43 mg. of mycelium per flask. Addition of 0.1 per cent succinic
acid to the medium increased the yield to 192 mg. (Leonian and Lilly,
1940).

Nitrogen utilization by the fungi has been studied for almost a century,
but many of the problems involved are not yet solved. Brenner (1914)
has reviewed the early work in this field, especially with reference to the
divergent views of Raciborski and Czapek on the mode of utilization of
amino acids. Raciborski held that amino acids were deaminat^d before

108 PHYSIOLOGY OF THE FUNGI

utilization, while Cznpek believed that amino acids were utilized directly.
Both processes arc doubtless involved, and only prolonged study of
specific fungi and various nitrogen sources will permit elucidation of these
questions.

One of the main uses of nitrogen is in the synthesis of proteins. With
the exception of certain amino acids (primary amino acids) and ammonia,
most nitrogen sources undergo modification before entering the synthetic
metabolic pathways. Nitrates, nitrites, and hydroxylamine are pre-
sumably reduced to ammonia before assimilation. Those amino acids
(secondary amino acids) which do not enter directly into the metabolic
pathways leading to the synthesis of protein are probably deaminated.
Burk and Horner (1939) have listed the types of deamination performed
by fungi as follows:

1. Deamination by hydrolysis:

H2O
R— CH(XH.:)— COOH > R— CH(OH)— COOH + NH3

2. Deamination by hydrolysis followed by decarboxylation:

H2O
R— CHCNHo)— COOH > R— CH2OH + CO2 + NH3

3. Oxidative deamination:

MO,
R— CHCNHa)— COOH > R— CO— COOH + NH,

The production of higher alcohols, "fusel oil," is due to hydrolytic
deamination and decarboxylation of various amino acids, especially
leucine, which yields isoamyl alcohol. Various species of filamentous
fungi, especially those which produce alcohol, are capable of the same
reactions. The following amino acids are converted by yeasts into alco-
hols having one less carbon than the parent amino acid: leucine, isoleucine,
phenylalanine, trytophane, and valine. Wirth and Nord (1942) indicate
that Fusarium lini oxidatively transforms alanine into pyruvic acid.
For further information on the process of deamination by yeast, see Thorn
(1937). The process of deamination releases nitrogen in the form of
ammonia, which is utilized by most fungi.

It seems probable that the synthesis of amino acids is the next step in
protein formation. The formation of primary amino acids may result
from the reaction of ammonia with certain alpha-keto acids (pyruvic,
oxalacetic, and ketoglutaric) ; this is essentially the reverse of oxidative
deamination. This process may be fo.'mulated as follows:

R— CO— COOH + NH3 -> R— C(=NH)— COOH + H. -^ R— CHCNH.)— COOH

In addition, yeasts are able to add ammonia to fumaric acid to form
aspartic acid (Haehn and Leopold, 1937). The role of the four-carbon
dicarboxylic acids in nitrogen assimilation may be explained on the basis
that these acids are transformed into kcto acids. Brian et al. (1947) have
assumed that those fungi, such as Phycomyces hlakesleeanus and Myro-

ESSENTIAL NONMETALUC ELEMENTS

109

thecium verrucaria, which make hmited growth on ammonium nitrogen do
so because they are unable to synthesize in adecjuate amounts the neces-
sary three-, four-, and five-carbon keto acids. The interrelation among
various dicarboxylic acids is shown in schemes IV, VIII, and IX.

The reactions discussed above account for the synthesis of only a few of
the 20 or so amino acids found in fungus protein. Another type of reac-
tion may account for the synthesis of secondary amino acids. This is
called the transamination reaction and may be represented as follows :

R— CO— COOH + R'— CH(NH2)C00H -^ R— CH(NH2)C00H+R'— CO— COOK

According to Roine (1947), Torulopsis utilis has the necessary enzymatic
mechanisms for the synthesis of the following amino acids by transamina-

30 40 50

Time in minutes

Fig. 19. Amounts of soluble nitrogen compounds found in the trichloroacetic acid
extract as a function of time. Data are based on 100 ml. of yeast suspension, or
about 5 g. fresh yeast. Curve 1 represents total soluble nitrogen, curve 2 total
amide nitrogen, curve 3 alanine nitrogen, and curve 4 dicarboxylic-amino-acid nitro-
gen. (Courtesy of Roine, Ann. Acad. Sci. Fennicae 26: 63, 1947.)

tion: aspartic acid, glutamic acid, alanine, valine, leucine, and isoleucine.
For a general review of the transamination reaction, see Herbst (1944).
Roine (1947) has obtained experimental evidence which indicates that
in Torulopsis utilis the primary amino acids are formed first and that the
secondary amino acids are then formed from them. This evidence w^as
obtained by analyzing the nonprotein nitrogen fraction which was
extracted from cells of various ages with trichloroacetic acid (a protein
precipitant) . Nitrogen-starved cells of T. utilis were suspended in carbo-
hydrate-free medium which contained ammonium nitrogen. The culture
was aerated. Every 10 min. a portion of the crop was harvested, and the
distribution of nitrogen compounds in the trichloroacetic acid extract was
determined. Figure 19 show clearly that the first stages of protein syn-
thesis consist in the formation of monoamino dicarboxylic acids, their

no PHYSIOLOGY OF THE FUNGI

amides, and alanine. It may be assumed that the amides of gkitamic and
aspartic acid function in yeast as nitrogen carriers, as they do in green
plants.

Preformed amino acids are probably used in protein synthesis. In
principle this process is the reverse of hydrolysis. Many complex chem-
ical reactions are involved. Proteins vary in complexity, the simplest
having molecular weights in the neighborhood of 16,000 to 17,000. The
molecular weight of some proteins is said to be greater than 1,000,000,
and tobacco mosaic virus protein is estimated to have a molecular weight
of 40,000,000. In spite of these enormous molecular weights, a good
deal is known about the structure of proteins. Fundamentally, a pro-
tein consists of amino-acid residues joined together by peptide linkages,
— CH2 — NH — CO — . Since different proteins have highly specific
properties which depend upon the molecular structure, the synthesis of
these compounds involves a systematic linking together of amino-acid
residues in a definite pattern. For reviews of protein structure the reader
is referred to Bull (1941) and Astbu^y (1943).

The general pathways of nitrogen utilization by fungi are shown in
scheme III.

Scheme III. Possible Pathways of Protein Synthesis from Various Sources

OF Nitrogen
Nitrates — ■ > Ammonia > Primary amino acids

Secondary amino acids
Ammonia —

i
Secondary amino acids

Primary amino acids-

Peptides

i
Polypeptides

i.
Protems

OTHER NONMETALLIC ELEMENTS

It is not known whether fungi require nonmetallic elements other than
hydrogen, oxygen, sulfur, phosphorus, and nitrogen. Boron and iodine
are frequently added to culture media, but good evidence of their essen-
tiality for fungi appears to be lacking. Sodium chloride is frequently
added to media, but neither sodium nor chlorine, so far as is known, is
essential for the fungi.

In nature fungi come in contact with many nonessential elements.
Some of these may be metabolized. Others may modify the life processes
of the fungi by their toxic action or by other means. Chlorine is found in
various compounds synthesized by fungi, e.g., non-ionic chlorine is found
in chloramphenicol, one of the newer antibiotics. Many species of fungi
metabolize arsenic. Penicillium hrevicaule, among other species, pro-

ESSENTIAL HON METALLIC ELEMENTS 111

duces a volatile, toxic, organic arsenic compound, trimethylarsine,
(CH3)3As, which has an odor resembling garlic. In the past P. hrevicaule
has been recommended for the detection of arsenic compounds in forensic
medicine. This microbiological test for the presence of arsenic is said to
))e many times as sensitive as the ]\Iarsh test. The early work on the
utilization of arsenic compounds by fungi is reviewed by La Far (1911)
and more critically by Challenger et al. (1933). P. hrevicaule also pro-
duces dimethyl selenide from selenium compounds (Challenger and

North, 1934).

SUMMARY

The classification of essential elements as structural or functional may
be misleading in that an element usually plays many roles. This is
especially true of the essential nonmetallic elements.

With the exception of carbon dioxide all the organic compounds used
by or contained in fungi contain hydrogen. One of the most important
hydrogen-containing compounds is water. This compound is associated
with proteins in the form of bound water, and it functions as a solvent in
which most if not all biochemical reactions take place. Water enters
into many reactions, particularly in the hydrolytic processes of ''diges-
tion." Apparently fungi do not utilize free hydrogen.

None of the fungi appear to be obligate anaerobes. Many are faculta-
tive anaerobes, while some appear to be strict aerobes. Free oxygen is
used by the fungi in respiration, chiefly as an acceptor of hydrogen. The
facultative anaerobes have another mechanism of oxidation which does
not involve free oxygen. This is called anaerobic respiration, or fermen-
tation. The rate and amount of growth and sporulation and the meta-
bolic by-products of a given fungus are affected by the oxygen supply.

The problem of specificity arises in connection with the form of sulfur
utilized. Most fungi utilize sulfate sulfur, but some require reduced sul-
fur. Other species are unable to synthesize specific sulfur-containing
amino acids, especially methionine. Sulfur enters into the composition
of enzymes and other proteins, peptides, and at least two vitamins.

The fungi utilize phosphorus in the form of phosphate salts and esters.
Some specificity in the different sources of phosphate has been found
Phosphate esters enter into a wide variety of enzymatic reactions, and
many coenzymes are phosphate esters.

It is thought that certain phosphate esters act to transfer chemical
energy to certain enzymatic reactions. Phosphorus enters into the com-
position of proteins, especially the nucleoproteins, which are found in the
nucleus or cytoplasm of every cell. Viruses and genes are thought to
consist largely of nucleoproteins.

Fungi differ in ability to utilize different forms of nitrogen. A few
utilize atmospheric nitrogen; many utilize nitrate nitrogen; and a still

112 PHYSIOLOGY OF THE FUNGI

greater number utilize ammonium nitrogen. All species are abletoutilize
some form of organic nitrogen. Other constituents in media, especially
the four-carbon dicarboxylic acids, modify the availability of ammonium
nitrogen and certain amino acids. Not all amino acids are of equal value
in fungus nutrition. The primary amino acids are those which enter
directly metabolic pathways, while secondary amino acids are deaminated
before the nitrogen is used.

Most of the nitrogen utilized by fungi enters into the synthesis of pro-
teins. The primary amino acids are formed first, and the secondary
amino acids are formed from primary amino acids. Proteins are the
most complex compounds synthesized by living cells. Many of the
vitamins and other essential metabolites also contain nitrogen.

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ESSENTIAL NON METALLIC ELEMENTS 113

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114 PHYSIOLOGY OF THE FUNGI

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ESSENTIAL NON METALLIC ELEMENTS 115

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CHAPTER 7
CARBON SOURCES AND CARBON UTILIZATION

Carbon occupies a unique position among the essential elements
required by living organisms. Almost half of the dry weight of fungus
cells consists of carbon. Protoplasm, enzymes, the cell wall, and reserve
nutrients stored within the cells are compounds of carbon. Carbon com-
pounds are equally important in fugus nutrition. Fungi secure energy
by oxidizing organic compounds. In addition to being the main struc-
tural elements, carbon compounds play an equally important functional
role. The number of carbon compounds known far exceeds the total
of known compounds of all the other elements, because of the property of
carbon of forming compounds in which carbon is linked to carbon in the
form of chains and rings. Various other elements such as nitrogen,
oxygen, and sulfur may serve as linking elements. While many carbon
compounds are stable at ordinary temperatures, others are extraordinarily
sensitive to a wide range of chemical reagents and to slight changes in the
physical environment.

Organic compounds differ in composition, structure, and configuration.
These are key factors which must be considered in relation to utilization
of organic compounds by fungi. Since more is known about carbohy-
drates and related compounds as carbon sources, and about the manner
in which they are dissimilated and assimilated, than about any other
class of organic compounds, most of the discussion in this chapter will
be devoted to these topics. In the main, only naturally occurring organic
compounds will be considered.

MONOSACCHARIDES AND RELATED COMPOUNDS

The simple sugars, or monosaccharides, have the general formula
C„(H20)n. The carbon chain is unbranched except in a few, very rare
sugars. The functional groups present are primary ( — CH2OH) and
secondary ( — CHOH — ) alcohol groups, and an aldehyde ( — CHO) or
ketone ( — CO — ) group, actual or potential, is always present. The
primary alcohol and aldehyde groups are restricted to the end positions
of the carbon chain, while the ketone group is usually on the second

116

CARBON SOURCES 117

carbon in the chain. Sugars having an aldehyde group are called aldoses,
those having ketone group, ketoses; the ending -ose denotes a sugar. In
addition, the sugars are further classified according to the number of
carbon atoms in the chain, e.g., pentoses, hexoses, or more specifically
as aldopentoses, ketohexoses, etc. While it will be necessary in the
discussion to follow to include some information about the chemistry
and structure of the sugars, the reader is advised to consult suitable
texts for further information. Those of Oilman (1943) and Pigman and
Goepp (1948) are recommended.

Compounds which have the same composition and the same molecular
weight are called isomers. There are 16 aldohexoses (32, if the alpha
and beta forms are considered), which have the same percentage com-
position and the same functional groups as glucose (dextrose). There
are eight possible ketohexoses isomeric with fructose. Two kinds of
isomers exist among the sugars: First, there are those which have the
same physical properties but differ in the direction in which they rotate
plane-polarized light (enantiomorphs) . Isomers of this kind occur in
pairs, and the configuration of the functional groups of one isomer is the
mirror image of the configuration of the other. Enantiomorphs usually
differ physiologically. One such isomer may be utilized and the other
not, or one may be utilized much more rapidly than the other. Pasteur
(1860) was the first to demonstrate that fungi are able to distinguish
between such isomers. Penicillium glaucum utilized c?-tartrate more
rapidly than Z-tartrate {d and / refer to optical rotation). Second, there
are those isomers which, although they have the same functional groups,
have these groups arranged in a different order, so that one isomer is not
the mirror image of the other (diastereoisomers). It is usually safe to
assume that one member of a pair of enantiomorphs will be better
utilized than the other, but such an assumption about utilization of
diastereoisomers is not possible.

Since not all sugars of a group such as the aldohexoses are utilized by
fungi, it is of interest to compare chemical structure or configuration with
utilization. Not all fungi are able to utilize exactly the same sugars
(Fig. 20). Whether a sugar is utilized or not depends upon both the
configuration of the sugar and the particular abihties of the specific
fungus. By configuration is meant the spatial arrangement of the
hydrogen and hydroxyl groups. The long history of chemical investi-
gation which established the configuration of the simple sugars must be
passed by. Inasmuch as glucose is the key compound in sugar chemistry,
as well as in physiology, particular emphasis will be devoted to this
aldose.

The structures of the glucose enantiomorphs are given at the top of
page 119.

118

PHYSIOLOGY OF THE FUNGI

A B CD

Fig. 20. Growth of three fungi on four sugars. A, gkicose; B, fructose; C, sucrose;
D, maltose. Top row, Monilinia Jructicola (8 days); middle, Mucor ramannianus
(8 days); bottom, Ustilago striiformis, fragmenting strain (20 days).

CARBOS SOURCES

119

CHO

H— C— OH

3. HO— C— H

4. H— C— OH

5.
6.

H— C— OH

CHoOH
D-Glucose

CHO

I
HO— C— H

I
H— C— OH

HO— C— H

CH.OH
L-Ghicosc

The letters d and l indicate that these sugars belong to different series;
they do not indicate optical rotation. The small letters d and / have
been used in the past to express two separate ideas, optical rotation or
configuration. The use of d and I in the old literature makes it difficult
at times to discover which enantiomorph was meant. The configuration
of the secondary hydroxyl group farthest from the carbonyl group deter-
mines to which series a sugar belongs. D-Glucose is the form which
occurs naturally and is meant when glucose is used without qualification.
Not all naturally occurring sugars belong to the d series; e.g., L-arabinose.
For the sake of clearness and accuracy, the series designation should
always be used where there is any chance of confusion and misinterpre-
tation. Pigman and Goepp (1948) point out that only sugars of the
galactose type occur naturally as both enantiomorphs. D-Galactose is
fermented by some yeasts, while L-galactose is not.

Hexoses. The following hexoses occur naturally: D-glucose, D-man-
nose, D-galactose, L-galactose, D-fructose, and L-sorbose. It is doubtful
if L-sorbose occurs in green plants, but it is formed from sorbitol by
bacterial {Acetobacter suboxydans) oxidation (Bertrand, 1904).

CHO CHO CHoOH

H— C— OH

HO— C— H

C=0

HO— C— H

H— C— OH

CH2OH

D-Glucose

CH2OH
D-Mannose

CH2OH

D-Fructose

CHO

CH2OH

H— C— OH

HO— C— H

C=0

HO— C— H

H— C— OH

HO— C— H

H— C— OH

HO— C— H

CH2OH
D-Galactose

CH2OH

ly-Galactose

CH2OH
L-Sorbose

120

PHYSIOLOGY OF THE FUNGI

The configuration of glucose, mannose, and fructose is the same for
carbons 3 to 6. In the presence of dilute alkali these sugars undergo
enolization to produce the same enol form.

D-Glucose
CHO

H— C— OH

D-Mannose
CHO

HO— C— H

I
R

it
CHOH

II
COH

D-Fructose
CH,OH

C=0

I
R

R ±:

Common enol form

Other effects of alkali and heat on sugars were noted in Chap. 2.

Many fungi will utilize these three sugars if configuration is important
in determining availability. However, these sugars are not equivalent
for all fungi. The fact that galactose is not utilized by all fungi which
utilize the three closely related sugars is illustrated by the data in Table
22.

Glucose is utilized by more fungi than any other sugar and is nearly a
universal carbon source. In attempting to culture fungi of unknown
nutritional requirements on synthetic or semisynthetic media, glucose
should be the first carbon source used. However, there are a few fungi
which are unable to utilize glucose, or any sugar, as a carbon source.
Leptomitus lacteus (Schade, 1940; Scliade and Thimann, 1940) is unable to
utilize glucose, fructose, galactose, or sucrose. Skoog and Lindegren
(1947) have reported the behavior of 12 strains of Saccharomyces cere-
visiae which did not utilize glucose when first isolated. These strains
became adapted to glucose on sufficiently long exposure to this sugar.
Cheo (1949) found certain isolates of Ustilago striiformis to be unable to
grow on glucose when freshly transferred from a sucrose medium. After
2 to 4 weeks these isolates began to grow. This behavior suggests the
formation of an adaptive enzyme which was not formed when these
isolates were cultured on sucrose medium. Some fungi, such as L. lacteus,
apparently lack the ability to form adaptive enzymes for glucose utili-
zation and must be classed as absolutely incapable of glucose utilization,
while the yeasts of Skoog and Lindegren and the isolates of U. striiformis
are facultatively able to utilize glucose. The differences among these
fungi probably lie in the ability to form adaptive enzymes.

No carbon source can be utilized if the medium is lacking in any
essential element or compound. Kinsel (1937) and Stevens and Larsh
(1939) reported that Diplodia macrospora would grow only on disacchar-
ides and not on media containing glucose or other monosaccharides.

CARBON SOURCES 121

The explanation of this anomalous situation was given by Margolin
(1940) and confirmed by Wilson (1942), who found that D. macrospora
was deficient for biotin. It is probable that other vitamin-deficient
fungi have been reported in the past as unable to utilize certain sugars
owing to the absence of specific growth factors. Negative results
reported in the literature are therefore to be viewed with caution.

Wolf and Shoup (1943) studied the oxidation of carbohydrates by
Allomyces arhuscula, A. javanicus, A. moniliformis, and A. cystogenus.
All four species oxidized dextrin (degraded starch), while A. arhuscula
oxidized maltose and sucrose in addition. The other common naturally
occurring sugars, including glucose and fructose, were not oxidized. It
has since been shown that A. arhuscula is deficient for methionine and
thiamine (Yaw, 1950).

While there is an immense amount of information scattered throughout
the literature to the effect that a certain sugar is utilized by various
species, much of this information deals with relatively few sugars.
Critical studies on the utilization of the sugars are rare. Margolin
(1942) studied the amount of growth of 21 fungi on four hexoses. These
data (Table 22) were obtained under uniform conditions. A mixed
nitrogen source (ammonium nitrate and amino acids) was used, and
vitamins were supplied to the deficient fungi. The time chosen for
harvest in this study was the time maximum weight was attained on
glucose. This work suffers from the common defect that the yields are
compared on the basis of a fixed time of harvest. The ideal way of
determining the value of different sugars for fungi would be to study
both the rate and amount of growth as a function of time of incubation.

The following generalizations about utilization of the common hexoses
may be drawn from the data in Table 22: (1) There is no single sugar
which supports the maximum amount of growth for all of these fungi.
(2) All of these fungi utilize glucose, although the maximum amount of
growth was not always attained on this sugar. (3) The more closely the
configuration of another sugar approaches that of glucose, the more
fungi utilize it. It is believed that these generalizations are valid for all
fungi which utilize sugars.

Steinberg (1939) found D-glucose, D-fructose, D-mannose, L-sorbose,
and sucrose to be equally effective in the nutrition of Aspergillus niger
while D-galactose, lactose, glycerol, and mannitol were poor sources of
carbon for this fungus. Herrick (1940) reported that two isolates of
Stereum gausapatum grew on glucose, fructose, mannose, and galactose.
One isolate made significantly better growth on fructose; the other grew
equally well on all four sugars. This indicates that not all isolates of a
species are alike in ability to utilize a given sugar. The utilization of
different carbon sources by A. oryzae was investigated in detail by Tamiya

122

PHYSIOLOGY OF THE FUNGI

(1932). This paper should he consulted for the experimental details
and references to the literature. One hundred twenty-three carbon
compounds were investigated, and of the hexoses, mannose supported

Table 22. Milligrams of Mycelium Produced by 21 Fungi Grown on Media

Containing Different Sugars
AL the sugars were used at a rate which supphed 8 g. of carbon per liter. Each
125-ral. flask contained 20 ml. of medium. Cultures were incubated at 25°C. Each
weight in the table is the average of 12 cultures. (Margolin, thesis, West Virginia
University, 1942.)

Fungus

Blakeslea trispora

Diplodia macrospora

D. natalensis

Fusarium lycopersici

Helicostylum pyriforme

Helmintiwsporum sativum. . .

Mucor ramannianus

Pilaira moreaui

Phycomyces blakesleeanus . . .

Phytophthora cactorum

P. erythroseptica

P.fagopyri

Pythiomorpha gonapodyoides

Pythium ascophaUon

Rhizopus nigricans

R. suinus

Rosellinia arcuata

Sordaria fimicola

Syncephalastrum racemosum.

Thielavia basicola

Typhula variabilis

Days of
incu-
bation

Mg. mycelium

D-

Glu-
cose

199

108

126

138

119

152

121

130

121

131

181

D-

Fruc-
tose

55
154
101

81
128
118

32
130

51
122

56
114
128

58
162
141

54
122

D-

Man-
nose

100

126

115

139

117

136

147

126

113

D-

Ga-
lac-
tose

123
55
50

126
99
46

116
44
74
11
17
11
14
10

121

135
33
28

140
78
23

Mal-
tose

113

190

119

102

128

101

157

114

111

121

127

132

202

Suc-
rose

10
58

199
74
11

100
12
11

111
77
93

130

142

116
7
12
38
16
15
61

126

Lac-
tose

124

the most growth. Quantitative data on the utilization of L-sorbose by
fungi is less abundant than for the other hexoses. Observations in this
laboratoiy indicate that many fungi either do not utilize sorbose or do so
slowly.

Pentoses. The pentoses shown below occur naturally, mostly in the
form of polysaccharides or other complex compounds. L-arabinose and
D-xylose are the most easily available and have been more extensively used
than the other pentoses. The formulas for the naturally occurring
pentoses are given below:

CARBON SOURCES

CHO

HO— C— H

H— C— OH

HO— C— H

H— C— OH

HO— C— H

H— C— OH

CH2OH

D-Arabinose

CH2OH

L-Arabinose

CH2OH
D-Ribose

CHO

CH2OH

CHO

H C OH

1
C— 0

H— C— OH

HO— C— H

H— C— OH

HO— C— H

CH2OH
D-Xylose

CH2OH

L-Xylulose

CH2OH
ir-Lyxose

123

Aspergillus niger utilizes D-xylose and L-arabinose but not their
enantiomorphs, as is shown in Table 23. Many of the pentoses listed
in Table 23 are difficult to obtain in quantity, which accounts for the
varied amounts used per culture.

Table 23. The Amount of Growth and Sporulation of Aspergillus niger on

Various Pentoses
Time of incubation, 4 days. Cultures incubated at 35°C. (Steinberg, Jour. Agr.
Research 64, 1942.)

Pentose

Pentose, g.
per culture

Mg. mycelium

Sporulation

D-Lyxose

D- Xylose

L-Xylose

D-Arabinose

L-Arabinose

D-Ribose

L-Ribose

1.0

2.0

0.5

2.0

0.25

0.2
860.2

6.2

0
205.1

5.2

0
10

1
0
6
0
0

Herrick (1940) found Stereum gausapatum to utilize xylose better than
arabinose, while Aspergillus oryzae utilizes arabinose better than xylose,
(Tamiya, 1932). Lentinus lepideus utilizes xylose (Nord and Vitucci,
1947). A comparative study of five fungi on xylose and arabinose indi-
cated that xylose was utilized either more completely or more rapidly
than arabinose (Margolin, 1942). The data of Margolin are given in
Table 24; for comparable growth of these species on other sugars, see
Table 22.

124

PHYSIOLOGY OF THE FUNGI

Methylpentoses. D-Isorhamnose, L-fucose, and L-rhamnose are related
to D-glucose, L-galactose, and L-mannose in that carbon G with its primary
alcohol group in these hexoses has been replaced by a methyl group.
These methylpentoses have not been thoroughly investigated in nutri-
tional studies involving many fungi. Aspergillus niger utilizes L-rham-
nose to some extent, but L-fucose is not utilized (Steinberg, 1942). A.
oryzae makes much poorer growth on L-rhamnose than on D-xylose or

Table 24. Milligrams of IMycelium Produced by Five Fungi Grown upon

Xylose and Arabinose
These sugars were used at concentrations which suppHed 8 g. of carbon per liter.
Each 125-ml. flask contained 20 ml. cf medium. Cultures were incubated at 25 °C.
(Margolin, thesis, West Virginia University, 1942.)

Fungus

Blakeslea trispora

Mucor ramannianus

Phycomyces blakesleeanus . . . .
Phytophihora erythroseptica . .
Pythiomorpha gonapodyoides .

Days of
incubation

6
8
7
12
6

D-Xylose '

77
77
126
15
33

L-Arabinose

49
74
85
7
18

* This sugar was called Z-xylose in the earlier literature.

L-arabinose (Tamiya, 1932). Of the five fungi listed in Table 24 only
Mucor ramannianus utilizes L-rhamnose (Margolin, 1942). Stereum
gausapatum utilizes rhamnose about as well as arabinose (Herrick, 1940),
Sugar alcohols. Reduction of the aldehyde or keto group of the
simple sugars converts them into alcohols. Several sugar alcohols are
widely distributed in nature. Only the formulas for three of the natu-
rally occurring sugar alcohols will be given.

CHoOH

CH2OH

H— C— OH

HO— C— H

H— C— OH

[0— C— H

HO C H

HO— C—H

H— C— OH

HO— C—H

H— C— OH

CH2OH

Sorbitol

CH2OH
Mannitol

CHoOH
Galactitol (Dulcitol)

Most fungi appear to utilize the corresponding sugars with greater
facility than the sugar alcohols. Data for the comparative growth of
five fungi on these sugar alcohols and the parent sugars are given in
Table 25.

CARBON SOURCES

125

Sugar acids. Three types of sugar acids may be produced from aldoses
by oxidizing the terminal groups. Oxidation of the aldehyde group
yields aldonic acids, such as D-gluconic acid from glucose, while oxidation
of the primary alcohol group yields uronic acids, such as D-galacturonic
acid from D-galactose. Oxidation of both the aldehyde and primary
alcohol groups yields saccharic acids. The uronic acids are widely
distributed in natural polysaccharides such as plant gums and mucilages
and in pectin. The fungi in nature must frequently come in contact
with uronic acids, but data on utilization of these and other sugar acids
are rare. Steinberg (1942) cultured Aspergillus niger on media which con-

Table 25. Milligrams op Mycelium Produced by FrvE Fungi Grown upon
Glucose, Mannose, and Galactose and the Corresponding Sugar Alcohols
These compounds were used at a rate which supplied 8 g. of carbon per liter. Each
125-ml. flask contained 20 ml. of medium. Cultures were incubated at 25°C. (Mar-
golin, thesis, West Virginia University, 1942.)

Fungus

D-Glu-
cose

Sor-
bitol

D- Man-
nose

Man-
nitol

D-Ga-

lactose

Galac-
titol

Blakeslea trispora

Mucor ramannianus

89
138

79
152

12
93
59
10
13

98
115
139

81
79

9
149
108

8
9

123

116

10
6

Phycomyces blakesleeanus

Phytophthora erythroseptica

Pythiomorpha gonapodyoides . . .

tained 1 g. of the calcium salts of the following sugar acids per culture
(the weight of mycelium in milligrams is given in parentheses) : 2-keto-D-
gluconic (201), 5-keto-D-gluconic (25), D-gluconic (32), D-glucuronic (206),
and mucic (102). Tamiya (1932) reports that A. oryzae utilizes
D-gluconic acid. While such compounds as the sugar acids are little
used in making media, they are of interest in attempting to discover the
relation between structure and configuration on the one hand and
utilization on the other.

Mixed carbon sources. In nature the fungi usually come in contact
with mixed carbon sources rather than a single source of carbon. Certain
fungi make more growth when supplied with a mixture of carbon sources.
This increased utilization may be expected only if one or both carbon
sources are poorly utilized. Horr (1936) investigated the growth of
Aspergillus niger upon mixtures of glucose and galactose. Some of these
data are given in Table 26. If these two carbon sources were utilized
independently, and without one affecting the utilization of the other, the
weight of mycelium produced on the combination of 18 g. of galactose
and 2 g. of glucose should be 42.4 -\- 145.6, or 188 mg. The actual
yield was 577.4 mg. The experiment indicates that A. niger is able to

126 PHYSIOLOGY OF THE FUNGI

utilize galactose to better advantage in the presence of glucose. The
experiments of Steinberg (1939) on the effect of two poor carbon sources
on the growth of A. niger were made at 35°C. Some combinations of
poor carbon sources supported more growth than when these sources
were used singly. Thus, the calculated weight of mycelium for the
combination, D-mannitol-lactose was 21.4; the actual yield was 233.6 mg.
Some combinations of poor carbon sources resulted in a decrease in
amount of mycelium formed (glycerol-D-galactose : calculated yield,
243.7 mg.; actual yield, 154.7 mg.). The effect of mixed carbon sources
in the amount of growth of Phy corny ces blakesleeanus and Pythiomorpha
gonapodyoides appeared to be purely additive (Margolin, 1942).

Table 26. The Effect of Galactose and Glucose, Singly and in Combination,
UPON the Amount of Growth of Aspergillus niger
Cultures incubated 7 days at 20°C. (Horr, Plant Physiol. 11, 1936.)
Grams of Sugars Used per Liter Yield, Mg. per Culture

10 galactose 45 . 1

18 galactose 42.4

20 galactose 44.3

2 glucose 145 . 6

10 glucose 411 .0

18 galactose + 2 glucose 577.4

10 galactose + 10 glucose 1,151.6

All these results indicate that the effect of mixed carbon sources is
highly specific. A mixture of poor carbon sources may or may not
result in increased growth, depending on the carbon sources involved as
well as the fungus concerned.

The favorable effects of mixtures of poor carbon sources on the rate
and amount of growth have been ascribed to the ease with which a fungus
is able to synthesize certain key intermediates. If the synthesis of
intermediate X from carbon source A is slow and difficult, and the syn-
thesis of X is rapid from carbon source B, it is probable that growth will
be more rapid on media which contain both carbon sources.

ORGANIC ACIDS

An organic acid is characterized by having one or more carboxyl
( — COOH) groups. Some organic acids are utilized as sources of carbon
and in other ways. Two series of organic acids are especially interesting
from the standpoint of physiology. The fatty acids are monocarboxylic
acids; the higher members, when esterified with glycerol, form fats.
The dicarboxylic acids, especially those which contain four carbon atoms,
enter into the metabolic pathways of the fungi in various ways; e.g.,
utilization of ammonium nitrogen (Chap. 6).

CARBON SOURCES 127

The form in which an organic acid exists (free acid or salt) is a function
of the pH of the medium or cells. The free acid is the predominant form
at low pH values. The terms for an acid and its salt {e.g., fumaric acid,
fumarate) are used in the literature somewhat loosely. The effect of a
free acid and its anion may be different (Chap. 8).

Leptomitus ladeus, w^hich does not utilize sugars, grows on various
fatty acids — acetic, butyric to capric — but not on formic or propionic
acids (Schade, 1940). Apodachlya hrachynema utilizes the same fatty
acids as L. ladeus and also, fumarate, succinate and malate. Aspergillus
niger, according to Steinberg (1942), makes some growth on acetate,
lactate, tartrate, malate, and fumarate. Growth was very poor com-
pared with that on sucrose. Dulaney (1949) reported that little strepto-
mycin was produced when organic acids were used by Streptomyces
griseus. Yeasts use acetate to synthesize fat (White and Werkman,
1947). Tamiya (1932) investigated the utilization of many organic acids
by Aspergillus oryzae. Growth w^as poor on most of these compounds
except quinic acid. While an organic acid may serve as the sole source
of carbon for fungi, in general acids do not allow as much or as rapid
growth as carbohydrates.

An amino acid may serve as a source of both nitrogen and carbon.
Peptone may serve as a source of carbon and nitrogen for many fungi.
Aspergillus niger, when grown on peptone as the sole source of carbon,
deaminates the peptides and amino acids and releases ammonia in
quantities greater than the fungus can use. The utilization of amino
acids as carbon sources by A. niger w^as investigated by Steinberg (1942a),
who found certain combinations of "primary" amino acids to be utilized
about three-fourths as efficiently as sucrose.

The utilization of individual amino acids by Penicillium roqueforti and
Fusarium oxysporum var. lycopersici was studied by Gottlieb (1946).
Not all the naturally occurring amino acids were utilized as carbon sources
by these fungi. The six-carbon straight-chain amino acids norleucine and
lysine and the sulfur-containing amino acids cysteine and methionine
were not utilized as carbon sources. Glycine and valine were poor
carbon sources for P. roqueforti, while F. oxysporum var. lycopersici grew
well on these amino acids. Alternaria solani, Helminthosporium sativum,
Rhizoctonia solani, Fusarium moniliforme, Chaetomium globosum, and
Aspergillus niger were unable to utilize the naturally occurring sulfur-
containing amino acids as a source of carbon.

Yeasts differ in ability to utilize different amino acids as the sole
source of carbon (Schultz et al., 1949). Glutamic acid and proline were
available to more species than other amino acids. It is characteristic of
fungi cultivated on amino-acid media as the sole source of carbon that
the medium becomes alkaline. This is probably due to accumulation of

128 PHYSIOLOGY OF THE FUNGI

ammonia which results from deamination. In general, the amino acids
appear to be poor sources of carbon.

GLYCOSIDES

The carbon sources to be discussed in this and the next two sections
differ from those previously considered in that they undergo hydrolysis.
The complex carbohydrates and carbohydrate-like compounds yield
simple sugars w^hen hydrolyzed. In some instances, other compounds
are also formed. In most instances, fungi utilize these compounds only
after hydrolysis. Therefore, utilization will be dependent upon the pro-
duction of the necessary hydrolytic enzymes. If a fungus is unable to
perform this preliminary "digestion," such complex carbohydrates will
be unavailable.

Many of the compounds to be considered in this section are isomers.
The simple sugars exist mainly in the form of ring structures, rather than
the open-chain forms which were depicted in the previous sections of this
chapter. The chemical evidence may be reviewed in Pigman and
Goepp (1948) or other text dealing with the sugars. Glucose exists in
aqueous solution as an equilibrium mixture of a-D-glucose and /3-D-glu-
cose. These formulas contain a six-membered ring of w^hich one atom is
oxygen (pyranose). Some sugars, however, contain a five-membered
ring (furanose).

The formulas for these two forms of glucose are given below:

H OH HO H

\ / \ /

c , c-

H— C— OH
HO— C— H
H— C— OH
H— C— O—

H— C— OH

I
HO— C— H

I
H— C— OH

I
H— C— O—

CH2OH CH2OH

a-D-Glucose /3-D-Glucose

The simple glycosides are a widely distributed group of naturally
occurring compounds which contain a sugar moiety and an alcohol or
phenol moiety. The form glucoside was formerly used to designate com-
pounds of this type irrespective of the sugar moiety. Specific glycosides
are designated by adding the ending oside to the name of the sugar
involved; e.g., glucoside, mannoside, etc.

Two glucosides are formed when glucose is treated with methanol
under appropriate conditions. The formulas are given below:

CARBON SOURCES 129

H OCH3 H3CO H

C , C-

H— C— OH

I
HO— C— H

I
H— C— OH

I
H— C— O—

H— C— OH
HO— C— H

H— C— OH

I
H— C— O—

CH,OH CH2OH

a-Methyl-D-glucoside /3-Methyl-D-glucoside

These formulas correspond to the alpha and beta isomers of glucose.
The proof that a-methylglucoside and a-glucose have the same structure
was furnished by Armstrong (1903), who followed the enzymatic
hydi'olyses of these glucosides polarimetrically.

Our interest in the glycosides is not in the chemical structure per se,
but in the fact that utilization of these and other compounds having the
same type of glycoside linkage is dependent upon configuration. Differ-
ent enzymes are required for the hydrolysis of the a- and /3-glycoside
linkages. Some fungi possess both types of hydrolytic enzymes, others
but one, and some fungi appear to lack both. Thus, certain yeasts
ferment a-methylglucoside but not /3-methylglucoside. These yeasts
have an enzyme or enzymes which catalyze the hydrolysis of the a-gly-
coside linkage but not the /3-glycoside linkage (lactose-fermenting yeasts
are able to hydrolyze ;S-glycosides).

The use of the methylglucosides is not always a safe guide in pre-
dicting which complex sugars will be utilized by fungi. Aspergillus niger
utilizes /3-methylglucoside rapidly and completely, while a-methyl-
glucoside is poorly utilized (Dox and Neidig, 1912). Attempts to adapt
A. niger to utilize a-methylglucoside as a sole source of carbon were
without much success, although the fungus apparently utilized this
compound in the presence of sucrose (Dox and Roark, 1920). A. niger
utilizes lactose poorly. Tamiya (1932) found A. oryzae made only a
trace of growth on a-methylglucoside. /3-Methylglucoside was not
tested. This fungus grows well on maltose. These results are, perhaps,
not unexpected in view of the specificity of enzymes. The utilization of
the naturally occurring simple glycosides by fungi has been investigated
but slightly.

OLIGOSACCHARIDES

These sugars are derived from two, three, or four hexose sugars by the
elimination of water. On hydrolysis, the individual sugars are regener-
ated. Five factors which determine the structure of the oligosaccharides
are (1) the component sugars; (2) the component sugar which functions

130

PHYSIOLOGY OF THE FUNGI

as the alcohol; (3) the stereochemical nature of the glycoside linkage; (4)
the carbon of the alcohol moiety which forms the glycoside linkage; and
(5) the ring structure of the component sugars (see Oilman, 1943),

Maltose. It is doubtful whether this disaccharide occurs free in
nature. It is formed when starch is enzymatically hydrolyzed; on
further hydrolysis two molecules of glucose are formed. This disac-
charide is utilized by many fungi. The glycoside linkage is alpha in
maltose.

H OH

H-C-OH

I
HO-C-H

I
H-C-0

CH2OH

Maltose

Cellobiose. The occurrence of this sugar as a repeating unit in cellu-
lose makes it important. Cellobiose differs from maltose only in the
nature of the glycoside linkage. With few exceptions only fungi which
produce enzymes which attack the /3-glycoside linkage will utilize this
sugar.

HO-C-H

CH2OH

Cellobiose

Since cellobiose and maltose differ only in the nature of the glycoside
linkage, it would be interesting to compare the utilization of these two
sugars by a large number of fungi. Cellobiose has been studied so infre-
quently that the necessary data are lacking.

CARBON SOURCES

131

Lactose. This sugar is probably present in the milk of all animals.
Hydrolysis of lactose by acids or lactase yields a molecule each of glucose
and galactose. This sugar is hydrolyzed by emulsin and is therefore a
/3-glycoside.

CH2OH

Lactose

Sucrose. This sugar is of common occurrence in plants. On hydroly-
sis one molecule of glucose and one of fructose are formed; a mixed a- and
j8-glycoside linkage unites the sugar moieties. Sucrose apparently is
utilized by fewer fungi than maltose, but more extensively than lactose
(see Table 22).

CH2OH

Sucrose

In addition to the three common disaccharides (maltose, lactose,
and sucrose), many other oligosaccharides are known. Owing to cost
and relative unavailability, these sugars have not been studied inten-
sively. Some of these "rare" sugars are used in differential media in
bacteriology. Brief mention will be made here of some of these sugars.
The nonreducing disaccharide trehalose (mushroom sugar) is syn-
thesized by various fungi and is fermented by many yeasts. Trehalose
on hydrolysis yields glucose ; it differs from maltose in the position of the

132

PHYSIOLOGY OF THE FUNGI

glycoside linkage. Tamiya (1932) reported that Aspergillus oryzae
utilized trehalose and raffinose. The trisaccharide raffinose is obtained
as a by-product of beet-sugar manufacture. On complete hydrolysis
galactose, glucose, and fructose are formed in equivalent amounts. The
structure for raffinose is given below.

Volkonsky (1934) found raffinose to be utilized readily by Pythmm
deharyanum and a species of Sporotrichum. One isolate of Phytophthora
parasitica utilized raffinose rapidly, while another isolate utilized this
sugar slowly. Phytophthora cactorum and P. palmivora utilized this
sugar slowly. The great majority of fungi tested by Volkonsky did not
utilize raffinose.

CH2OH
O— C

HO— C— H

H— C— OH

I
H— C— O—

H-

H— C— OH

HO— C— H

I
H— C— OH

H— C— O—

CHoOH

D-Fructose

CH2O-

D-Glucose

H— C

H— C— OH

I
HO— C— H

H— C— O—

CH2OH

u-Galactose

Raffinose

Oligosaccharides and polysaccharides are utilized by fewer fungi than
is glucose. All microorganisms which can utilize a given polysaccharide
are also able to utilize its hydrolytic products (Van Niel, 1944). Not all
polysaccharides yield glucose on hydrolysis, but the majority of them do.

While the evidence at hand does not exclude the direct utilization
of disaccharides by some fungi, it is probable that these sugars are
hydrolyzed before utilization in most instances. Smith (1949) suggests
that Marasmius chordalis attacks cellobiose by a route that involves
neither preliminary hydrolysis nor phosphorylation.

The failure of a fungus to utilize an oligosaccharide may be due either to
the lack of the necessary hydrolytic enzyme or to inability to utilize the
component sugars. Failure to synthesize the necessary hydrolytic
enzymes appears to be by far the most common cause of nonutilization.
This is borne out by the data in Table 22. Of the 21 fungi studied by
Margolin, two failed to grow on maltose, while eight did not utilize
sucrose. Since all these fungi grew well on glucose and fructose, it is
evident that failure to utilize maltose and sucrose was due to the fact that
these fungi could not hydrolyze these sugars. The nonutilization of
lactose by Syncephala strum racemosum is evidently due to the failure of

CARBON SOURCES 133

this fungus to synthesize lactase, for this fungus makes good growth on
either glucose or galactose. The same argument applies to Blakeslea
trispora, Fusarium lycopersici, Rhizopus nigricans, and R. suinus. Non-
utilization of a complex carbohydrate is usually due to the lack of the
necessary hydrolytic enzymes.

The hydrolysis of oligosaccharides by fungi is easily demonstrated.
Phycomyccs blakesleeanus utilizes sucrose while Mucor ramannianus does
not. If the mycelium of P. blakesleeanus is removed from a flask of
sucrose medium after several days' incubation and the flask reinoculated
with M. ramannianus, the latter fungus will grow. P. blakesleeanus
excretes sucrase, which catalyses the hydrolysis of sucrose to D-glucose
and D-fructose, both of which are utilized by M. ramannianus.

A complex carbohydrate and its hydrolytic products are not necessarily
equivalent in all respects. Hawker (1947) reported that the amount of
mycelium produced by Melanospora destruens was different when this
fungus was grown on equivalent amounts of glucose, fructose, mixtures of
glucose and fructose, and sucrose. More mycelium was produced from
glucose than from an equivalent amount of sucrose, and this was true
whether the concentrations of these sugars were low or high. On the
other hand, perithecia were produced more abundantly on sucrose than
on glucose media. Indeed, hydrolysis of the same lot of sucrose to
glucose and fructose allowed the production of no more perithecia than
other samples of these sugars. The conclusion seems inescapable that
the particular structure of sucrose was in some way favorable for the pro-
duction of perithecia. While a fungus may utilize an oligosaccharide and
its hydrolytic products, it is not safe to assume that both are used with
the same efficiency for all purposes.

POLYSACCHARIDES

The chemistry of the polysaccharides resembles that of the oligosac-
charides except that the number of sugar residues is much larger. These
substances constitute the bulk of carbohydrate materials synthesized by
plants and animals. The most important polysaccharides are cellulose,
starch, and glycogen. On hydrolysis simple sugars are formed. The
molecular weights of polysaccharides may be very large; cellulose from
different sources may have a molecular weight ranging from 200,000 to
400,000. The molecular weights of many polysaccharides are much less
than that of cellulose. In general, polysaccharides are insoluble or only
colloidally soluble. The utilization of these substances by fungi is
dependent upon the excretion of the necessary hydrolytic enzymes. Pig-
man and Goepp (1948) classify polysaccharides on the basis of the
hydrolytic products as homopolysaccharides, which yield only one mono-
saccharide on hydrolysis, and heteropolysaccharides, which yield two or

134 PHYSIOLOGY OF THE FUNGI

more monosaccharides or related compounds on hj^drolysis. Cellulose,
starch, and glycogen are members of the first class and yield glucose on
hydrolysis. Polysaccharides are frequently named b}^ replacing the end-
ing -ose of the parent monosaccharide by -an. Fructan (le\ailan) desig-
nates a polysaccharide which yields fructose on hydrolysis. A hexosan
is a polysaccharide which yields hexose sugars on hydrolysis, and a pen-
tosan yields pentoses. Pectins are polymers of galacturonic acid.

The heteropolysaccharides occur in lesser amounts than the homopoly-
saccharides. Among them are the hemicelluloses, which on hydrolysis
yield D-xylose as the principal sugar, the plant gums, and agar.

Cellulose. Chemically, cellulose is a linear polymer of o-glucose. The
glucose residues are joined together through /3-glycoside linkages as in
cellobiose, and cellulose may be thought of as consisting of repeating
cellobiose units. Norman and Fuller (1942) postulate that the majority
of fungi are able to utilize cellulose. In spite of the importance of cellu-
lose utilization by fungi in the economy of nature much remains to be
learned about this process.

It is commonly accepted that the first stage in utilization of cellulose is
hydrolysis, although Campbell (1932) has suggested oxidation. The
hydrolysis of cellulose may be expressed schematically as follows: cellu-
lose — > cellodextrins -^ cellotetrose -^ cellobiose -^ D-glucose. Fungus
cellulases appear to have been infrequently studied. Grassmann et al.
(1933) separated cellulase and cellobiase from Aspergillus oryzae. This
cellulase was inactive in hydrolyzing cellulose degradation products hav-
ing a molecular weight less than 1,000 (six glucose residues), while the
cellobiase hydrolyzed cellulose fragments containing from two to six
glucose residues.

Fungi differ widely in ability to utilize cellulose. In general, the rate of
utilization of cellulose is less than that of glucose. This is probabty due
to the insolubility of cellulose, which limits the action of cellulase to the
surface, or to an inadequate rate of enzyme synthesis.

The principal source of cellulose available to fungi in nature is wood and
other plant remains, ^^^lile cellulose is the chief constituent in such
materials, hemicelluloses, gums, tannins, and lignin are also present.
The wood-rotting fungi have been classified according to whether they
cause white or brown rots. The fungi which cause brown rots attack
cellulose in preference to lignin. The fungi which preferentially attack
the noncellulosic constituents of wood cause white rots. The latter
species are apparently more numerous than those which cause brown rots.
The following are some of the fungi listed by Nobles (1948) as causing
white rots: Armillaria mellea, Ganoderma lobaturn, Lenzites hetulinus,
Pleurotus ostreatus, Polyporus ahietinus, P. cinnabarinus, P. pargamenus.
A few fungi causing bro^^^l rots are Daedalea quercina, Lentinus lepideus,

CAR BOX SOURCES

135

Lenzites trabea, Merulius lacrymans, Polyporus betulinus, and Trametes
americana.

The effect of a typical fungus causing white rot on the composition of
wood is given in Table 27. Polyporus pargamenus was allowed to act on
blocks of aspen wood for 20 months. At the end of this time the wood
block showed three degrees of attack. The tan-colored portion was
altered least. The pink-colored portion was intermediate, while the
white portion had lost the most lignin. P. pargamenus also degraded the
cellulose somewhat, as shown by lower degree of polymerization.

T.\BLE 27. The Effect of Polyporus pargamenus ix Altering the Composition of

Aspen Wood
Time of incubation 20 months. (Heuser et al., Arch. Biochem. 21, 1949. Published
by permission of Academic Press, Inc.)

Portion of
wood block

Lignin, %

Pentosans, %

CeUulose, %
(calculated)

Original

Tan

Pink

\Miite

17.5

10.4

4.5

3.4

19.3

12.8

8.3

8.4

GO. 68
73.84
84.20
85.32

The effect of fungi causing brown rots on the composition of coniferous
woods has been studied by Schubert and Xord (1950). Lenzites saepiaria
in 13 months caused a decrease in cellulose in pine sawdust from 45.5 to
18.5 per cent. During this period the apparent lignin content increased
from 33.9 to 50.1 per cent. Similar results were obtained with Lentinus
lepideus and Poria vaillantii. For a recent review of the microbiological
degradation of cellulose see Nord and Vitucci (1948).

Starch. Like cellulose, starch is a polymer of D-glucose. The glucose
residues are j oined through a-glycoside linkages, and starch (and glycogen)
may be thought of as consisting of repeating units of maltose. Starch
consists of two tj-pes of molecules. The linear portion of starch is called
amylose, while the branched-chain fraction is known as amylopectin.
Starch is sjTithesized by green plants, while glycogen is formed by
animals and fungi. The enzymes which catatyze the hydrolysis of starch
are known as amylases and were discussed in Chap. 4. The enzymatic
hj'drolysis of starch may be represented schematically as follows : starch -^
dextrins— ^ maltose -^ D-glucose. The branched-chain dextrins are incom-
pletely hydrolyzed by amylase, while the straight-chain dextrins are
completely converted to maltose (]\Iyrback, 1948).

Starch is insoluble in water. Only those furgi which produce amylase
are able to utilize starch. This ability is common among fungi but not
universal. Volkonsky (1934) found 26 isolates and species of the

136 PHYSIOLOGY OF THE FUNGI

Saproliginales to utilize starch and its hydrolytic products (dextrin,
maltose, and glucose). Thirteen other carbon sources, including fructose,
were not utilized. Margolin (1942) found that 19 out of 21 fungi which
utilized maltose also utilized dextrin.

The nonutilization of starch by Sclerotinia libertiana has been suggested
as the basis of a method of preparing potato starch (Kakeura, 1946).
Few yeasts utilize starch, although maltose and glucose are readily
utilized.

All the fungi listed in Table 22 except Pythiwn ascophallon and Phy-
tophthora jagopijri utilized dextrin. A comparison of the ability of fungi
to utilize glycogen and starch has not been investigated thoroughly.
Tamiya (1932) found the yield of mycelium of Aspergillus oryzae to be
greater on glycogen than on dextrin. Dextrin was a better carbon source
than starch.

The role of the pectin-destroying enzymes in parasitism and the rotting
of fruits and vegetables is discussed in Chap. 17. Presumably these fungi
utilize some or all of the hydrolytic products of pectin (n-galacturonic acid
and methyl alcohol). None of the fungi, in so far as is known, utilize agar
as a source of carbon. A . niger utilizes the arabo-galactan from western
larch as a source of carbon (Ratajak and Owens, 1942).

HETEROTROPHIC UTILIZATION OF CARBON DIOXIDE

The assimilation of carbon dioxide is not restricted to green plants.
Carbon dioxide fixation has been demonstrated in bacteria, fungi,
protozoa, liver slices, barley roots, and intact green plants in the absence
of light. The basis for classifying organisms according to the way they
utilize carbon dioxide is discussed by Werkman and Wood (1942), By
the use of carbon isotopes an elegant method is available for demonstrating
carbon dioxide assimilation. In addition, the mechanism of fixation can
be studied. This involves isolation and degradation studies of the com-
pounds synthesized Avhile the organisms were exposed to isotopic carbon
dioxide. Either stable or radioactive carbon isotopes may be used. The
finding of isotopic carbon in compounds synthesized is proof of assimilation.

Aspergillus niger and Rhizopus nigricans were shown to assimilate car-
bon dioxide (Foster et al., 1941). Radioactive carbon dioxide (C^i02)
was used in these experiments. Mycelium of R. nigricans was suspended
in 5 per cent glucose solution and agitated in a closed system containing
isotopic carbon dioxide. At the end of the experiment the mycelium and
the medium were analyzed for radioactivity. More than one-third of
the carbon dioxide assimilated was incorporated into cell constituents
which were not decomposed by boiling for 1 hr. with 2M hydrochloric
acid. Carbon dioxide was assimilated under aerobic and anaerobic
conditions. The data of such an experiment are given in Table 28.

CARBON SOURCES

137

Table 28. Distribution of Radioactive Carbon (C^O in the Culture Medium
AND Mycelium of Rhizopus nigricans Exposed to C^'02 in the Gas Phase for

30 Minutes
Results are expressed as percentage of C^Oo assimilated. (Foster et ah, Proc.

Natl. Acad. Sci., U.S. 27, 1941.)

Substance tested

Total C* in supernatant solution after removing cells.

Fumaric * acid in this solution

C* in neutral volatile distillate

Total C* in acid extract of cells

Fumaric * acid in this solution

C* remaining in cells after acid extraction

Aerobic

Anaerobic

29.0
25.0
0.2
30.0
12.0
41.0

* Designates radioactive carbon.

It is probable that carbon dioxide enters into various metabolic proc-
esses. Foster and Davis (1948) postulate that strains of Rhizopus
nigricans which produce fumaric acid anaerobically do so according to
scheme IV. Cantino (1949), in studying the metabolism of Blastocladia

Scheme IV. A Scheme for the Anaerobic Transformation of Glucose into

Fumaric Acid by Rhizopus nigricans*

nC6Hi206

CH3CHOHCOOH
Lactic acid

+2H yf-nAU

nCHsCOCOOH

Pyruvic acid

+ CO2

HOOCCH2COCOOH
Oxalacetic acid

■^ CO2+ CH3CHO
Acetaldehyde

+ 2H

CH3CH2OH

Ethyl alcohol

+ 2H

HOOCCHgCHOHCOOH
Malic acid

* Courtesy of Foster and Davis, Jour. Bad., 56 : 335, 1948,
& Wilkins Company.

-HOH

HOOCCH-.CHCOOH
Fumaric acid

Published by permission of The Williams

pringsheimii, found that, by increasing the carbon dioxide in the gaseous
phase, the formation of lactic acid was decreased, while the amount of

138

PHYSIOLOGY OF THE FUNGI

succinic acid was increased. It was surmised that this fungus utilizes
carbon dioxide, since none was set free.

The formation of oxalacetic acid by the reaction between pyruvic acid
and carbon dioxide suggests that heterotrophic carbon dioxide fixation
may play a role in amino-acid synthesis. Support of this hypothesis may
be found in the work of Ajl and Werkman (1949), who found the carbon
dioxide requirement of Aerohacter aerogenes could be replaced by oxal-
acetic, a-ketoglutaric, fumaric, or aspartic acid. For further information
on carbon dioxide utilization by fungi see Foster (1949).

UTILIZATION OF CARBON

Carbon compounds are utilized by fungi for two general purposes, as a
source of energy and as a source of the chief structural element. These
two processes may be the same until a number of chemical transforma-
tions have taken place but may then diverge after certain intermediate
compounds are formed. The over-all use of carbon is quite easily
determined, but it is a problem of a different order to trace all the chemical
transformations which occur when a compound is utilized.

Table 29. The Distribution of Carbon from Arabinose among the Products

OF Metabolism of Fusarium lini
(White and Willaman, Biochem. Jour. 22, 1928. Published by permission of
Cambridge University Press.)

Age of

Mycelium,

CO2,

Alcohol,

Lead pre-

Sugar,

Total

culture, days

cipitate, %

carbon, %

0.8

0.6

7.6

90.6

99.6

3.4

4.4

7.6

0.6

85.2

101.2

4.6

6.1

6.6

1.0

80.4

98.7

4.0

9.4

3.3

1.5

81.2

99.4

10.4

20.8

9.9

1.7

55.2

98.3

Carbon balances. A general idea of the way a carbon source is utilized
may be gained by following the amounts of mycelium synthesized, carbon
dioxide evolved, and other metabolic products formed. If the initial
amount of carbon is known, its distribution can be followed by analysis.
From 95 to 99 per cent of the carbon is usually accounted for in such
experiments. The accompanying data from White and Willaman (1928)
illustrate this distribution of carbon from arabinose by Fusarium lini
(Table 29).

While the analytical difficulties in experiments of this kind are con-
siderable, chemical analysis of the mycelium and the other metabolic
products reveals how the carbon originally present in the carbon source is
distributed. Such analyses are useful in detecting the major metabolic

CARBON SOURCES 139

products. Carbon balances are especiall}^ useful in determining the effi-
ciency with which a fungus produces metabolic products of value, such as
alcohol and citric acid. For further examples see Raistrick ct al. (1931).

Utilization ratios. The relations of the amounts of fungus metabolic
products to the amount of carbon soiu'ce (or other substance) used are
frequently expressed as ratios. However, these ratios are valid only for
the fungi and the experimental conditions used. These ratios should be
considered as absolute values only for the conditions under which they
were obtained. The various utilization ratios are of less value than
complete carbon balances, but the analytical determinations are fewer.
To be of most value, these ratios should be determined at various intervals
during incubation, because these ratios change with age.

The most useful of these ratios is the economic coefficient, which is
obtained by dividing the weight of mycelium and spores by the weight of
sugar or other carbon source used. The residual carbon source in the
medium must be determined at the end of an experiment. In general an
efficient fungus will convert half the weight of sugar supplied in the
medium into cellular material. The efficiency of most fungi when grown
on laboratory media is much less. This is due in part to the use of
unbalanced media and to the type of carbon metabolism taking place.
The carbon which is not utilized for the synthesis of cellular material
appears either as carbon dioxide or as intermediate metabolic products,
such as alcohol and organic acids. In industrial applications it is desir-
able to employ cultural conditions which divert a large part of the carbon
used into the desired intermediate products, rather than into the produc-
tion of mycelium and carbon dioxide.

The economic coefficient of Fusarium sambucinum under various
cultural conditions has been studied by Holzapfel (1925). This fungus
utilized sucrose (0.33) and fructose (0.36) more efficiently than glucose
(0.24). The economic coefficient varied with the concentration of the
carbon source and w^ith the source of nitrogen, as w'ell as with the age of
the cultures.

For a discussion of other utilization ratios and examples, see Steinberg
(1942), Peterson et al. (1922), White and Willaman (1928), and Fries
(1938).

Intermediary metabolism. The problem, to be considered here is the
way fungi utilize the various sources of carbon available to them. From
the data and discussion in the earlier part of this chapter it is clear that
structure and configuration play an important role in determining which
compounds may serve as a source of carbon for a given fungus. The
availability of complex natural compounds, such as the carbohydrates,
was found to depend upon the production of the necessary extracellular
hydrolytic enzymes. The utilization of simple compounds, such as the

140 PHYSIOLOGY OF THE FUNGI

monosaccharides, is likewise an enzymatically catalyzed chain of meta-
bolic processes. It may be assumed that the chemical composition of the
fungus will be about the same, irrespective of the carbon source utilized.
Therefore, at some place along the path of synthesis the initial carbon
sources are converted into the same compounds. It is probable that the
original compounds are converted into the same intermediate compounds
before synthesis. Thus, galactose is apparently transformed by Sac-
charomyces fragilis into galactose- 1-phosphate, which is then converted
into glucose- 1-phosphate (Caputto et at., 1949). These intermediate
compounds then enter the various metabolic reaction chains which lead
to the production of materials which make up the fungus. We may
suppose that the first steps in utilization are those which transform a
carbon source into key intermediates.

The intermediate metabolic products should also serve as a source of
carbon for the fungus in question. If a fungus transforms compound A
into compound B, then compound B should serve as a source of carbon.
Nonutilization of compound B indicates that this compound is not part of
the metabolic pathway. This simple hypothesis neglects two important
considerations : compound B may not enter the fungus cells with the same
facility as compound A, or compound B may be toxic in the concentra-
tions present. As an example of this approach, the work of Steinberg
(1942) may be consulted. Since Aspergillus niger made only a trace of
growth on D-gluconic acid, it seems probable that the first step in the
utilization of glucose by this fungus is not the oxidation of the aldehyde
group. The isolated enzymes from a fungus may also be studied to
determine the reactions catalyzed, or the effect of specific enzyme
inhibitors on the intact fungus may be studied.

In some instances intermediates of sugar dissimilation are excreted into
the medium and may be isolated. Thus, the production of acetaldehyde
may be demonstrated by adding bisulfite to the medium. Acetaldehyde
forms an insoluble addition product with this reagent. The excretion
of intermediate metabolites may be due to slowness of the next step in the
metabolic process. These products are usually utilized in the course of
time. Among such intermediates which have been identified are acet-
aldehyde, ethyl alcohol, and pyruvic acid.

On the basis of the evidence now available we may not assume that all
fungi utilize a sugar or other carbon source in exactly the same way, or
that a fungus has only one metabolic pathway for the utilization of a
sugar. Nord and Mull (1945) consider that species of Fusarium dis-
similate carbohydrates by oxidation, by splitting the carbon chain, and
by a phosphorylation mechanism. The relative importance of these
three methods of attack depends upon the fungus involved and upon the
environmental conditions. Identity of a metabolic product formed by

CARBOX SOURCES

141

two fungi is not proof that the reaction mechanism is the same in both
instances. Yeast and certain species of Fusarium produce alcohol, but
the pathways from glucose to alcohol appear to be different. The
mechanism of carbohydrate dissimilation by Fusarium lini, when grown
upon a nitrate medium, is believed to take place as shown in scheme V.
An essential feature of this scheme is the formation of pyruvic acid from
both pentoses and hexoses. The intermediate steps in this biosynthesis
by Fusarium lini have not been elucidated. A portion of the hydrogen
derived from the dissimilation of carbohydrate is enzymatically trans-
ferred and used for the reduction of nitrate ion which acts as a hydrogen
acceptor. The nitrite produced inhibits the carboxylase enzyme system
which transforms pyruvic acid into carbon dioxide and acetaldehyde.
Pyruvic acid does not accumulate in the culture medium when ammonium
nitrogen is used.

Scheme V. The Pathway of Hexose and Pentose Utilization by Fusarium lini

Grown on Nitrate Medium
(Courtesy of Wirth and Nord, Arch. Biochem. \ : 155, 1942. Published by permis-
sion of Academic Press, Inc.)

Acceptor
(Nitrate)

Nitrite

Carboxylase system

Hydroxylamine
+
->- Pyruvic acid ^- Alcohol

X reduction
Amino acid

Utilization

Hexoses

Pentoses

Pyruvic acid is the key intermediate compound formed in the dis-
similation of hexoses and pentoses by F. lini. The transformation of
pyruvic acid into alcohol by F. lini and yeasts appears to follow the same
pathway and to require the same coenzymes, cocarboxylase and code-
hydrogenase I.

The anaerobic dissimilation (fermentation) of glucose by yeast and
the comparable process in muscle (glycolysis) have been intensively
studied. These are perhaps the best understood of all metabolic proc-
esses. Although it does not function in glucose dissimilation by F. lini in
the same way as in yeast, phosphate plays a role in all these transforma-
tions until pyruvic acid is formed. Many investigators have contributed

142

PHYSIOLOGY OF THE FLi.XGI

to the scheme of gUicose dissimilation presented in schemie VI (Meyerhof,
1938, 1949). Further information about these reactions may be found in
Sumner and Soraers (1947), Tauber (1949), and Prescott and Dunn

(1949).

Scheme VI. The Pathway of Glucose Dissimiliation by Yeast (Alcoholic
Fermentation) and Muscle (Glycolysis)
(Courtesy of Meyerhof, WaUerstein Labs. Communs. 12 : 256, 1949. Published by
permission of WaUerstein Laboratories.)
Glycogen, starch D-Glucose

± H,P04

+ H3PO4

Glucose- 1-phosphate (Cori ester)^=±GIucose-()-phosphate (Robison ester)

Fructose-6-phosphate (Neuberg ester)

+ H3PO4

'4'

Fructose-ljG-diphosphate (Harden- Young ester)

Dioxyacetone phosphate*

+ H2

CHaOHCiOCHrOPOsHa

/-Q^Glycero phosphate

[CHsOHCHOH-CHaOPOsHs

Glycerol + H3PO4

Aceta
Ethanol

dehyde + C02<

Kd) 3-GIyceraIdehyde phosphate (Fischer-Baer

ester)
tCHOCHOHCH2-0-P03H2

+ H2

+ H3PO4
(d) 1,3-Diphosphoglyceric jicid
+ H3PO4

'CO-O-POsHsCHOH-CHa-O-POsH,

(d) 3-Phosphoglvceric acid

TCOOH-CHOH-CHa-O-POsHs

(d) 2-Phosphoglvceric acid

* CH2dHCH(OP03H2)COOH

+ H2O

(Enol)-Phosphopvruvic acid

TCH2:C(OP03Ho)(COOH)

-Pyruvic acid + H3PO4 .

±H2

Lactic acid

Pyruvic acid is a key intermediate compound in metabolism. Pyruvic
acid serves as a source of carbon for many fungi, although the rate of
growth on this substance is frequently slow. This is in accord with the
hypothesis that intermediate metabolites are able to replace the original
carbon source. The accumulation of this compound in the culture
medium may be demonstrated by the formation of iodoform in the cold
by adding a solution of iodine and making the medium strongly alkaline.
The sensitive color test of Lu (2,4-dinitrophenylhydrazine) may also be
used [see Friedemann and Haugen (1943) for details]. Acetaldehyde also
yields iodoform under these conditions, but gentle heating will drive off

CARBON SOURCES

143

this substance. AVe have noted in this laboratory that pyruvic acid
ordinarily disappears from culture medium as the time of incubation is
increased. The disappearance of the pyruvic acid in the culture medium
is usually correlated with a rise in pH. Some typical reactions of pyruvic
acid are shown in scheme VII. For a review of pyruvate metabolism see
Stotz (1945).

Scheme VIIo Some Typical Transformations of Pyruvic Acid

CH3-CO— COOH

H ^^ Pl7ri11M^ rtmA ^\ NH3+H

CH3-CHOH-COOH

Lactic acid

Pyruvic acid

-CO2

CH3— CHO
Acetaldehyde

CH3-CH(NH2)-COOH

ot - Alanine

CH3-COOH
Acetic acid

CH3-CH2OH

Ethyl alcohol

It is probable that most intermediates used in the synthesis of proto-
plasm are synthesized from low-molecular-weight compounds. Acetate
is used by yeasts for the synthesis of fats and other cellular constituents.
Weinhouse and Millington (1947) studied the metabolism of isotopic
acetate by yeast depleted of endogenous nutrients. Acetate was rapidly
utilized. The distribution of the carbon from the isotopic acetate was

Scheme VIII. Oxidation of Acetate by Yeast by Means of the Krebs Citric

Acid Cycle*

COOH

Hooc— CH2— c— CHj— coon

CH3— COOH -I HOOC— CHr— CO— COOH -

2CH3— COOH

HOOC— CH2— CHO H— COOH

HOOC— CH=CH— COOH

HOOC— CH2—C Ho— COOH <—

HOOC— CH:

-CO2

—CH— CHO H— COOH

COOH
CO2

HOOC— CH2—CH2— CO— COOH

* Original scheme modified according to Weinhouse. Courtesy of Weinhouse and Millington, Jour.
Am. Chem. Soc. 69: 3093, 1947. Published by permission of the American Chemical Society.

determined by analysis. A portion of the acetate was oxidized; another
portion was found in the lipide fraction and cell residue; some was con-
verted to citric acid. It was calculated that from one-fourth to one-third
of the lipides found in the yeast cells at the end of the experiment (a 7-hr.
period) were newly synthesized from acetate. The cell residue (after
extraction of the fats) contained only a little isotopic carbon. This is not
surprising, since nitrogen was not furnished during these experiments.

144

PHYSIOLOGY OF THE FUNGI

The mechanism of acetate oxidation by yeast is postulated by these
authors to follow a modified Krebs citric acid cycle (scheme VIII). The
oxidation of acetate is thus the result of a rather complex cyclic process.
While the four-carbon dicarboxylic acids of the Krebs cycle are poor
sources of carbon for most fungi, they are important in intermediary
metabolism. These acids are readily interconvertible. The role of the
keto acids in amino-acid synthesis was noted in Chap. 6. Lewis (1948)
studied the metabolism of mutants of Neurospora crassa which were
unable to synthesize either aspartic or glutamic acids. These amino

Scheme IX. A Generalized Krebs Isocitric Acid Cycle Proposed to

Illustrate the Pathways of Conversion of Carbohydrate into

Aspartic and Glutamic Acids by Neurospora*

Carbohydrate

Pyruvate -^

(CHg-CO-R)

Cis-aconitate
n

Isocitrate
A
A

oi -Ketoglutarate

Oxalacetate

Succinate

* Courtesy of Lewis, Am. Jour. Botany 36: 294, 1948.

acids could be replaced by a-ketoglutaric, succinic, malic, or fumaric
acids. A generalized Krebs cycle was proposed by Lewis which indicates
the pathway of synthesis of aspartic and glutamic acids from glucose
(scheme IX). Compounds utilized by the Neurospora mutants are
printed in italics. The probable location of the genetic block which
prevents the biosynthesis of aspartic and glutamic acids is indicated by A.

SUMMARY

Organic compounds are utilized by fungi for the synthesis of structural
and functional compounds and as sources of energy. The fungi utilize a
wide range of natural organic compounds including those of great com-

CARBON SOURCES 145

plexity. Not all fungi utilize all natural organic compounds, nor do all
species utilize a given compound with the same facility. The composi-
tion, structure, and configuration of organic compounds affect utilization,
but the effect of these factors may be different for different fungi.

The carbohydrates are the most common and important sources of
carbon for the fungi. Sugars (and other compounds) having the same
structure, but with mirror-image configuration, differ physiologically.
Usually only one enantiomorph is utilized, or one is utilized much more
rapidly than the other. Glucose is utilized by more fungi than any other
sugar. Few fungi are unable to utilize glucose. A few species are appar-
ently unable to utilize any sugar; e.g., Leptomitus lacteus. The species
that utilize the pentoses, sugar alcohols, acids, and other simple organic
compounds are fewer in number than those which utilize glucose.

The oligo- and polysaccharides are utilized by fewer species than is
glucose. The nature of the glycoside linkage as well as the sugar residues
is important in determining whether these compounds are utilized by a
given fungus. It is probable that most fungi hydrolyze oligosaccharides
before utilization occurs. This does not exclude direct utilization in some
instances. An oligosaccharide and its hydrolytic products are not always
physiologically equivalent. The general order of availability of the three
common disaccharides appears to be maltose, sucrose, and lactose.
Among the polysaccharides, cellulose and starch are the most abundant.
These compounds are insoluble and must be hydrolyzed or otherwise
degraded to low-molecular-weight compounds before utilization. Only
those fungi which form cellulase and amylase are able to utilize these
compounds. This "digestion" is accomplished by enzymes. Ability to
utilize other polysaccharides is also dependent upon possession of the
necessary hydrolytic enzymes.

Some fungi utilize carbon dioxide, but not as a sole source of carbon.
It is postulated that carbon dioxide combines with pyruvic acid and other
keto acids to form key intermediate products which are necessary for the
formation of amino acids.

The fate of the carbon supplied to a fungus is best determined by
carbon-balance studies; i.e., by complete chemical analyses of the myce-
livim and other metabolic products, including the carbon dioxide produced.

The first step in utilization of sugars and other carbon sources is the
formation of certain key intermediate metabolic compounds. These key
compounds are in part utilized for synthesis and in part oxidized to pro-
vide energy. The metabolic pathways leading to the formation of key
intermediates differ, depending upon the environmental conditions and
the fungus involved. Among the key intermediates pyruvic acid is
especially noteworthy. Reduction of this compound yields lactic acid,
while amination and reduction leads to alanine. Decarboxylation pro-

146 PHYSIOLOGY OF THE FUNGI

duces acetaldehyde, which in turn may yield either ethyl alcohol or acetic
acid. Acetate is utilized by yeast and other fungi for the synthesis of
fats and other cellular constituents. A fungus utilizes a compound by a
series of step-by-step transformation. Among the best understood of
these metabolic activities is the transformation of glucose into alcohol by
yeasts.

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artificial media, thesis. West Virginia University, 1949.
Dox, A. W., and R. E. Neidig: Spaltung von a- und /3-Methylglucosid durch

Aspergillus nigcr, Biochem. Zeit. 46: 397-402, 1912.
Dox, A. ^Y., and G. W. Roark, Jr.: The utilization of a/p/ia-methylglucoside by

Aspergillus nigcr, Jour. Biol. Chem. 41: 475-481, 1920.
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growth and streptomycin production, Mycologia 41: 1-10, 1949.
Foster, J. W. : Chemical Activities of Fungi, Academic Press, Inc., New York, 1949.
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as an indicator of carbon dioxide utilization. VII. The assimilation of carbon

dioxide by molds, Proc. Natl. Acad. Sci. U.S. 27: 590-596, 1941.
Foster, J. W., and J. B. D.wis: Anaerobic formation of fumaric acid by the mold

Rhizopus nigricans. Jour. Bad. 56 : 329-338, 1948.
Friedemann, T. E., and G. E. Haugen: Pyruvic acid. II. The determination of

keto acids in blood and urine. Jour. Biol. Chem. 147: 415-442, 1943.
Fries, N.: Ucber die Bedeutung von Wuchsstoffen flir das Wachstum verscheidener

Pilze, Symbolae Botan. Upsalienses 3 : 2, 1938.
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Gottlieb, D. : The utilization of amino acids as a source of carbon by fungi, Arch.

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Grassmann, W., L. Zeichmeister, G. Toth, and R. Stadder: Ueber den enzyma-

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CARBOX SOURCES 147

■*Ha\vker, L. E.: Further experiments on growth and fruiting of Melanospora

destruens Shear in the presence of various carbohydrates, with special reference

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Ohio Jour. Sci. 40: 123-129, 1940.
*Heuser, E., B. F. Shema, W. Shockley, J. W. Appling, and J. F. McCoy: The

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■*HoRR, W. II.: Utilization of galactose by Aspergillus niger and Pcnicillium glaucum,

Plant Physiol. 11: 81-99, 1936.
Kakeura, M.: Starches by using parasitic fungi, Japanese patent 172,381, Mar. 6,

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KiNSEL, K.: Carbohydrate utilization by the corn Diplodias, Phytopathology 27:

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Lewis, R. W. : Mutants of Neurospora crassa requiring succinic acid or a biochemi-
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Margolin, A. S.: The carbohydrate requirements of Diplodia macrospora, Proc.

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Margolin, A. S. : The effect of various carbohydrates upon the growth of some

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Meyerhof, O: The intermediary reactions of fermentation. Nature 141: 855-858,

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Meyerhof, O.: Glycolysis of animal tissue extracts compared with the cell-free

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Nobles, M. K.: Studies in forest pathology. VI. Identification of cultures of

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NoRD, F. F., and R. P. Mull: Recent progress in the biochemistry of Fusaria,

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Prescott, S. C, and C. G. Dunn: Industrial Microbiology, 2d ed., McGraw-Hill

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148 PHYSIOLOGY OF THE FUNGI

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Yaw", K. : Personal communication, 1950,

CHAPTER 8
HYDROGEN-ION CONCENTRATION

The growth of fungi and bacteria may be inhibited or prevented by
media which are too acidic or too alkaline. A completely satisfactory
medium may be made useless by the addition of relatively small amounts
of strong acids or bases but may have its former usefulness restored if the
excess acid or base is neutralized. This suggests that the ions which
characterize acids and bases are particularly active in life processes. It is
necessary to understand certain fundamental ideas about acidity and
ways of measuring concentration of these ions before discussing in detail
the effects of acids and bases on the activities of the fungi.

IONIZATION OF COMPOUNDS

Since water is the universal solvent for all life processes, our discussion
will be confined to aqueous solutions. The chemical compounds which
comprise natural and synthetic media may be divided into two classes,
those which form ions in solution (acids, bases, and salts), and those
which do not ionize (organic compounds in general, except organic acids
and bases). Water is a compound of the first class, although it forms ions
to a very slight degree. The ionization of water may be represented by
the following equation:

(1) HOH ^ H+ + OH- or HOH + HOH ^ H3O+ + OH"

For each molecule of water ionized one hydrogen and one hydroxyl ion
are formed.

In any aqueous solution the product of the concentrations of the
hydrogen and the hydroxyl ions (in moles) is equal to a constant (K^),
Water is a neutral compound, i.e., the concentrations of hydrogen and
hydroxyl ions are equal. A solution which contains a greater concentra
tion of hydrogen than of hydroxyl ions is acidic ; a solution which contains
a greater concentration of hydroxyl ions than of hydrogen ions is basic, or
alkaline. Since all aqueous solutions contain hydrogen and hydroxy]
ions, the deleterious effects of these ions must be due to their relative
concentrations. At room temperature (23 to 25°C.) the concentration of
hydrogen and hydroxyl ions in water is 1 X 10"^ mole per liter, or 1 mole
each of these ions in 10 million liters. The degree of ionization of water
increases with temperature. However, water is a neutral substance at

149

150 PHTSIOLOGT OF 71- 1 FVXGI

all lempersTures because equal number? of hydrogen and hydrox\-l ions
are pi^sent. The value of K^ at 23 to 2o^C. is obtained by multiphing
the conceniraticais irf hydrogwi and hydroxyl ic«is present.

(3j K, = : > :>-- 1 X icr- = 1 X lO-^*

For ihe parpoee of this discussicsa. we will consider an acid to be a com-
pound. whcKe aqueous scJuticHi contains a greater concentration of hydro-
gen rhxn at hydroxyl ioais. A t-o^ is a compound whotse aqueous solution
contains a greater concentraticai of hydro^l ions than of hydrc^n itms.
Tbese deaniiioas vdll include a number ci compounds which are ordinarily
con^doed as salts. A solmion of an acid c«Mitains a greater concentra-
tion of hydrogen ions th.--^- pure water by Tirtue of the ionization of the
acid. Hydrochloric acid ionises as follow?:

C3) Ha ^ H- - a-

In a sofaxtica of hydrochloric acid there are two sources <rf hydrogen ions.
the acid and 'irater. Enou^ of the hydrogen icais will combine with the
hvdrasyi ions to reduce the concentration erf this ion so that Eq. (2) will
b* satined. The ionization o£ a base may be represented as follows:

, i XaOH = Xa- - OH"

- ^ c- - - -- es^ctly the same as gi"r«i above fca* an acid except that
._- , _ _ :>ii C'f hy<irogen ions is less in a solution of a base than in

pure

A s~.: i-^ : i- soch as Lyir >;h-oric. is considered to be completely itm-

ixed even lu ; rated solutions- A weak acid, such as acetic, in

IX s-i-u : I- - uly sli^tly about 1 per cent . The perc-entage

of iooixi" : : i ^ increases as the dHuticm increases. The c<»-

ccntri " : _ liS in. equal volumes of normal hydrochloric and

acetic - - -^ — r chief differraice between these acids. Thus the

stoaiiT r ' ~ ~ ' - -^pressed in tw^o ways. (1 < the total acidity

A*^^ ■ . \ includes both the ionized and nonionized

mofecuks CH iL i.e.. the titratab'ie acidity, and (2 ) the actwal acidity
at any instant. - :_ ^i is a functicHi of the cc«icentration of hydrogen ions
jwesBQt. T: - -tration c^ hydrogen ions is a nincrion of the con-
centre I. - : i-izatitHi of the acid involved. It is the actual
acadity ~ - esses. It is also necessary to consider
the phy- . riieii- : ; z^ or cations which are associated
wz- ^- - : -". i= impossibie to add just hydrogen

7 HZ MIAXC^G OF pH

T: - ::. I. : UL : - _ ^-:i : ui in a soluticai can be expressed in
Ti —ays. A derived unit pH is most used in biological work. The

HYDROGES-IOS COSCESTRA TIGS'

151

calculated c<Hic«itrati<Mis of hydrogen and hydroxyi ions in sohition; <rf
hydrochloric acid and potasdum hydroxide are given in Tat4e 30. C<«n-
plete ionization was assumed in these falmlatioPiS.

Table 30. Calcxxated Coxcest^atioks or Htoso^cix axd Htdeoiti. loss rs
Prsz Watee axd cf SoLmoxs of Htdeoceuckic Acid axd PotasctctiC

Vonaaiit.

'?*;c -r^'r ;."._- j_

- JL ; -:i.*ji-_:_

1.0

1 X10»

1 X 10-^

0.1

1 X10--

1 X 10-^

0.01

1 xio-=

1 X 10--

0.001

1 xio-»

1 X 10-^

0.0001

1 X 10-*

1 X io-»

0.00001

1 X10-*

i X 10-*

0.000001

1 xio-^

1 X 10-*

H20

1 X10--

KOH

0.000001

1 xio-«

1 X10-*

0.00001

1 X io-»

1 X 10-*

0.0001

1 X 10--'

1 X 10-*

0.001

1 X 10--

1 xia-»

0.01

1 X lO-i^

1 X 10-s

0.1

1 X 10--^

1 X 10-^

1.0

1 X 10- *

-IX

I ^ Iff

S<$rensen devised a logariiiunic ~ ^ - ~-^:
hydrc^en icHis in solutions. The ij^:.. _ . - .
tial of hydrogen.'" which is abreviated to pH.
and hydrc^en-ion concentration is grven bdow.

- -V:.c -^1= ;c

in-e "I r.i--
i^nseei pH

pH=logi

•^H="]

In words this equation means pH is equal t : _ - . ' of

the reciprocal (rf the hydrogaMon : r. r _ : :_ x^wk oO li £

seen that the hydrogen-itm ctMicentrtiiioii ci •j.l,'^»juu'UV z.ydrorhloric add
is 1 X 10~* mole per liter. Suhstitutiiiz "his value in Eq. ^5), "we hxve

(6)

1 .X. l\f~*

-. = lie 10* = 6

One obvious advantage <rf the pH scale lies in the fact that hydrogen-ion
concentrations are expressed as positive numbers rather than as decimal

152 PHYSIOLOGY OF THE FUNGI

fractions. If a solution contains 4.23 X 10"" mole per liter of hydrogen
ion, this concentration of hydrogen ions may be expressed in terms of pH
by use of Eq. (5).

(7) pH = log ^^3 ^ ^Q_, = log 1 + log 10« - log 4.23 = 5.37

By reversing the above calculations, the hydrogen-ion concentration may
be calculated if the pH value is known.

The pH scale has three features which may be perplexing: (1) alkaline
solutions are designated on the same scale as acidic solutions; (2) increas-
ing acidity is expressed by smaller pH values; and (3) the logarithmic
nature of the scale. A difference of one pH unit indicates a tenfold
difference in hydrogen-ion concentration, while a difference of 0.3 pH unit
means a twofold difference. A solution having a pH value of 4 contains
10,000 times as many hydrogen ions per unit volume as a solution having
a pH of 8. A solution having a pH value of 6.2 has twice the hydrogen-
ion concentration of a solution having a pH value of 6.5. The student
should remember that pH is the name of a logarithmic scale used to
measure hydrogen-ion concentration and not an entity in itself.

BUFFERS AND BUFFER CAPACITY

A medium having pH values between 5 and 6 at the time of inoculation
is suitable for most fungi, but more acidic or more alkaline media are used
at times. It is often important that the pH of the culture medium does
not change too greatly as the result of metabolic activities of the organism.
The ease with which the pH of a medium is modified depends upon the
composition of the medium.

Substances which tend to maintain the pH of a solution relatively
constant when an acid or base is added, or when the solution is diluted,
are called buffers. In general, the kinds of compounds which act as
buffers are mixtures of weak acids or bases and their salts, acid salts of
polybasic acids, basic salts of polyacidic bases, and amphoteric compounds
such as amino acids and peptides. As an example of a buffer we may
consider a solution which contains equivalent amounts of acetic acid and
sodium acetate. If a strong acid such as hydrochloric is added to this
buffer solution, a reaction will occur between the sodium acetate and the
hydrochloric acid.

(8) CH3— COONa + HCl -^ CH3— COOH + NaCl

The net effect of adding hydrochloric acid is the formation of an equiv-
alent amount of acetic acid. Inasmuch as acetic acid is only slightly
ionized, no great change in hydrogen-ion concentration will take place
until most of the sodium acetate has been converted into acetic acid. If

HYDROGEN-ION CONCENTRATION 153

sodium hydroxide is added to an acetate-acetic acid buffer, the sodium
hydroxide will react with the acetic acid.

(9) CH3— COOH + NaOH -* CHsCOONa + H2O

This reaction will convert sodium hydroxide, which is a strong base,
into a salt of a weak acid and water. A slight increase in pH will occur,
owing to the hydrolysis of the sodium acetate formed. After most of the
acetic acid has reacted with sodium hydroxide, the addition of more of
the base will result in a rapid increase in the pH value of the solution.

In culturing fungi, it is important to choose buffers which retain the
pH of the medium in the desired range. The effective yH range of buffers
prepared from weak acids and their salts is related to the degree of ion-
ization of the acids. The more highly an acid ionizes, the lower will be
the pH range of a buffer prepared from it and one of its salts. The degree
of ionization of weak acids is designated by a term called the ionization
constant {Kg). Mixtures of weak bases and their salts are also buffers.
A few ionization constants of w^eak acids are acetic, 1.8 X 10~^; carbonic
(first hydrogen), 3.5 X 10"^; phosphoric (first hydrogen), 1.1 X 10"^;
phosphoric (second hydrogen), 7.5 X 10^^. Extensive data of this kind
may be found in various handbooks of chemistry.

The ionization constants of weak acids may be used to calculate the
effective pH range of buffers prepared from these compounds and their
salts by means of the following relation:

[salt]

(10) pH = p2v„ + log

[acid]

The symbol p/C is equivalent to log {l/Ka). When the mole concen-
trations of the weak acid and its salt are equal, Eq. (10) becomes:

(11) pH = log -^ = p/Va

The p/va value of a weak acid is thus the pH of a buffer which contains
equivalent quantities of a weak acid and one of its soluble salts. The
pH of an acetate buffer containing equivalent amounts of acetic acid and
an acetate may be calculated using Eq. (11).

(12) pH = log ^ g ^ ^Q_, = log 1 + log 105 - log 1.8 = 4.74

In an analogous manner it can be shown that the pH of a buffer com-
posed of equivalent amounts of a weak base and one of its salts is related
to the ionization constant of the base {Kh) by the following equation:

(13) pH = 14 - log ^ = pA^t,

For a derivation of the formulas relating pH and ionization constants

154

PHYSIOLOGY OF THE FUNGI

see Umbreit et al. (1945). The useful range of a buffer extends one pH
unit above and below the p/v^ (or p/v^) value. The data in Table 31
giving the pH range of a number of buffer systems of biological interest
were calculated using Eq. (11) and (13). The information in Table 31 is
useful in selecting buffers which are active in certain pH ranges. Direc-
tions for preparing buffer solutions of definite pH are given by Gortner
(1949). In practice, media are diluted with these buffers (Lindeberg,
1944).

Table 31. The Useful pH Range of a Number of Buffer Systems of Biological
Interest Calculated by the Use of Eqs. (11) and (13)

Acid or base

Equilibrium reaction

pH range

pA'a or pA'a

Acetic

CH3— COOH ^ CH3— COO-

3.7- 5.7

4.7

Phosphoric

H3PO4 ^ H2PO4-

1.0- 3.0

2.0

H2PO4- ^ HPO4-

5.7- 7.7

6.7

Carbonic

H2CO3 ^ HCO3-

5.5- 7.5

6.5

Fumaric

HOOC— CH^CH— COOH ^

HOOC CH— CH COO-

2.0- 4.0

3.0

HOOC— CH=CH— COO- ?=i

3.5- 5.5

4.5

-OOC— CH=CH— COO-

Malic

HOOC— CH2— CHOH— COOH ^

HOOC— CHo— CHOH— COO-

2.4- 4.4

3.4

HOOC—CH2— CHOH— COO- <=±

4.1- 6.1

5.1

-OOC— CHo— CHOH— COO-

Ammonia

NH4OH ^ NH4+ + OH-

8.3-10.3

9.3

A number of other considerations enter into the choice of buffers.
They must be nontoxic, but even a buffer composed of an essential
nutrient such as phosphate may be inhibitory if used in high concentra-
tions. The use of calcium compounds in media was found to reduce the
toxicity of phosphate and citrate buffers to species of Marasmius (Linde-
berg, 1944). The amount of a buffer required to achieve a given degree
of immobilization of pH during growth cannot be specified without con-
sidering the fungus involved and the medium used. In experiments
where it is desired to maintain the pH of the culture medium essentially
constant, the concentration of nutrients, especially the sugar, in the
medium should be low, and an organic source of nitrogen should be used
in preference to nitrates and ammonium salts. Lindeberg (1944) used
Af /25 phosphate buffer in a medium which contained 10 g. of glucose and
1 g. of asparagine, with satisfactory results.

In general, the usefulness of highly buffered media is restricted to deter-
mining pH limits and for special problems. Many fungi would not
develop "normally" in media having a fixed pH, especially in certain pH
ranges.

H YDROGEN-ION CONCENT RA TION

loo

If a medium contains several buffer systems, as is frequently the case,
each buffer system will play its role over the pH range in which it is active
alone. If two buffers with overlapping pH ranges are present, it will
require more acid or alkali to effect a change of unit pH than if one buffer
were present. The effect of buffer concentration has not been considered
in the above discussion. ^Vhile the pH of a buffer depends solely upon
the ratio of the concentrations of the weak acid and its salt, it is obvious
that the amount of an acid or base required to change the pH value of a

7.0

6.0

5.0

^4.0

• o" \

5 f

.*• "

t
1

•^4--'fT

1.0 0.8 0.4

Ml O.IN HCl

0.4

2.0

0.8 1.2 1.6

Ml. O.IN NAOH

Fig. 21. Buffer-capacity curve.s of two media. The dotted line was obtained by
titrating 20 ml. of glucose-asparagine medium with O.IjV hydrochloric acid and O.liV
sodium hydroxide. The pH was determined after each addition of acid or base. The
solid line was obtained in the same way on the above medium to which 10 mg. of
neutralized glutamic acid had been added, (Courtesy of Robbins and Schmitt, Am.
Jour. Botany 32 : 324, 1945.)

buffer one unit wdll depend upon the concentrations of the buffer acid and
salt present. The hufer capacity of a medium is measured by titrating a
definite volume of medium (usually 100 ml.) with standard acid and
alkali. The pH is measured after each addition of acid or alkali. From
the curve drawn from these data the buffer capacity for any range of pH
A^alues may be obtained. The curves in Fig. 21 illustrate the buffering
capacity of two media (Robbins and Schmitt, 1945). These media
differed in the buffers present. The unsymmetrical nature of the curves
is due to the presence of overlapping buffers.

The pH of culture media may be controlled within desirable limits, in
some instances, by adding calcium carbonate to the medium. Calcium
carbonate is essentially insoluble in neutral and alkaline media but acts
as a neutralizing agent for acids. The calcium carbonate is used up as
acid is produced by a fungus. The degree of neutralisation achieved
depends upon the amount of calcium carbonate added and whether the

156

PHYSIOLOGY OF THE FUNGI

cultures are agitated. See Foster (1949) for a discussion of the use of
calcium carbonate in industrial microbiological processes.

For fungi which have an extremely narrow pH range, the special cul-
ture flask devised by Cantino (1949) for culturing Blastodadia pring-
sheimii may be used (Fig. 22). A base (or acid) is placed in the side arm

and an internal indicator of the desired pH
range is added to the medium. A little of the
base is tipped into the culture flask as desired.
Flasks with two side arms may be used so that
either acid or base maybe added to the culture
medium.

Fig. 22. Flask designed for
the study of glucose dis-
similation by Blastodadia.

A, the side arm containing
NaOH for neutralization;

B, sintered-glass aerator; C,
inlet for aeration with differ-
ent gas mixtures; D, the
outlet for removal of media.
(Courtesy of Cantino, Am.
Jour. Botany 36 : 100, 1949.)

METHODS OF DETERMINING pH VALUES

Only two general methods of measuring pH
values will be discussed. The colorimetric
method is simple, inexpensive, and sufficiently
accurate for most purposes, but it cannot be
used with highly colored or turbid media.
The potentiometric method using the glass
electrode is more accurate and is often the
preferred method.

Colorimetric methods. The use of indi-
cators w^hich change color in response to
varying concentrations of hydrogen ion is
the basis of this method. Indicators may
be considered as weak acids or bases, and as
such they act as buffers, but the amounts used are so small they do not
affect the accuracy of a determination. For methods of measuring the
pH of unbuffered solutions see Snell and Snell (1948). The property of
these indicator buffers which distinguishes them from other buffers is
that the colors of the salts and free acids or bases (nondissociated) are
different. Within the usable pH range, the color of the indicator is a
function of the hydrogen-ion concentration of the medium. For exam-
ple, bromocresol purple (p/va, 6.3) is yellow in solutions having pH values
of 5.2 or less and purple at pH 6.8 or more. Within the pH range 5.2
to 6.8 the color changes from yellow to purple. To determine the pH
value of an unknown solution within this range, the indicator is added to
equal amounts of standard buffers and the unknown solution, and from
the color of the standard buffers of known pH, the pH value of the
unknown may be estimated to within 0.1 pH unit. By a suitable choice
of indicators the pH range of interest may be covered. A few indicators
with their pH ranges are listed in Table 32.

Two methods of color comparison are in general use. The first involves

HYDROGEN -ION CONCENTRATION

157

the use of the familiar comparator block. A slight color or turbidity
of the medium may be compensated for by the use of suitable blanks. A
porcelain spot plate may be used instead of a comparator block with
considerable saving of time and materials, although the accuracy is some-
what less. Drops of the indicator are added to the depressions in the
spot plate. A drop of the medium is added to one depression, and drops
of standard buffers to the other depressions. The pH of the medium
is estimated from the pH of the buffer which yields a color matching that
developed in the medium.

Table 32. The pH Range and Color Changes of Various Indicators

(Courtesy of Eastman Kodak Company.)

Indicator

Bromophenol blue .
Bromocresol green.
Chloro phenol red . .
Bromocresol purple
Bromothymol blue .
Phenol red

pH range

3.0-4.7
3.8-5.4
4.8-6.8
5.2-6.8
6.0-7.6
6.8-8.4

Color change

Yellow-blue

Yellow-red

Yellow-purple

Yellow-blue

Yellow-red

All colorimetric methods of measuring pH require the use of standard
buffers (buffers of known pH) or permanent standards. Buffers may be
prepared in the laboratory or purchased from laboratory supply houses.
It is convenient to use prepared buffer tablets, which need only to be
dissolved in a measured amount of water before use. Potentiometric
pH meters also require the use of a standard buffer for calibration.
The easiest of these to prepare is a saturated solution of potassium hydro-
gen tartrate (pH 3.57). The use of this buffer was recommended by
Lingane (1947). It is simple to prepare, and temperature affects the pH
very little.

From Table 32 it will be noted that the pH range of a single indicator
is less than two pH units. Much time can be saved in pH determinations
by the use of a wide-range indicator to determine the approximate pH
before using a single indicator for the final measurement. Wide-range
indicators (pH range 2 to 10) may be purchased or prepared by mixing
suitable indicators (Snell and Snell, 1948). The pH value of a medium
may easily be determined within 0.5 pH unit by the use of a wide-range
indicator. Either the comparator block or the spot-plate method may
be used. For detailed information about indicators, see Kolthoff and
Rosenblum (1937).

Potentiometric methods. The potential difference which develops
between certain pairs of electrodes when they are dipped into a solution
is a function of the hydrogen-ion concentration. Solutions which give

158 PHYSIOLOGY OF THE FUNGI

rise to the same potential difference have the same pH value. Modern
pH meters are calibrated in pH units so that direct readings are obtained.
Color or turbidity does not affect potentiometric measurement of pH.

The glass electrode in conjunction with the calomel half cell is the most
commonly used for liquids of biological interest. The glass electrode
consists of a bulb blown from a special glass. The bulb is filled with
O.IA'^ hydrochloric acid. A potential difference develops between the
inside and the outside of the electrode; the magnitude of this potential
difference depends upon the hydrogen-ion concentration of the liquid
in which the bulb is dipped. Measuring the potential difference which
develops between the glass electrode and the calomel half cell is equivalent
to determining the pH value of the unknown solution. Sensitive auxiliary
electrical equipment is required to measure this potential difference.
For a discussion of the glass electrode, see Dole (1941).

Many suitable pH meters are available. The trend appears to be
toward instruments which use alternating current rather than batteries
as a source of power. Since the details of operation are somewhat dif-
ferent for the various makes, the directions of the manufacturer should
be consulted.

The pH of media should be determined before autoclaving and the
reaction adjusted by the addition of acid or alkali if necessary. The pH
of a sample of a medium should also be determined after autoclaving and
before inoculation. The pH value at this time is known as the initial
pH. Alkaline media absorb carbon dioxide from the atmosphere, causing
a slow decrease in pH, Pritham and Anderson (1937) reported that the
pH of uninoculated alkaline media may decrease as much as two units
during the course of an experiment. This factor is of particular impor-
tance when upper pH limits are being investigated. For methods of
adjusting pH, see Suggested Laboratory Exercises.

EFFECTS ON FUNGI

Hydrogen and hydroxyl ions are present in all media and in substrates
upon which fungi grow in nature. The pH of the medium exerts a
decided effect upon the rate and amount of growth and many other life
processes. A medium may have a pH which is favorable for growth and
unfavorable for sporulation or other processes. The production of pig-
ments, vitamins, and antibiotics may be influenced by the pH of the
medium. As a result of metabolic activity a fungus ordinarily changes
the pH of the medium upon which it grows.

pH limits. The upper and lower pH values between which a fungus
grows form the pH range of that species. The pH values which inhibit
growth vary with the species. Between the limiting pH values there is
a pH range which allows optimum growth. An initial pH of 5 to 6 is

HYDROGEN-ION CONCENTRATION

159

satisfactory (not necessarily optimum) for the majority of the fungi.
The optimum pH ranges for Blastocladia pringsheimii, Allomyces arhus-
cula, and Blastocladiella simplex are rather narrow (Emerson and Cantino,
1948) (see Fig. 23). Most of the pH optima reported in the literature
are less than 7. Meacham (1918) reported pH 3 to be optimum for
Lenzites saepiaria, Fomes roseus, Merulius lacrymans, and Coniophora
cerebella. Wolpert (1924) found the pH optimum of various Basidio-
mycetes to be in the neighborhood of 5.5. Johnson (1923) reported that
the upper pH limit of Penicillium varidbile is 10.1 to 11.1, which is con-

Blastocladfa

Allomyces

Blastocladiella

Fig. 23. Relation of pH of the medium to growth of Blastocladia -pringsheimii,
Allomyces arbuscula, and Blastocladiella simplex. (Courtesy of Emerson and Cantino.
Am. Jour. Botany 35: 162, 1948.)

siderably higher than that of most fungi. The lower pH limits reported
vary from 5.3 for B. simplex (Emerson and Cantino, 1948) to 0.5 for
Acontium velatum and an unidentified imperfect fungus (Starkey and
Waksman, 1943).

The method used to determine the pH limits of a fungus is to inoculate
a series of nutrient solutions having pH values spaced 0.2 to 0.4 unit
apart. Growth may be observed visually, or the mycelium may be
weighed. Such media should be well buffered. The pH limits for a
given fungus as determined in different laboratories are frequently at
variance. This is not unexpected, since the composition of the medium
and the nature of the buffer influence the tolerance of fungi to hydrogen
and hydroxyl ions. The behavior of Marasmius graminum is revealing
(Lindeberg, 1944). Calcium ion was effective in overcoming the toxic
effect of an initial pH of 3.3. The weight of M. graminum after 12 days
was 0.4 mg., but when calcium sulfate was added to the medium, the
yield was 8.0 mg. Tamiya (1928) also found calcium ion to protect
Aspergillus oryzae to some extent against high concentrations of hydrogen
ion. The optimum pH for Gibber ella saubinetti is lower when calcium is
present in the medium (Lundegardh, 1924). Wolpert (1S24) also found

160

PHYSIOLOGY OF THE FUNGI

the pH range of many fungi to vary on different media and concluded
that the widest pH range was obtained on favorable media.

The temperature of incubation may influence the optimum pH as well
as the pH range of a fungus. The optimum pH for Phacidium infestans
is 4.5 at 5°C., 5.0 at 10°C., 5.5 at 15°C. and 6.0 at 20°C. (Pehrson, 1948).
The pH range of Armillaria mellea on a sucrose-peptone medium was
reported to be 2.5 to 7.5 at 15°C., 2.0 to 7.8 at 25°C., and 2.5 to 7.4 at
35°C. (Wolpert, 1924).

21 0

180

150

.120

■o

.^ 90

.y^""^*

^^^0--^

—

^ -.•

,.-'-'

^/-'-

■'"/

ucose-).

oofossiui

77 nitrate

^ — ^Gi

\
\
\

ST —

um sulfa

1 X

lucose-ammoni

£. xJ

8.0

7.0

5,0

4.0

3.0

0 5 10 15

Doys of incubation

Fig. 24. Rate of mycelial growth of Sordaria fimicola and accompanying changes
in pH of two media. Media contained biotin but no thiamine. Sohd hnes indicate
growth, and the broken lines represent pH values.

Two pH optima have been reported for a number of fungi. Rhizopus
nigricans, when grown on potato-glucose liquid medium, has two optimum
pH ranges, one on either side of the isoelectric point of the mycelium,
which was about pH 5.5 (Robbins, 1924). Scott (1924) reported
Fusarium lycopersici to have two optimum pH ranges for growth on
glucose-nitrate medium: pH 4.5 to 5.3 and 5.8 to 6.8. Mathur et al.
(1950) obtained evidence that there are two optimum pH ranges for the
sporulation of Colletotrichum Undemuthianuni.

In addition to the use of media having low initial pH values, the lower
pH limit may be determined in some instances by choosing a medium
in which the fungus produces sufficient acid to inhibit growth completely.
This is illustrated by the pH and growth curves of Sordaria fimicola in
Fig. 24. This fungus was grown upon a glucose-ammonium sulfate
medium having initial pH 6.0; after a few days the pH of the culture
medium fell to 3.3 and remained there for 5 weeks. More difficulty may
be experienced in determining the upper pH limit. If a fungus is able

HYDROGEN-ION CONCENTRATION

161

to make a trace of growth in an alkaline medium, the carbon dioxide
produced will lower the pH. Organic acids may also be produced. Car-
bon dioxide from the air will be absorbed by alkaline media.

0.08

t 0.06

■I 0.04

_>i

•o
>«

% 0.02

•a
m

' '

Urease

■J

7
pH

Fig. 25.

of urea

25. The shift of optimum pH for urease activity due to change in concentration
sa. (Courtesy of \'an Slyke, Advances in Enzymol. 2: 41, 1942. Pubhshed by

permission of Interscience Publishers, Inc.)

pH
Pig. 26. The effect of pH on the rate of linear growth of Neurospora crmsKi.
tesy of Ryan, Beadle, and Tatum, Am. Jour. Botany 30: 790, 1943.)

(0,11. 1 V

It was pointed out in Chap. 4 that pH affects the activity of a.i/jymes.
In general, there is a striking correlation between the optimum pH range
for most enzymes and the optimum pH range for gro^vth. In a survey
of the literature Haldane (1930) found all but 9 of 105 enzymes to have
optima between pH 4 and 8. Most fungi have pH optima for growth

ief2

PHYSIOLOGY OF THE FUNGI

between these limits. The effects of pH upon the activity of urease
(Van Slyke, 1942) and upon the rate of growth of Neurospora crassa
(Ryan et al., 1943) are shown in Figs. 25 and 2G. From the general
similarity of these two curves it appears probable that pH affects the rate
of growth of fungi, at least in part, by modifying the rate of certain
enzymatic reactions.

pH changes in media during growth. Fungi, as a result of their
metabolic activities, ordinarily change the pH of the media in which
they grow. These changes cannot be studied by making a single deter-
8.0r

2 3 4 5 6 7

Doys of incubation

Fig. 27. Changes in pH during incubation of Sordaria fimicola in different volumes of
liquid glucose-casein hydrolysate medium at 25°C.

mination of pH at any fixed time. Just as it is necessary to study growth
as a function of time of incubation (growth curve), it is necessary to deter-
mine the pH changes in an inoculated medium day after day to obtain a
complete representation of these changes (pH curve). The pH of the
medium should be followed in connection with the other functions being
studied. Since fungi differ in their metabolic activity and rate of growth,
the pH changes produced in the culture medium will differ. The pat-
terns of pH changes for the same fungus will depend upon the composition
and concentration of the media used.

As an illustration of the effect of the composition of the medium upon
the pH changes, some of our data for Sordaria fimicola are given in Fig.
24. The correlation of the pH changes with the rate and amount of
growth of this fungus may be obtained by comparing the growth curves
obtained at the same time. From Fig. 27 it is evident that the hydrogen-

HYDROGEN-ION CONCENTRATION 163

ion concentration of a nutrient solution may change 10,000-fold during a
few days as a result of the metabolic activities of a fungus. These
changes in pH are due to changes in the relative amounts of acids and
bases formed or withdrawn and to the ionization constants of these
compounds. Some of the metabolic processes which result in a change in
pH of a nutrient solution are discussed below.

The utilization of cations, such as ammonium ion, for the synthesis of
protoplasm or for any other purpose whereby essentially non-ionic com-
pounds are formed, leaves an equivalent number of anions in the nutrient
solution. Since solutions are electrically neutral, an equivalent number
of both cations and anions must be present. Thus, when an equivalent
of ammonium ion is transformed into non-ionic compounds, an equivalent
of some other cation or cations will be formed in the nutrient solution.
These ''new" cations are usually hydrogen ions, which are formed from
water. If it is assumed that both cations and anions are adsorbed on
the protoplasmic membrane, the process may be thought of as replace-
ment. The production of acid would result from the utilization of other
cations as well.

The utilization of nitrate ion or other anion such as phosphate or
sulfate for the formation of non-ionized compounds has the effect of
increasing the hydroxyl-ion concentration of the medium. We may
assume the same type of mechanism as before, except that the anion
released to the nutrient solution is the hydroxyl ion.

Fungi produce acids from nonacidic nutrients such as carbohydrates.
Among these acids are carbon dioxide and various organic acids such as
pyruvic, citric, and succinic acids. Carbon dioxide combines with water
to form carbonic acid, which is unstable in the presence of stronger acids
and decomposes to set free carbon dioxide. Under alkaline conditions
carbonic acid reacts with bases to form bicarbonates. Pyruvic acid
accumulates in the nutrient solution in which many fungi are grown, and
in some instances the formation of this acid accounts for a considerable
part of the early depression of pH. The eventual utilization of pyruvic
acid causes the pH of the nutrient solution to rise. Other metabolizable
acids behave similarly. Ammonia is, perhaps, the most common basic
substance produced by fungi. Piricularia oryzae produces ammonia in
considerable amounts (Henry and Andersen, 1948). The production of
ammonia results from the deamination of amino acids and proteins. The
processes discussed above may occur simultaneously. Whether a culture
solution becomes more acid or alkaline depends upon the extent of these
various processes. In general, the processes which produce acid pre-
dominate during early growth, especially when ammonium nitrogen is
used.

The importance of the composition of the medium in determining what

164 PHYSIOLOGY OF THE FUNGI

changes in pH will take place during growth is illustrated by the work of
Dimond and Peltier (1945), who studied the pH changes produced by
Penicillium notatum as a function of the carbon and nitrogen nutrition.
When the initial pH was 6.0 and sodium nitrate was the nitrogen source,
the lowest pH values attained on different sugars were glucose, 5.1;
sucrose, 4.0; lactose, 3.2; maltose, 4.8; and galactose, 4.8. These were
the lowest pH values attained under these conditions. In another experi-
ment a mixture of tryptophane, asparagine, and cystine w^as used as the
nitrogen source. The pH again varied with the sugar used in the medium
The lowest pH attained with fructose was 5.3; glucose 3.5; sucrose, 4.0;
and an equimolecular amount of fructose and glucose, 3.5. When lactose
w^as used in combination with these amino acids, the pH of the culture
medium remained essentially constant at 7.0

Any changes in environmental factors which affect the rate of growth
of a fungus may also affect the changes in pH of the culture medium.
Robbins and Schmitt (1945) found that the time required for Phycomyces
blakesleeanus to lower the pH of a glucose-asparagine medium to a given
level was a function of temperature of incubation. Growth and the
production of acid were more rapid at 26°C than at 20°C. The rate at
which the pH of a culture medium is changed by a fungus is also depend-
ent upon the volume of medium used in flasks of the same size. Some of
our data which illustrate this for Sordaria fimicola are shown in Fig. 27.
The time required for these cultures to attain maximum weight and to
produce perithecia correlated with the changes in pH.

Effect of acidity on media. The composition of a medium may be
changed as a result of changing the pH. The various cations and anions
may combine to form insoluble compounds at certain pH values. Mag-
nesium and phosphate ions are compatible in acidic solutions, but as the
concentration of hydrogen ion is decreased, these ions combine to form
an insoluble compound, the solubility of which becomes less as the pH is
increased. Calcium phosphate is likewise less soluble in alakline solu-
tions. Ferric iron may be largely removed from media as either the
hydroxide or the phosphate, by making the media alkaline. If an alkaline
medium is filtered, certain constituents will be removed to a greater or
lesser extent. Lilly and Leonian (1945) found that by making a medium
alkaline to pH 8 and filtering, the iron concentration w^as lowered to such
levels that Rhizohium trifolii made about one-fifth as much growth as
when 250 jug of iron per liter was added to the medium. If a precipitate
is not removed by filtration, the situation is different. Any insoluble
precipitate is in equilibrium with the dissolved compound, as indicated
below.

FeP04 ^ FeP04 ^ Fe+ + + + PO4"
solid in solution ionized

HYDROGEN-ION CONCENTRATION 1C5

As the ions are utilized, more and more of the precipitate will dissolve.
The effect of a change in pH of the solution as a result of the metabolic
activities of the fungus must be considered. An acid reaction will hasten
solution of the precipitate, while an increase in alkalinity will delay the
process. It is possible that the harmful effects sometimes noted in
alkaline media may be due, in part, to an induced iron deficiency.

The influence of pH on the solubility of certain ions may be modified
by the presence of other compounds, especially those which form com-
plexes. The solubility of iron in alkaline solutions is greatly increased in
the presence of hydroxy organic acids such as citric, tartaric, and malic
acids. Ammonia and amino acids also form complexes with certain ions,
e.g., copper. The presence of any complex-forming compound may
modify the availability of the ions with which it forms complexes. The
chemical changes in media due to alteration of pH, whether imposed
from the outside or caused by the fungus, affect metabolic processes.
The pH of a culture medium changes during the growth of a fungus, and
these changes may affect the composition of the medium and thus the
response of the fungus.

pH and oxygen supply. The solubility of oxygen in water is slight,
being less than 10 mg. per liter at 20°C. The rate of diffusion of oxygen
into media is dependent upon the composition and the pH. Rahn and
Richardson (1941) have described a simple and elegant method of measur-
ing the rate of diffusion of oxygen into agar media. Methylene blue, an
organic dye which is colorless when reduced and blue when oxidized, w^as
used as an indicator. When this dye (1/200,000) is autoclaved with
media which contain easily oxidized constituents such as glucose, the dye
is reduced to the leuco, or colorless, form. As oxygen diffuses into the
medium, the reduced dye is oxidized, and the rate at which the blue zone
advances into the medium is a measure of the rate of oxygen diffusion.
The pH of the medium also affects the ease with which certain constitu-
ents are oxidized. Some data of Rahn and Richardson (1941) on the rate
of oxygen diffusion into a peptone medium are shown in Fig. 28. The
amount of oxygen available to submerged mycelium is greater in acidic
than in alkaline media.

Effect of pH on utilization of nutrients. Before any substance (ion
or molecule) is utilized, it must first pass through the cell wall and the
protoplasmic membrane. The cell wall is nonliving and consists of
polysaccharide-like compounds. For a discussion of the nature of the
cell wall and literature citations, see Brian (1949). The protoplasmic
membrane appears to be composed of proteins and lipoid-protein com-
plexes. Proteins are colloidal amphoteric compounds. An amphoteric
compound possesses both acidic and basic properties and may form salts
with either acids or bases. The protoplasmic membrane has acidic

166

PHYSIOLOGY OF THE FUNGI

properties due to carboxyl and sulfhydryl groups and basic properties by
virtue of having amino and other basic groups. The protophismic mem-
brane, therefore, should form salt-hke compounds with both cations and
anions.

Bacteria are considered by McCalla (1940) to act as ion-exchange sub-
stances, and fungus spores have been shown to act in the same manner.
McCalla investigated ion replacement by saturating cells of Escherichia

60
Time in hours

Fig. 28. The effect of hydrogen-ion concentration on the rate of diffusion of oxygen
into 1 per cent peptone in phosphate buffer. Leucomethylcne bkie was used as an
indicator. The rate of penetration of oxygen with time was followed by measuring the
depth of the blue zone. (Drawn from the data of Rahn and Richardson, Jour. Bad.
41 : 240, 1941. By permission of The Williams & Wilkins Company.)

coli with magnesium ion and tested the replacing effects of other cations.
Sodium and potassium ions replaced only a little magnesium, while hydro-
gen and calcium ions were much more effective.

From this viewpoint the relative amounts of the various cations
adsorbed from a medium would be a function of the concentration of the
ions present and the relative affinity of the membrane proteins for the
different cations. The concentrations of the hydrogen and hydroxyl ions
in a culture medium change during growth and may act to regulate the
adsorption of other ions. The pH of the culture medium may alter the
relative adsorption of other ions which are essential to nutrition or which
are toxic. At the lower pH limit the protoplasmic membrane may be so
thoroughly saturated with hydrogen ions that the essential cations are
unable to enter the cell in adequate amounts. The same situation would

HYDROGEN-ION CONCENTRATION 1G7

exist at the limiting alkaline pH values, except that it is the adsorption of
essential anions which would be hindered by hydroxyl ions.

A satisfactory explanation of all the phenomena involved in cell perme-
ability is lacking. It is known that the external pll affects the absorption
of various compounds, particularly those which ionize. The mycelium
of Aspergillus niger takes up acid dyes, such as light green and methyl
orange, when the external pH is 3.1 or less. Basic dyes such as methylene
blue and neutral red are absorbed only when the external pH is greater
than 3.1 (Biinning, 193G). These dyes escaped from the cells only when
the external pH was in the same range in which these dyes were absorbed.

Wyss ct al. (1944) found the utilization of p-aminobenzoic acid by a
deficient mutant of Neurospora crassa to be greatly increased in acidic
media. The ionization constant of p-aminobenzoic acid is about
2 X 10'"'' {pKa, about 4.8). Therefore, at pH 3.8 about 90 per cent of
the metabolite would exist in the form of the free acid, and at pH 5.8 only
10 per cent would be in this form. It was found that about eight times
as much of this vitamin was required at pH 6.0 as at pH 4.0 to support the
same amount of growth (see Fig. 41). On theoretical grounds, it is
probable that the pH of the medium would affect the utilization of other
vitamins which are weak acids (biotin, pantothenic and nicotinic acids).

The external pH of the medium has been shown to affect the internal
pH of fungus cells. By changing the external pH and by using indica-
tors, Biinning (1936) found the internal pH of Aspergillus niger cells could
be changed between 4.2 and 5.0 without injuring the cells. Greater
changes in internal pH were possible, but injury and death ensued.
Armstrong (1929) crushed the fruit bodies of a number of fleshy fungi
and measured the pH of the expressed juice. The pH range of these
liquids was 5.9 to 6.2. At best these are but average values.

It is well known that the external pH may affect certain processes
within the fungus cells. For example, growth of Sordaria fimicola in
glucose-casein hydrolysate medium was slow when the initial pH of the
medium was 4.0, but when the initial pH of the medium was 3.6 to 3.8,
normal development did not occur (Lilly and Barnett, 1947). This
failure to grow was traced to a thiamine deficiency, for when thiamine
was added to the medium (initial pH 3.6 to 3.8), normal growth and
perithecial formation took place. It appears possible that the low exter-
nal pH may have lowered the internal pH to such an extent that the
synthesis of thiamine was prevented (this fungus is self-sufficient for
thiamine when the pH of the medium is 4 or greater). These effects are
shown in Figs. 38 and 40. Additional evidence indicated that these
conclusions are correct, for pyruvic acid accumulated in the culture
medium when the initial pH was 3.6 to 3.8. On the addition of thiamine
this acid disappeared from the culture medium.

168 PHYSIOLOGY OF THE FUNGI

SUMMARY

All aqueous solutions contain hj^drogen and hydroxyl ions. Hydrogen-
ion concentration is most often expressed in terms of S0rensen's scale of
pH. The pH scale is logarithmic. Acidity and alkalinity are expressed
on the same scale. A pH of 7 indicates equivalent concentrations of
hydrogen and hydroxyl ions. Values of less than 7 indicate acidity, and
pH values greater than 7 indicate alkalinity. The smaller the pH values,
the greater the concentration of hydrogen ions.

Buffers are substances which tend to maintain the pH of a solution
constant when either strong acid or strong alkali is added or when the
solution is diluted with water. Mixtures of weak acids or bases and
their soluble salts, and amphoteric compounds such as amino acids and
proteins act as buffers. The pH range over which a given buffer is effec-
tive is a function of the ionization constant of the weak acid (Ka) or base
(Kb) from which the buffer is made. The effective pH range of a simple
buffer is two pH units.

The upper and lower pH values between which a fungus is able to grow
is called the pH range. The pH ranges of various species are different.
Fungi generally tolerate more acid than alkali. The optimum pH range
may be different for growth and sporulation. The pH of a medium in
which a fungus is growing may change. High buffer concentration and
limited growth may keep the changes in pH at a minimum. To follow
the changes in pH of a culture medium, frequent determinations should
be made.

Four metabolic processes operate to change the pH of a culture medium :
(1) utilization of cations, (2) utilization of anions, (3) formation of acids
from neutral metabolites (especially carbohydrates), and (4) formation
of bases (especially ammonia) from amino acids and proteins. The net
change in pH is the result of the interaction of all of these processes,

REFERENCES

Armstrong, J. I.: Hydrogen-ion phenomena in plants. Hydrion concentration and

buffers in the fungi, Protoplasma 8: 222-260, 1929.
Brian, P. W.: Studies on the biological activity of griseofulvin, Ann. Botany (N.S.)

13:59-77, 1949.
BiJNNiNG, E, : Ueber Farbstoff- und Nitrataufnahme bei Aspergillus niger, Flora

131:87-112, 1930.
Canting, E. C: The physiology of the aquatic Phycomycete, Blastocladia Pring-

sheimii, with emphasis on its nutrition and metabolism, Am. Jour. Botany 36:

95-112, 1949.
*DiMOND, A. E., and G. L. Peltier: ControUing the pH of cultures of PenicilUum

notatum through its carbon and nitrogen nutrition. Am. Jour. Botany 32 : 46-50,

1945.
Dole, M.: The Glass Electrode. Methods, Applications, and Theory, John Wiley &

Sons, Inc., New York, 1941.

HYDRUUEN-ION CONCENTRATION 169

^Emerson, R., and E. C. Canting: The isolation, growth, and metabohsm of Blasto-
cladia in pure culture. Am. Jour. Botany 35: 157-171, 1948.
Foster, J. W.: Chemical Activities of Fungi, Academic Press, Inc., New York, 1949.
GoRTNER, R. A.: Outhnes of Biochemistry, 3d ed., John Wiley & Sons, Inc., New-
York, 1949.
Haldane, J. B. S.r Enzymes, Longmans, Roberts and Green, London, 1930.
Henry, B. W., and A. L. Andersen: Sporulation by Piricularia oryzae, Phytopathol-
ogy 38: 265-278, 1948.
Johnson, H. W.: Some relationships between hydrogen ion, hydroxyl ion and salt

concentration and the growth of seven soil molds, loiva State Coll. Ayr. Mech,
Arts Research Bull. 76, 1923.
KoLTHOFF, I. M., and C. Rosenblum: Acid-base Indicators, The Macmillan Com-
pany, New York, 1937.
*LiLLY, V. G., and 11. L. Barxett: The influence of pH and certain growth factors

on mycelial growth and perithecial formation by Sordaria fimicola, Am. Jour.

Botany 34: 131-138, 1947.
Lilly, V G., and L. LI. Leonian: The interrelationship of iron and certain accessory

factors in the growth of Rhizobium trifolii strain 205, Jour. Bad. 50: 383-395,

1945.
Lindeberg, G.: Ueber die Physiologie Ligninabbauender Bodenhymenomyzeten,

Symbolae Botan. Upsalienses 8(2): 1-183, 1944.
LiNGANE, J. J. : Saturated potassium hydrogen tartrate solutions as a pH standard.

Anal. Chem. 19: 810-811, 1947.
LuNDEGARDH, H. : Dcr Einfluss der WasserstofRonenkonzentration in Gegenwart

von Salzen auf das Wachstum von Gibherella Saubinetti, Biochem. Zeit. 146:

564-572, 1924.
McCalla, T. M.: Cation adsorption by bacteria. Jour. Bad. 40: 23-32, 1940.
Mathur, R. S., II. L. Barnett, and V. G. Lilly: Sporulation of Colletotrichum

Imdemuthianum in cultvire. Phytopathology 40: 104-114, 1950.
Meacham, M. R.: Note upon the hydrogen ion concentration necessary to inhibit

the growth of four wood-destroying fungi, Science 48: 499-500, 1918.
Pehrson, S. O.: Studies on the growth physiology of Phacidium infestans Karst.,

Physiologia Plantar um 1: 38-56, 1948.
Pritham, G. H., and A. K. Anderson: The carbon metabolism of Fusarium lyco-

persici on glucose, Jour. Agr. Research 55 : 937-949, 1937.
Rahn, O., and G. L. Richardson: Oxygen demand and oxygen supply. Jour. Bad.

41: 225-249, 1941.
RoBBixs, W. J.: Isoelectric points for the mycelium of fungi, Jour. Gen. Physiol.

6: 259-271, 1924.
*RoBBiNS, W. J., and M. B. Schmitt: Factor Z2 and gametic reproduction by Phy-

comyces, Am. Jour. Botany 32 : 320-326, 1945.
Ryan, J. F., G. W. Beadle, and E. L. Tatum: The tube method of measuring the

growth rate of Neurospora, Am. Jour. Botany 30 : 784-799, 1943.
*Scott, I. T.: The influence of hydrogen-ion concentration on the growth of Fusarium

lycopersici and on tomato wilt, Missouri Coll. Agr. Research Bull. 64, 1924.
Snell, F. D., and C. T. Snell: Colorimetric INIethods of Analysis, 3d ed., D. Van

Nostrand Company, Inc., New York, 1948.
Starkey, R. L., and S. A. Waksman: Fuiigi tolerant to extreme acidity and high

concentrations of copper sulfate, Jour. Bud. 4C : 509-519, 1943.
Tamiya, H.: Studi^n liber die Stoffwechseiphysiologie von Aspergillus oryzae. II.

Acta Phyiochim. {Japan) 4: 77-213, 1928.
Umbreit, W. W., R. IL Burris, and -J. F. Staufj^es? Manomptrjc Techniques and

170 PHYSIOLOGY OF THE FUNGI

Related Methods for the Study of Tissue MetaboUsm, Burgess Publishing Co,

Minneapolis, 1945. , , . , . .1 j

Van Slyke, D. D.: The kinetics of hydrolytic enzymes and their bearmg on methods

of measuring enzyme activity, Advances in Enzymol. 2 : 33-47, 1942.
WoLPERT F. S.: Studies in the physiology of the fungi. XVII. The growth of

certain wood-destroying fungi in relation to the H-ion concentration of the

media, Ann. Missouri Botan. Garden 11: 43-97, 1924.
Wyss, O., V. G. Lilly, and L. H. Leonian: The effect of pH on the availability of

p-am'inobenzoic acid to Neurospora crassa, Science, 99 : 18-19, 1944.

CHAPTER 9
VITAMINS AND GROWTH FACTORS

It is kno^\^l that, for normal growth and development, animals and
man require in their diet minute amounts of certain organic compounds,
in addition to those which yield energy or are used for structural purposes.
Similarly, certain fungi must obtain from the substrate some of the same
substances for growth, reproduction, and other vital functions. Other
fungi are able to synthesize these compounds, which are called groivth
factors, or vitamins. Both terms have often been applied to the same
compounds, although the terms are not always synonymous. Originally,
the term vitamin was applied to the accessory factors in animal nutrition,
and some workers would restrict its use to animals and man. The term
growth factor has a somewhat broader connotation than vitamin. It
includes the components and derivatives of some vitamins, as well as
other compounds which cannot be classified otherwise at present. The
chemical names of the vitamins also may be used.

GENERAL CONSIDERATIONS

A number of vitamins, such as thiamine and biotin, have been shown
to perform definite functions in fungi as well as in animals, and there is
no reason to assume that the fundamental functions in the two groups
of organisms are essentially different. The characteristic features of a
growth factor (vitamin) include the following: (1) its organic nature; (2)
its activity in minute quantities; (3) its catalytic action; (4) the specificity
of its action. It is known that some vitamins are components of enzyme
systems, and it may be assumed that all act in this way.

In the fungi the relative effects of the presence of vitamins in the
medium usually are measured by the resultant vegetative growth,
although vitamins are known to affect reproduction and other processes.
Needless to say, studies of vitamin deficiencies must be carried out under
carefully controlled conditions, using clean glassware, purified chemicals,
and precaution against contamination. Despite all precautions possible,
variable results often occur, and tests may need to be repeated several
times before the vitamin deficiencies of some fungi can be definitely
determined.

SYNTHESIS OF VITAMINS BY FUNGI

Many fungi are able to grow and develop normally on a substrate con-
taining no vitamins. For example, Aspergilhis niger grows well on a

171

172

PHYSIOLOGY OF THE FUNGI

synthetic medium composed of pure chemicals (glucose, asparagine, salts,
and micro elements). Phycomyces hlakesleeaniis makes no growth on
this medium unless thiamine is added. We may conclude that A. niger
either does not need thiamine in its metabolism or is capable of synthesiz-
ing from the compounds of the medium all vitamins in sufficient quantities
to meet its needs. The growth of P. blakesleeanus on the culture filtrate
of A. niger is proof that thiamine is synthesized by the latter species.
Thus, A. niger may be called a self-sufficient fungus with respect to
vitamins. Schopfer (1943) has applied the term autotrophic with respect
to vitamins to this group of organisms. The detection of self-sufficient
fungi in the laboratory is dependent upon their ability to grow on vitamin-
free synthetic media containing suitable sources of carbon and nitrogen.
A discussion of the economic importance of certain vitamins as metabolic
products of fungi is given in Chap. 13.

Some fungi which have been reported to be self-sufficient with respect
to vitamins are listed below:

Aspergillus (most species tested)
Basisporium gallarum
Botrytis allii
Cercospora apii
C. beticola

Chaetomium globosum
Cordyceps militaris
Daldinia concentrica
Fusarium (most species tested)
Glomerella cingulata
Helminthosporium gramineum

H. victoriae

Monascus purpurea

Monilinia fructicola (some isolates)

Neocosmopara vasinfecta

Penicillium (most species tested)

Phoma betae

Rhizopus nigricans

Sclerotinia sclerotiorum

Septoria nodorum

Sphaeropsis malorum

Ustilago striiformis

Growth curves of Chaetomium globosum are presented in Fig. 29, as an
example of a self-sufficient fungus. It is evident that good mycelial
growth was made in the vitamin-free medium and that the addition of
four vitamins caused no significant increase in the rate of growth at any
time.

VITAMIN DEFICIENCIES IN FUNGI

As pointed out above, some fungi do not grow on synthetic media
composed of pure chemicals, because they are unable to synthesize certain
vitamins. These fungi have been called variously vitamin-deficient,
vitaminless, or heterotrophic with respect to one or more specific vitamins.
We prefer to use the term vitamin-deficient, following Robbins and
Kavanagh (1942). Vitamin deficiencies among the fungi have been
detected only for certain members of the water-soluble B-complex group.
The most common vitamins involved are thiamine, biotin, inositol,
pyridoxine, nicotinic acid, and pantothenic acid. Vitamin deficiencies
can be detected accurately only on synthetic media which, other than

VITAMINS

173

-^^■"^^

^^^— — 1

250

•'^

^"^

y^'*

4 vitamins— _^
present

200

r/^

150

>
/

//"

~ Without vitamins

100

^
^

0 2 4 6 8

Days of incubation

Fig. 29. Growth curves of Chaetomiurn globosum, a self-sufficient fungus, in 25 ml. of
liquid glucose-casein hydrolysate medium in the absence of vitamins and when
thiamine, biotin, inositol, and pyridoxine were added.

Fig. 30. Mutualistic symbiosis with regard to vitamins. Phycomyces blakesleeanus,
thiamine-deficient, inoculated on the right and Sordaria fimicola, biotin-deficient,
inoculated on the left. Both fungi made only slight growth until the two colonies met.
Note the perithecia of Sordaria on the right and the sporangiophores of Phycomyces on
the left. Each fungus excreted into the medium the vitamin which the other could not
synthesize.

174 I'HYSIOLOGY OF THE FUNGI

vitamins, meet all the requirements for normal growth and development
of the fungus under study. The effect of one deficient fungus on another
is shown in Fig. 30.

Methods of detecting vitamin deficiencies. Tests for vitamin deficiencies
of fungi are not difficult to perform, but they do require clean glassware
and careful preparation of media. It is convenient to conduct pre-
liminary experiments using only the four vitamins (thiamine, biotin,
inositol, pyridoxine) for which fungi are most frequently deficient. A
greater number of vitamins may be included in subsequent tests if a
fungus does not grow well on any of the media first used. Either agar
or liquid media may be used, and the visual measure of growth is satis-
factory for the screening tests. A simple and convenient method for
preliminary tests for deficiencies in filamentous fungi isolated from nature
is by the use of agar media in test tubes, as shown in Fig. 31. Slight
growth on agar media without added vitamins may be due to impurities
in the medium. A high percentage of the deficiencies will be detected
by this method, since deficiencies for only one or two vitamins are com-
mon among the filamentous fungi. After the deficiencies have been
identified by preliminary experiments, it is then highly desirable to grow
a fungus in liquid media, so that the mycelium may be harvested and
dry weights determined (see Suggested Laboratory Exercises for direc-
tions). The casein hydrolysate-glucose medium, given in Chap. 10, has
proved quite satisfactory for accurate vitamin studies. From the dry
weights of cultures determined at intervals throughout the growth period
of a fungus, growth curves may be plotted. Such curves are necessary
for accurate interpretations of the effects of vitamins in the medium.

A somewhat different method is used by Burkholder (1943) for defi-
ciency studies of yeasts, where deficiencies for more than two vitamins
are common. This method is illustrated in Fig. 32. A deficiency is
detected by the inability to grow in a medium which is complete except
for one vitamin. Failure to grow in a medium indicates a deficiency for
the vitamin omitted. Liquid media in test tubes are used for yeasts,
so that growth may be measured by photoelectric colorimeter.

Total and partial deficiencies. Phycomyces hlakesleeanus was widely
used in the early studies of thiamine. Schopfer established the deficiency
for thiamine and determined the requirements for this vitamin. Schopfer's
graph (Fig. 33) shows the growth curves of the fungus over a period when
different amounts of thiamine were added to the basal culture medium
(Schopfer, 1943). The fact that no growth occurred in the medium
lacking thiamine is not shown by the graph. An increase in both the
rate and the total amount of growth, as the amount of thiamine is
increased, is clearly shown between the fifth and seventh days. Thus
P. hlakeslseanus is unable to synthesize thiamine, which it must obtain

VITAMINS

175

A B C D E

Fig. 31. Method of detecting common vitamin deficiencies of filamentous fungi.
Deficiencies are evident by failure to grow on media lacking the necessary vitamin or
vitamins. The above media contained: A, no vitamins; B, thiamine; C, bio tin; D,
thiamine and biotin; E, thiamine, biotin, inositol, and pyridoxine. The fungi, from
top to bottom, are Ceratostomella fimbriata, Sordaria fimicola, Pleurage curvicolla, and
C. ulmi.

176

PHYSIOLOGY OF THE FUNGI

Fig. 32. Method of detecting multiple vitamin deficiencies of yeasts. Failure to
grow in the absence of a particular vitamin indicates a deficiency if the culture grew in
a medium supplied with a combination of vitamins. Growth of a strain of Saccharo-
myces cerevisiae (above) and Mycoderma valida (below) after 5 days at 25°C. From
left to right the vitamin supplements were: Tube 1, none; 2, less thiamine; 3, less
pantothenic acid; 4, less pyridoxine; 5," less inositol; 6, less bio tin; 7, less nicotinic
acid ; 8, all six vitamins.

VITAMINS

177

from its substrate. Figure 33 emphasizes two important features which
must be considered in vitamin studies: (1) the effects of different amounts
of the vitamin in the medium, and (2) the response of the fungus over a
period of time sufficiently long to allow maximum growth. The three-
dimensional graph permits one to plot dry weight against both variables.
The failure of a fungus to make an appreciable amount of growth even
after an extended period of incubation on a medium essentially free of a

Asparagine 0.1 %

r, 9 ^

Mg.
^90

Fig. 33. Three-dimensional graph showing growth of Phycomyces blakesleeanus on a,
synthetic medium as a function of thiamine concentration and time. (Courtesy of
Schopfer, Protoplasma 28: 383, 1937; also from the book "Plants and Vitamins,"
p. 102, 1943. PubUshed by permission of Chronica Botanica Co.)

particular vitamin, like the case illustrated by P. hlakesleeanus and
thiamine, indicates that the deficiency is total; i.e., the synthesis of that
vitamin is zero. Vitamin deficiencies of many fungi are only partial, as
shown by a slower rate of growth in a vitamin-free medium than in the
presence of added vitamins. The degree of partial deficiency may vary
M^dely, from slight to nearly total. Partial deficiencies may be easily
overlooked by terminating an experiment too soon. An incubation
period of 1 or 2 months is often required to distinguish between partial
and total deficiencies of some fungi.

An example of partial thiamine deficiency is illustrated by Lenzites
trabea (Fig. 34). In a medium containing thiamine, maximum Aveight

178

PHYSIOLOGY OF THE FUNGI

was attained in 20 days, while, in medium lacking thiamine, the fungus
required approximately 40 days to reach the maximum weight. This is
attributed to the slow rate of synthesis of thiamine. Other isolates of
L. trahea showed varying degrees of partial deficiency (Lilly and Barnett,
1948).

Single and multiple deficiencies. The above discussion has dealt with
examples of single deficiencies (for a single vitamin). For example,
Sordaria fimicola is deficient only for biotin (Fig. 31), Lenzites trahea.

EO 30 40

Incubation (days)

Fig. 34. Growth of a haploid isolate of Lenzites trahea and change in pH of liquid
glucose-casein hydrolysate medium at 25°C., with and witliout the addition of
thiamine. These curves illustrate a partial deficiency for thiamine. (After Lilly and
Barnett, Jour. Agr. Research 77: 290, 1948.)

Ceratostomella Hmhriata (Fig. 31), and Phycomyces blakesleeanus for
thiamine only. On the other hand, some fungi have multiple deficiencies
(for two or more vitamins). These may be total or partial. An illustra-
tion of multiple deficiency is furnished by Sclerotinia camelliae (Fig. 35).
Little or no growth occurred on vitamin-free medium or that containing
either thiamine or biotin alone; the fungus grew well only in media con-
taining both thiamine and biotin. When inositol also was added, growth
was consistently better than in the presence of the two vitamins. This
indicates a partial deficiency for inositol, in addition to the total, or near
total, deficiencies for thiamine and biotin. Pyridoxine, when added to
the other three vitamins, had little or no effect on growth under these
conditions.

Other examples of multiple vitamin deficiencies are common. Pleurage
curvicolla (Fig. 31), Chaetomium convolutum, Coemansia interrupta, and

VITAMINS

179

400

10 12 14 16

Days of incubation

Fig. 35. Growth of Sclerotinia camelliae in 25 ml. of liquid glucose-casein hydrolysate
medium at 25°C. Note the nearly total deficiency for biotin and the partial deficiency
for inositol. Failure to grow in thiamine alone and in the absence of vitamins indicates
total deficiency for thiamine.

6 12 18 24

Days of incubation

Fig. 36. Growth of Lambertella pruni in 25 ml. of liquid glucose-casein hydrolysate
medium containing various vitamins. Partial deficiencies for both thiamine and
biotin are evident, being greater for thiamine. Note that the addition of inositol
and pyridoxine to media containing thiamine and biotin depressed growth.

180

PHYSIOLOGY OF THE FUNGI

Ophiobolus graminis are highly or totally deficient for both thiamine and
biotin. Partial deficiencies for both thiamine and biotin are illustrated
by Lambertella pruni (Fig. 36). Slight growth in the control and excellent
growth only in media containing both thiamine and biotin identify the
deficiencies. Intermediate growth in thiamine alone and in biotin alone
shows that the deficiencies are partial. The synthetic capacity is rela-
tively greater for biotin than for thiamine. The deficiencies of Endothia
parasitica are similar to those of L. pruni. Blastodadia pringsheimii was
30

-^ 20
E
o>

E
^^

.5*

A 10

•

Biotin

• <
Thiamin

weight (mgms)
D o c

♦'

^^* ■ — 1

y^ Nicotinamide

.. 1 1

— 1 — 1 — t

•

05 1.0 1.5
Micrograms per 75 cc.

' 1 1 1 1

2.0

0.002

0.015

0.005 0.01

Micrograms per 75 cc.

Fig. 37. The effect of concentration of essential vitamins on dry weight of Blasto-
dadia pringsheimii. (Courtesy of Cantino, Am. Jour. Botany 35: 241, 1948.)

reported (Cantino, 1948) to be partially deficient for thiamine and biotin
and nearly totally deficient for nicotinic acid (Fig. 37). Cerotostomella
ips No. 255 was shown to be completely deficient for thiamine, biotin, and
pyridoxine (Ptobbins and Ma, 1942a).

Multiple vitamin deficiencies are more common among the yeasts than
among the filamentous fungi, and some yeasts show deficiencies not
known to exist in filamentous fungi isolated from nature. For these
reasons the yeasts as a group have received much attention in vitamin
investigations. The vitamin requirements of 38 species and strains of
yeast were reported by Burkholder (1943), and for 110 additional named
species and varieties by Burkholder et at. (1944). A summary of the
deficiencies reported in these two papers is as follows: biotin, 114; thia-
mine, 48; pantothenic acid, 44; inositol, 19; nicotinic acid, 19; pyridoxine,
19. No deficiency for riboflavin was found. Several isolates were
deficient for three or more vitamins. Saccharomyces oviformis was
deficient for biotin, pantothenic acid, and pyridoxine, while S. mace-
doniensis Y-91 showed complete or partial deficiencies for thiamine,

VITAMINS

181

pantothenic acid, nicotinic acid, and biotin. S. ludwigii Y-974 and
Kloeckera brevis were totally or partially deficient for six vitamins (thia-
mine, biotin, inositol, pyridoxine, nicotinic acid, and pantothenic acid).
Growth of all of the 38 yeasts reported by Burkholder (1943) was increased
by the addition of liver extract to the medium containing the seven
vitamins.

The preceding discussion of the effects of added vitamins on the growth
of fungi has been based on the assumption that near-optimum amounts
of the vitamins were present in the media. However, the optimum

20 28

Days of incubation

Fig. 38. The effect of concentration of biotin on the rate and amount of growth ot
Sordaria fimicola in 25 ml. liquid glucose-casein hydrolysate medium, initial pH 4.4.
Growth in this medium containing biotin but no thiamine is evidence that this fungus
can synthesize thiamine under these conditions. (After Lilly and Barnett, Am. Jour.
Botany 34: 134, 1947.)

amount of a vitamin may vary with changes in other conditions and may
be different for different fungi. We have found that the following
amounts per liter of the four commonly needed vitamins are near optimum
for many filamentous fungi: thiamine, 100 Mg; pyridoxine, 100 jug; biotin,
5 /ig; inositol, 5 mg.

The effects of biotin concentration on the growth of Sordaria fimicola
are illustrated in Fig. 38, which shows a decided increase in growth rate
with greater amounts of biotin. Growth was most rapid in a medium
containing 6.4 jug biotin per liter (0.16 /zg per flask), but a steady slow
increase in dry weight is evident in as low as 0.1 ^g biotin per Hter.

Absolute and conditioned deficiencies. According to Robbins and
Kavanagh (1942), the deficiency of a fungus for a specific vitamin may be
absolute or conditioned. Phycomyces blakesleeanus and Ceratostomella

182

PHYSIOLOGY OF THE FUNGI

fimbriata show absolute total deficiencies for thiamine. No environ-
mental condition is known to allow the synthesis of this vitamin by these
fungi. In the case of a conditioned deficiency, the synthesis of the
vitamin may be influenced by certain environmental conditions, such as
temperature, composition, concentration, and pH of the medium.

Pythium hutleri failed to grow in a mineral salts-asparagine medium
containing 16.4 g. of salts per liter unless thiamine was added (Robbins
and Kavanagh, 1938). When the salt concentration was reduced to 1.64
g. per liter, this species grew without the addition of thiamine. A defi-

30 35 40 45

Temperature
Fig. 39. Growth-temperature relations for wild-type Neurospora and a temperature-
sensitive mutant deficient for riboflavin. Amounts of riboflavin are indicated on the
curves in micrograms per 20 ml. of medium. Below 25°C. growth was good without
riboflavin, while no growth occurred above 28°C. without added riboflavin. (Courtesy
of Mitchell and Houlahan. Am. Jour. Botany 33: 31, 1946.)

ciency for riboflavin conditioned by temperature was reported by Mitchell
and Houlahan (1946) for a mutant of Neurospora (Fig. 39). Growth was
poor or none at temperatures above 25°C. unless riboflavin was added.
Below 25°C. the fungus was able to synthesize riboflavin. The partial
deficiency of Sclerotinia camelUae for inositol was influenced by tempera-
ture, particularly in the above-optimum range (Barnett and Lilly, 1948).
Low pH of the medium resulted in partial thiamine deficiency of
Sordaria fimicola, while no deficiency for thiamine was apparent at initial
pH 4.0 or above (Lilly and Barnett, 1947). Within the range of 3.8 to
3.4, growth was quite slow, but the addition of thiamine overcame the
inhibition due to the high acidity (Fig. 40). These results indicate that
pH 3.8 or lower inhibits the synthesis of thiamine by S. fimicola. In a
similar way, the availability of p-aminobenzoic acid to a mutant of

VITAMINS

183

10 14

Days of incubation
Fig. 40. Effect of concentration of thiamine on the rate and amount of growth of
Sordaria fiynicola in 25 ml. Hquid glucose-casein hydrolysate medium, initial pH 3.8.
Compare with Fig. 38. (After Lilly and Barnett, Am. Jour. Botany 34: 134, 1947.)

O20

PH4.0

pHj^

— •■

:—

—

T *

.^ f

(

pH 6.0

, -<

pHTCL

i —

0.05 0.1 0.2 0.3 0.4

Microgroms p-aminobenzoic ocid per 25 ml. medium

0.5

Fig. 41. Effect of concentration of p-aminobenzoic acid at different pH values on the
growth of a mutant of N eurosj}ora crassa deficient for this vitamin. The fungus was
grown on liquid glucose-casein hydrolysate medium for 3 days. (Drawn from the
data of Wyss, Lilly, and Leonian, Science 99: 18, 1944.)

184 PHYSIOLOGY OF THE FUNGJ

Neurospora crassa deficient for this vitamin was found to be influenced
by the pH of the medium (Wyss et al, 1944) (Fig. 41).

The abiHty of a mutant of Neurospora sitophila to synthesize pyridoxine
was shown to be dependent not only on the pH of the medium but also
on the source of nitrogen (Stokes et al., 1943). When nitrate, amino,
amide, or certain other nitrogen compounds served as the nitrogen source,
no growth occurred without the addition of pyridoxine. However, in
the presence of ammonium salts, growth occurred at an initial pH range
of 5.6 to 7.3, without added pyridoxine. In this pH range, free ammonia
is formed. In the absence of free ammonia, the pyridoxine synthesized
is unavailable to this mutant (Strauss, 1951).

According to Fromageot and Tschang (1938), the red yeast, Rhodotorula
sanniei, requires thiamine when the carbon source is glucose, but when
redistilled glycerol replaces glucose, thiamine is not needed. It is inter-
esting to speculate whether this fungus is better able to synthesize thia-
mine in a glycerol medium or whether much less thiamine is required to
metabolize glycerol than glucose.

The concentration of the micro essential elements has also been shown
to influence the synthesis of vitamins by microorganisms (see Chap. 13
for specific information).

INHIBITORY EFFECTS OF VITAMINS

In certain cases vitamins may have an inhibitory effect on growth,
particularly when present in excessive dosages. The interrelated effects
of temperature and amount of inositol were described (Barnett and Lilly,
1948) for Sclerotinia camelliae. At a temperature below 26°C. the partial
deficiency was overcome by adding 5 mg. inositol to the medium. Above
26°C. the fungus was highly sensitive to small changes in temperature
and in amounts of inositol in the medium (Fig. 42). The same amount
of inositol which stimulated grow^th at or below 26°C. was strongly inhibi-
tory at 27°C. Increased amounts of inositol caused greater inhibition
of growth. Since the maximum temperature for growth is slightly above
27°C., it is believed that, as the temperature approaches this point, the
fungus becomes highly sensitive to the increased amounts of inositol in
the medium.

Some vitamins are known to have a depressing effect on growth of
certain fungi not deficient for these particular vitamins. For example,
Fig. 36 shows that Lambertella pruni produces more dry weight in the
presence of both thiamine and biotin than when inositol and pyridoxine
are also added to the medium. Similarly, it is reported (Elliott, 1949)
that, for a self-sufficient isolate of Fusarium avenaceum, both the rate of
growth and maximum amount of mycelium were greater in vitamin-free
medium than when vitamins were added. The presence of thiamine also

VITAMINS

185

Fig. 42. The interrelated inhibitory effects of high concentrations of inositol and
near-maximum temperatures on the growth of Sclerotinia camdliae, which is partially
deficient for inositol below 26°C. Cultures 19 days old. Thiamine and biotin were
added to all media. Temperatures ±0.3°C. Left to right: 26°C., 26.6°C., 27°C.
Amounts of inositol added per liter were: A, none; B, 1 mg.; C, 10 mg.; D, 100 mg.

186 PHYSIOLOGY OF THE FUNGI

has been reported to depress the growth of several fungi, including
Collctotrichum lindemuthianum (Mathur et al., 1950), Rhizopus suinus
(fechopfer and Guilloud, 1945), and Fusarium lini (Wirth and Nord,
1942). Other cases have been observed in our laboratory. In the case
of Rhizopus suinus, the addition of inositol overcame the inhibitory effects
of thiamine, and we believe it to be effective with certain other fungi.
On the basis of these reports, it would seem unwise to add vitamins
indiscriminately to media used for the study of fungi which are self-
sufficient for these vitamins.

VITAMERS

Certain microorganisms are less specific in their vitamin requirements
than are animals, owing apparently to their greater synthetic ability.
Some vitamin-deficient fungi may respond well to one of the vitamin
moieties, as in the case of thiamine, or to a compound similar to the
vitamin. The term vitamer w^as suggested by Burk et al. (1944) to denote
a compound having vitamin activity but differing in molecular structure
from the true vitamin. Usually the structure of a vitamer is closely
related to that of the vitamin. More specifically, these compounds are
known as thiamine vitamers, biotin vitamers, etc. In general, a vitamer
is active for fewer fungi than is the vitamin. Some vitamers are anti-
vitamins. This topic is discussed in Chap. 11.

UNIDENTIFIED GROWTH FACTORS

It is quite probable that some fungi wdll be discovered which are defi-
cient for vitamins or other growth factors w^hich are at present unknown.
Fungi which fail to grow in synthetic media to which all the known growth
factors have been added offer a challenge and an opportunity to the
investigator. Burkholder and Moyer (1943) reported that Candida
albicans 475 and Mycoderma vini 939 did not grow unless liver extract
was added to glucose-asparagine medium containing six vitamins. One
may speculate that the effect of liver extract was due to some amino acid
or to an undetermined growth factor, possibly vitamin B12, which is
known to be present in liver. In view of the common experience regard-
ing the stimulating effect of natural substances on growth of fungi, it is
evident that much more investigation on this phase of nutrition is needed.

SPECIFIC VITAMINS

In the first portion of this chapter the general aspects of vitamins and
growth factors were considered. Different types of vitamin deficiencies
and the methods of detecting deficiencies were discussed. The second
portion deals with the specific vitamins, their characteristics and
functions.

VITAMINS

187

THIAMINE AND ITS MOIETIES

Thiamine (vitamin Bi, aneurine) was the first vitamin shown to be
required by a filamentous fungus. Thiamine deficiency in man is known
as beriberi. Certain fungi and other microorganisms resemble man in
that they are unable to synthesize this vitamin. It is probably required
in the metabolism of all forms of life, and its function, to a large extent, is
believed to be the same in all organisms.

Schopfer (1934) demonstrated that Phycomyces blakesleeanus failed to
grow in a synthetic medium unless thiamine was added. This was a
stimulus for numerous studies on vitamin deficiencies of fungi. The
chemical synthesis of thiamine, in 1936, was another important step in
vitamin research. The student is referred to Williams and Spies (1938),
Rosenberg (1942), and Schopfer (1943) for information on the history,
synthesis, and natural occurrence of thiamine.

The structural formula for thiamine is

N=C— NHrHCl

CH,— C C

-CH,— N

CHs

c==c—

CH2— CH,OH

Cl CH — S
N— CH

Thiamine chloride hydrochloride

The thiamine molecule contains two ring structures, a substituted
pyrimidine and a substituted thiazole. The pyrimidine moiety has the
following formula:

N====C— NH2

CH,

C— CH2X

Thiamine pyrimidine
2-Methyl-4-amino-5-methylpyrimidine

X in the substituted methyl group on C5 may be hydroxyl, chlorine,
bromine, etc. The thiazole moiety has the following formula:

CH3

=C— CH2— CH2OH

CH-

Thiamine thiazole
4-Methyl-5-/3-hydroxyethylthiazole

188 PHYSIOLOGY OF THE FUNGI

These moieties are referred to in the Hterature as the thiamine pyrimidine
and thiamine thiazole, respectively.

Thiamine is somewhat unstable when exposed to alkali and heat, but
at pH 3.5 it is unaffected by autoclaving. Sulfur dioxide and sulfites
are destructive at pH 5 to 6. These factors must be taken into considera-
tion, and it is sometimes desirable to sterilize thiamine separately, either
by filtration or by autoclaving in an acidified solution. For most investi-
gations, however, it is permissible to autoclave thiamine with the medium.
For most fungi 100 fxg of thiamine per liter of medium is near optimum
for growth and sporulation. However, the optimum varies with the
amount of sugar in the medium and with other conditions.

Soon after pure thiamine became available, it was discovered that
certain treatments destroyed its activity for animals but did not greatly
affect the potency when certain fungi were used as test organisms. The
solution to this problem was reached when it became known that thia-
mine, when autoclaved in the presence of alkali, was broken down into
thiamine pyrimidine and thiamine thiazole.

Thiamine-deficient fungi differ in their ability to utilize or synthesize
the moieties of thiamine. These fungi may be classified into four groups
on this basis: (1) The intact molecule of thiamine is required by some
fungi which are unable to synthesize either moiety or to complete the
synthesis of thiamine, even when both moieties are supplied. Examples
of the group are species of Phytophthora. (2) Some other fungi, such as
Phycomyces hlakesleeanus, are capable of utilizing thiamine, or of syn-
thesizing thiamine when furnished with a mixture of the two thiamine
moieties. (3) The addition of thiamine or thiamine pyrimidine satisfies
the need of those fungi which are able to synthesize the thiazole moiety
and combine it with the pyrimidine moiety to make thiamine. Examples
are Parasitella simplex and Rhodotorula rubra. (4) Other fungi are able
to synthesize only the thiamine pyrimidine and complete the synthesis
of thiamine when furnished with the thiazole moiety. Mucor raman-
nianus and Stereum frustulosum are examples.

In the above discussion it was assumed that in every case the intact
molecule was the active product and that neither moiety nor the presence
of the two had any activity until thiamine was synthesized. Leonian
and Lilly (1940) found this hypothesis to be correct. The following fungi
Avere grown in a basal medium to which had been added the minimal
growth factor: Fusarium niveum (none), Pythiomorpha gonapodyoides
(pyrimidine), Mucor ramannianus (thiazole), Phycomyces hlakesleeanus
(both moieties of thiamine), and Phytophthora erythroseptica (thiamine).
After growth, the mycelium and the medium were tested for thiamine
and its moieties by growing fungi of known thiamine or thiamine-moiety
requirements upon media containing the mycelium extract and the
medium. Some of these data are collected in Table 33.

VITAMINS

189

Table 33. Assay for Thiamine and Thiamine Moieties in Mycelium and
Medium Extracts of Some Fungi after Growth on Media Containing

THE Minimum Growth-factor Requirements
Numbers refer to relative growth on the scale of 10. (Leonian and Lilly, Plant
Physiol. 15, 1940.)

Test fungi and substance tested for

Fungi tested and

minimum vitamin

requirements

Pythium
ascophallon
(thiamine)

Phycomyces

blakesleeanus

(both

moieties)

Pythiomorpha

gonapodyoides

(pyrimidine)

Mucor

ramannianus

(thiazole)

Fusarium niveum
(none)

Pythiomorpha gonapody-
oides
(pyrimidine)

Mucor ramannianus
(thiazole)

Phycomyces blakesleeanus
(both moieties)

Phytophthora erythroseptica
(thiamine)

10*
0*

10
0

10
2

3
2

10
8

8
6

5
3

10
8

10
6

10
10

* Upper figures refer to extract of mycelium, lower figures to extract of medium.

It is evident that Fusarium niveum was able to synthesize thiamine
from the basal medium because two test fungi which require thiamine
per se grew on extracts prepared from the hyphae. The same type of
proof shows that Pythiomorpha gonapodyoides synthesized thiamine when
thiamine pyrimidine was added to the basal medium. Mucor raman-
nianus synthesized thiamine when thiamine thiazole was added, and
Phycomyces blakesleeanus synthesized thiamine when both moieties were
added.

In all cases the greater portion of thiamine was stored within the
mycelium, and only small amounts were present in the medium. The
medium extract from three fungi contained no thiamine, although appre-
ciable quantities of the pyrimidine and thiazole moieties were present
in all media. This shows that Phytophthora erythroseptica, for example,
had broken down the thiamine molecule into its moieties, which diffused
into the medium and were later utilized by certain fungi, such as Phyco-
myces blakesleeanus. This suggests that in the process of its utilization
thiamine is slowly destroyed. The moieties may be recombined by
certain organisms but not by those which require the entire thiamine

190 PHYSIOLOGY OF THE FUNGI

molecule. Robbins and Kavanagh (1941) showed that P. blakesleeanus
destroyed the thiazole more rapidly than it did the pyrimidine moiety.
Thus, an excess of thiazole in the mixture of the two moieties was more
effective than equal quantities. They termed this the thiazole effect.

Some thiamine -deficient fungi. A deficiency for thiamine is by far
the most common vitamin deficiency among filamentous fungi isolated
from nature. Fries (1948) states than over 200 fungi are known to be
partially or totally deficient for thiamine. No doubt this is a modest
estimate. Deficiency for this vitamin is more common among certain
groups of fungi than others. For example, all species of Phytophthora
studied have been found to require the intact molecule of thiamine.
Only a few species of the true Basidiomycetes have been reported to be
self-sufficient for thiamine. Many of these fungi show only partial
deficiencies, while some are totally deficient. In most cases, however,
there seems to be little or no correlation between thiamine deficiency and
taxonomic relationship.

Some common filamentous fungi (other than Basidiomycetes) which
have been reported to be totally or partially deficient for thiamine or its
moieties, with other deficiencies (if any) indicated in parentheses, are
as follows : Blakeslea trispora, Ceratostomella fimbriata, C. ips (biotin and
pyridoxine), C. montium (biotin and pyridoxine), C. pini (biotin), Chae-
tomium convolutum (biotin), Choanephora cucurbitarum, Coemansia inter-
rupta (biotin), Dendrophoma obscurans, Endothia parasitica (biotin),
Hypoxylon pruinatum (biotin), Lambertella pruni (biotin), Lophodermium
pinastri (biotin and inositol), Melanconium betulinum (biotin and inositol),
Melanospora destruens (biotin), Mucor ramaymianus, Nectria coccinia,
Ophiobolus graminis (biotin), Phycomyces blakesleeanus, Phytophthora
spp., Piricularia oryzae (biotin), Pleurage curvicolla (biotin), Podospora
curvida (biotin), Pythiomorpha gonapody aides, Pythium arrhenomanes , P.
ascophallon, P. butleri, P. oligandrum, Sclerotinia camelliae (biotin and
inositol), S. minor, Sordaria fimicola, certain isolates only (biotin),
Thielaviopsis basicola, Valsa pini (biotin and inositol), and Xylaria
hypoxylon.

Reports of deficiencies for most of the above-named fungi may be found
in the references for this chapter. Some few of these fungi have been
studied in our laboratory and have not been previously reported as being
deficient for thiamine. For thiamine-deficient yeasts see the reports of
Burkholder (1943) and Burkholder et al. (1944).

Mode of action. One of the primary uses of thiamine in plants and
animals is for the regulation of carbohydrate metabolism. It is also
probable that thiamine may be involved in other processes. A vitamin
which constitutes a part of an enzyme system is known as a coenzyme.
Generally a vitamin must be combined with organic or inorganic com-

VITAMINS 191

pounds (or both) before it combines with the protein portion (apoenzyme)
of the enzyme system. The pyrophosphoric ester of thiamine is known
as cocarboxylase, or as thiamine pyrophosphate. This compound is the
coenzyme of carboxylase.

CH, O O

N=C— NH2 I II II

I C=C— CH2— CH2— O— P~0— P— OH

CH3— C C— CH2— N

H H

CI CH— S

N— CH

Thiamine pyrophosphate (cocarboxylase)

This substance is as active as thiamine (mole for mole) . Lilly and Leonian
(1940) compared the action of thiamine and thiamine pyrophosphate on
several thiamine-deficient fungi. No significant differences were found
in the maximum weights of mycelium formed in the presence of equivalent
quantities of these two growth factors. The rate of early growth was
greater with thiamine pyrophosphate than with thiamine for Phyco-
myces hlakesleeanus and less for Mucor ramannianus and Phytophthora
erythroseptica.

Pyruvic acid, one of the key intermediate products of carbohydrate
metabolism, is transformed into carbon dioxide and acetaldehyde by the
action of the enzyme carboxylase. Pyruvic acid accumulates in the
culture media of many thiamine-deficient fungi when insufficient thiamine
is present. Haag and Dalphin (1940) found that the maximum accumu-
lation in Phycomyces hlakesleeanus cultures occurred when about one-
twentieth of the optimum amount of thiamine was added. Wirth and
Nord (1942) studied the effect of added thiamine upon the accumulation
of pyruvic acid in cultures of Fusarium lini, a self-sufficient fungus with
respect to thiamine. Some of the data are presented in Table 34.

The accumulation of pyruvic acid in the culture medium is common,
especially during the early period of growth. Pyruvic acid may be
detected qualitatively by adding of iodine solution (KI3) to the culture
filtrate and making the solution strongly alkaline with sodium hydroxide.
Iodoform is produced instantly without heating. Acetaldehyde, which is
very volatile, also reacts with iodine and alkali in the cold to produce
iodoform. Sordaria fimicola, Lenzites trahea, or other fungi which produce
acid during the early stages of growth may be used to demonstrate the
production of pyruvic acid.

Specificity. So far as is known, thiamine which occurs in nature has
the structure given in the formula. This vitamin has been isolated from
only a few substances such as wheat germ and rice polish. The ethyl
homologue (ethyl in place of methyl in position 2) of thiamine is slightly
more active for certain fungi than ordinary thiamine. Higher homologues

192

PHYSIOLOGY OF THE FUNGI

have been reported to be less active or inhibitory. Whether ethyl thia-
mine occurs in nature is not known. The student is referred to Schopfer
(1943) for further information on thiamine specificity.

Table 34. The Effect of Added Thiamine upon the Accumulation of Pyruvic
Acid in the Culture Filtrate of Fusarium lint Grown on Glucose-
Nitrate Medium
(Wirth and Nord, Arch. Biochem. 1, 1942. Published by permission of Academic
Press, Inc.)

Days of

Glucose fermented,
g. per liter

Pyruvic acid accumulated,
mg. per liter

Mycelium produced,
mg. per 50 ml.

incuba-
tion

Thiamine added, ^g per liter

500

2
4
6
8

1.5
17.3
34.7
40.9

2.5
13.5
33.9
40.7

50
1,590
1,710
1,550

Trace

80
260
Trace

132

259

91
135

BIOTIN

Biotin (vitamin H) was originally isolated as a grow^th factor for yeast.
It is known to be the factor which prevents raw-egg-white injury to
animals and is the respiratory coenzyme (coenzyme R) for species of
Rhizohium. Biotin is active at greater dilutions than are the other
vitamins. Pure biotin methyl ester was first isolated by Kogl and Tonnis
(1936) w^ho obtained 1.1 mg. of this substance from 250 kg. of dried duck
eggs. The structure of biotin was determined by Du Vigneaud et al.
(1942a) and confirmed by the synthesis of this compound (Harris et al.,
1943). The structure of the biotin molecule is as follows:

NH NH

I I

CH CH

CH2 CH— (CH2)4— COOH

Biotin

Some fungi deficient for biotin. Biotin deficiency appears to be
characteristic of most yeasts (Burkholder, 1943 ; Burkholder and Moyer,
1943; Leonian and Lilly, 1942). Numerous filamentous fungi have been
reported to be deficient for biotin, but this number is not so great as that
for thiamine. Frequently biotin deficiency accompanies thiamine
deficiency.

VITAMINS 193

Among the first investigators to test the action of biotin on filamentous
fungi were Kogl and Fries (1937), who showed that Nematospora gossypii
did not grow in the absence of biotin. As httle as 0.4 ^g of biotin per
liter permitted almost as much growth as did ten times that amount. For
most filamentous fungi 5 /xg of biotin per liter is adequate. The effects
of biotin deficiency on the development of the ascospores of Sordaria
fimicola are shown in Fig. G8.

Some filamentous fungi reported as being partially or totally deficient
for biotin, with other deficiencies (if any) given in parentheses, are as
follows: Chaetomium convolutum (thiamine), Coemansia interrupta (thia-
mine), Diplodia macrospora, Endothia parasitica (thiamine), Hypoxylon
pruinatum (thiamine), Lamhertella pruni (thiamine), Melanospora destru-
ens (thiamine), Memnoniella echinata, Neurospora spp., Ophioholus
graminis (thiamine), 0. oryzinus, Ophiostoma catonianum, Penicillium
digitatum (thiamine, pyridoxine, pantothenate), Piricularia oryzae (thia-
mine), Pleurage curvicolla (thiamine), Podospora curvula (thiamine),
Pseudopeziza ribis, Rosellinia arcuata, Sclerotinia camelliae (thiamine,
inositol), Sordaria fimicola, Sporormia intermedia, Stachybotrys atra,
Thraustotheca clavata.

Specificity. The biotin molecule is not separable into moieties as is
thiamine. One of the first related compounds to be studied was desthio-
biotin. As the name indicates, the molecule no longer contains sulfur.
The structure of the desthiobiotin molecule is as follows:

/ \
NH NH

I I

CH3— CH CH— (CH2)6— COOH

Desthiobiotin

The removal of sulfur from the biotin molecule destroyed the tetrahydro-
thiophene ring and introduced a methyl group. In addition, the acidic
chain of desthiobiotin contains one more methylene group than does that
of biotin. Stokes and Gunness (1945) tested the growth of some biotin-
deficient microorganisms on desthiobiotin and found that this compound
was utilized by Neurospora sitophila and three strains of Saccharomyces
cerevisiae, but Rhizobium trifolii 209, Lactobacillus casei, and L. arabinosus
17-5 were unable to utilize desthiobiotin. From further experiments it
was concluded that the yeast synthesized biotin, or some other compound
which replaced it, from desthiobiotin, rather than utilizing desthiobiotin
directly. The source of sulfur in the medium was found to influence the
amount of desthiobiotin converted into biotin, with methionine and
sodium sulfate being better sources than cystine, sulfanilamide, or
thiamine thiazole.

Lilly and Leonian (1944) studied the effect of desthiobiotin on 45

194 PHYSIOLOGY OF THE FUNGI

biotin-deficient microorganisms and found that it replaced biotin for
some fungi, while it acted as an antibiotin for some few others. Desthio-
biotin replaced biotin quantitatively for Ceratostomella ips. Goldberg
et al. (1947) found some homologues of biotin to inhibit growth of yeast
139 and Lactobacillus casei. Whether any of these biotin homologues
will replace biotin for other microorganisms must await further testing.
These preliminary results indicate that the length of the acidic side chain
of the biotin molecule is of great importance in biological activity.

Oxybiotin is also known as 0-heterobiotin and has the same structure
as biotin except that the sulfur in the tetrahydrothiophene ring has been
replaced by oxygen. Pilgrim et al. (1945) found oxybiotin to be active
for Lactobacillus casei, L. arabinosus, and a strain of Saccharomyces cere-
visiae. Oxybiotin is apparently used as such and is not converted into
biotin by the organism (Axelrod et al., 1947). This is the only instance
that has come to our attention where a vitamer is used directly instead
of being converted into the vitamin. Rubin et al. (1945) had previously
reported that oxybiotin was converted into biotin. The cause of this
disagreement is unknown.

Pimelic acid is a growth factor for certain strains of the diphtheria
bacterium (Mueller, 1937). It is reported (Du Vigneaud et al., 1942)
that pimelic acid replaced biotin and was probably the precursor in the
synthesis of biotin by a strain of the diphtheria organism. The higher
and lower homologs of pimelic acid were ineffective. The formula for
pimelic acid is HOOC— CH2— CH2— CHa— CH2— CH2— COOH. At
present there is no evidence that pimelic acid replaces biotin for the
fungi. This observation is supported by the findings of Robbins and Ma
(1942), who studied 13 biotin-deficient fungi. A favorable effect of the
presence of pimelic acid was reported by Eakin and Eakin (1942), who
found that Aspergillus niger synthesizes much more biotin in the presence
of pimelic acid than in its absence. Cysteine and also cystine increase
the synthesis of biotin. The lower homologues of pimelic acid (adipic,
glutaric, and succinic) were without effect, while the higher homologs
(suberic and azelaic) were as effective as pimelic acid. This is interesting,
inasmuch as homobiotin and bishomobiotin are reported inactive for
yeast growth (Goldberg et al., 1947).

Mode of action. It has been assumed that biotin acts as a coenzyme
for various enzyme systems, but definite proof seems to be lacking,
Winzler et al. (1944) found that, when biotin was added to a biotin-
starved yeast, some time elapsed before any effect was noted. The order
of response was fermentation, respiration, and growth. The assimilation
of ammonia did not take place unless biotin was added.

The presence of aspartic acid in the culture medium has been shown
to reduce the amount of biotin required by Torula cremoris (Koser et al.,

VITAMINS 195

1942) and by Memnoniella echinata and Stachyhotrys atra (Perlman,
1948). There is also evidence (Stokes et al., 1947) that biotin plays a
role in the synthesis of aspartic acid by certain bacteria. Thus, it appears
probable that one of the functions of biotin is connected with the synthesis
of aspartic acid. When aspartic acid is added to the medium, it is
unnecessary for the organism to perform this synthesis and the need for
biotin is greatly reduced. However, it should be noted that, although
the absolute amount of biotin needed is reduced, exogenous biotin is still
required by these biotin-deficient organisms. From this it may be
deduced that biotin has a multiple role in the cell.

INOSITOL

meso-Inositol (also known as inactive inositol, isoinositol, inosite, or
dambose) is widely distributed in both plants and animals. It was first
isolated in 1850. It was not until 1928 that Eastcott (1928) showed that
it was a growth factor for a strain of yeast. Later, Woolley (1940)
recognized it as a vitamin for animals. meso-Inositol is a hexahydroxy-
cyclohexane. It has the following configuration:

H H

Q Q

OH/i i\ H

1/ OH 0H\|

c c

:\ H OH/1
H \| 1/ OH

C C

OH li
meso-Inositol

There are seven different cis-trans isomers, which are optically inactive,
and a pair of optically active d and I forms. The available evidence
indicates that the stereochemical configuration of weso-inositol is specific
for vitamin activity. Some of the isomers have only slight activity.
Inositol is active only in high concentrations as compared to the other
vitamins. The usual amount added is around 5 mg. per hter of medium.

Fungi deficient for inositol. Many strains of yeast are deficient for
this vitamin, while others are not. In most cases the deficiency appar-
ently is partial rather than total. Partial deficiencies for various yeasts
are reported by Leonian and Lilly (1942), Burkholder (1943), and Burk-
holder and Moyer (1943). In the last two references total deficiencies
for inositol are reported for Saccharomyces uvarum Y 969 and Schizosac-
charomyces pomhe.

Kogl and Fries (1937) were apparently the first to investigate the
action of inositol on various filamentous fungi. They found that Nemato-
spora gossypii was totally deficient and that Lophodermium pinastri was

196 » PHYSIOLOGY OF THE FUNGI

partially deficient for this vitamin. The partial deficiency of Sclerotinia
camelliae is shown in Fig. 35. Deficiencies for inositol are commonly
accompanied by deficiencies for thiamine and biotin. Trichophyton
discoides is reported as being totally deficient for inositol, pyridoxine, and
thiamine (Robbins et at., 1942). Totally deficient mutants of Neurospora
crassa have been developed. Their use in bioassays for inositol was
described by Beadle (1944) and by Leonian and Lilly (1945).

Some filamentous fungi reported to be partially or totally deficient for
inositol, with other deficiencies given in parentheses, are as follows:
Colletotrichum lindemuthianum (certain strains only), Epichloe typhina
(thiamine), Lophodermium pinastri (thiamine, biotin), Melanconium
hetulinum (thiamine, biotin), Nematospora gossypii (thiamine, biotin),
Sclerotinia camelliae (thiamine, biotin), Trichophyton discoides (thiamine,
pyridoxine), Valsa pini (thiamine, biotin).

The effects of temperature upon the synthesis of inositol by Sclerotinia
camelliae and upon the toxicity of high concentrations of inositol at high
temperatures were described by Barnett and Lilly (1948) and are illus-
trated in Fig. 42.

Mode of action. The addition of inositol overcame the inhibition of
growth of Rhizopus suinus due to excess thiamine (Schopfer and Guilloud,
1945). In part, the inhibition was due to an increased production
of alcohol (pyruvate ^ acetaldehyde—^ alcohol). Similarly, we have
observed in our laboratory the same favorable effect of inositol on growth
of certain fungi which are inhibited by the presence of excess thiamine.

NICOTINIC ACID

A deficiency for nicotinic acid, or nicotinic acid amide, leads to pellagra
in man and blacktongue in dogs. The structural formulas of these com-
pounds follow:

/\^r.f^c^vi /\

-CONH2

Nicotinic acid Nicotinic acid amide

Nicotinic acid was obtained by the oxidation of nicotine in 1867. Knight
(1937) and Mueller (1937o) recognized that nicotinic acid amide was a
growth factor for certain bacteria. So far as is known, the amide is the
form utilized by organisms. Some microorganisms can convert nicotinic
acid into its amide with ease, others with difficulty; still others are unable
to use nicotinic acid but require either nicotinic acid amide or a coenzyme
containing the amide.

Fungi deficient for nicotinic acid. Rogosa (1943) tested 114 strains of
yeast that ferment lactose and found that all of them required an exoge-

VITAMINS 197

nous supply of nicotinic acid for growth. Rogosa used the technique
of serial passage in a medium devoid of nicotinic acid. It is possible to
overlook a vitamin deficiency by failure to observe this precaution.
Yeasts found to be deficient for this vitamin include Saccharomyces
anamensis 154, S. lactis 131, S. fragilis 15, Zy go saccharomyces lactis (two
strains), Torida lactosa 168, T. sphaerica 13, T. cremoris 2, Torulopsis
kefyr 149, Mycotorula lactis 130. Strains of Saccharomyces cerevisiae
failed to show deficiency for nicotinic acid (Rogosa, 1943; Leonian and
Lilly, 1942; Burkholder, 1943).

Until recently, nicotinic acid deficiency among filamentous fungi iso-
lated from nature was unknown. Cantino (1948) has shown that Blasto-
cladia pringsheimii is completely deficient for nicotinamide and partially
deficient for thiamine and biotin. Some of Cantino's results are pre-
sented in Fig. 37. A second filamentous fungus, a strain of Microsporum,
audouini, is reported as deficient for nicotinic acid (Area Leao and Cury,
1949). Mutants deficient for this vitamin have been developed in
Neurospora by Bonner and Beadle (1946) and in Penicillium by Bonner
(1946).

Specificity. In so far as the fungi are concerned, nicotinic acid replaces
nicotinic acid amide, but few critical studies in this connection have been
made. Various studies have been made of the specificity for bacteria
of the compounds related to nicotinic acid. Bovarnick (1943) reported
that heating asparagine and glutamic acid together produced a compound
which replaced nicotinic acid or its amide for various species of bacteria.
This author later showed that this substance was nicotinic acid amide.
This is an unsuspected way of adding a vitamin to a basal medium.

Mode of action. Nicotinic acid amide is a constituent of two or more
coenzymes. Codehydrogenase I on hydrolysis yields adenine, nicotinic
acid amide, and two molecules of D-ribosephosphoric acid. Codehydro-
genase II yields the same products as codehydrogenase I except that three
molecules of phosphoric acid, instead of two, are produced. In the
literature codehydrogenase I is often referred to as DPN (diphospho-
pyridine nucleotide) and codehydrogenase II as TPN (triphosphopyridine
nucleotide). These coenzymes in combination with specific proteins
form enzyme systems which transfer hydrogen (oxidation-reduction).
Apparently the amide of nicotinic acid is reversibly oxidized and reduced
in the process.

One organism. Hemophilus parainfluenzae, requires codehydrogenase
I as a growth factor. This organism is unable to form the coenzyme
u'hen furnished with the moieties, nicotinic acid amide, adenine, D-ribose,
and phosphate. DPN is also known as factor V (Gingrich and Schlenk,
1944). Other bacteria are known which require preformed coenzymes
as growth factors. While no fungus isolated from nature has yet been

198 PHYSIOLOGY OF THE FUNGI

shown to require such growth factors, it is possible that some do exist.
Such requirements may be found among the artificially induced mutants.

PANTOTHENIC ACID

Pantothenic acid was first discovered (Williams et al., 1932) as a growth
factor for the Gebriide Mayer strain of Saccharomyces cerevisiae. The
isolation, identification, and synthesis of this compound was complete by
1940. It was later shown to be a vitamin for animals. Pantothenic
acid consists of two moieties joined together by means of an amide link-
age. The chemical formula for this vitamin is given below:

CH3

I
HO— CH2— C— CHOH— CO— NH— CH2— CH2— COOH

I
CH3

Pantothenic acid

Pantothenic acid may be hydrolyzed to form /3-alanine (jS-amino-
propionic acid), the formula of which is H2N — CH2 — CH2 — COOH, and
a,7-dihydroxy-/3,;5-dimethylbutyric acid, a substituted butyric acid that
forms a lactone by elimination of one molecule of w^ater between the
carboxyl and the gamma hydroxyl (pantoyl lactone). Pantothenic acid
is thus analogous to thiamine, in that the molecule may be split into two
moieties. We might expect to find different pantothenic acid-deficient
organisms which require the intact molecule or one or both moieties. It
was found that the Gebriide Mayer strain of Saccharomyces cerevisae was
stimulated by /3-alanine and that this yeast completed the synthesis of
pantothenic acid when furnished with )8-alanine in the medium (Wein-
stock et al., 1939). Most yeasts deficient for pantothenic acid are unable
to synthesize the j8-alanine moiety of this vitamin. In general, this
moiety is not used so efficiently as pantothenic acid, and more than an
equivalent amount is required to support the same amount of growth.
The composition of the medium affects utilization, since, in the presence
of sufficient asparagine, /3-alanine is not utilized (Atkin et al., 1944).

So far as is known, none of the fungi require pantoyl lactone as a growth
factor, but this compound was found (Ryan et al., 1945) to replace panto-
thenic acid for Clostridium septicum. It was shown by microbiological
tests that this bacterium completed the synthesis of pantothenic acid.

Fungi deficient for pantothenic acid. Of the 10 strains of Saccharo-
myces cerevisiae tested for vitamin deficiencies by Leonian and Lilly
(1942), 9 w^ere highly deficient for this vitamin. Burkholder (1943) found
14 of the 38 species and strains tested to be deficient for pantothenic acid,
9 of these being species of Saccharomyces. It appears that deficiency
for this vitamin is more common in Saccharomyces than in other genera
of yeasts. Varying degrees of pantothenic acid deficiency were found in

VITAMINS 199

species of Zygosaccharomyces (Lockhead andLanderkin, 1942). /S-Alanine
could be used in place of pantothenic acid for the deficient species of
Zygosaccharomyces.

Growth of Penicillium digitatum is reported (Wooster and Cheldehn,
1945) to be stimulated by pantothenate, as well as by pyridoxine, biotin,
and thiamine. To our knowledge this is the only report of a filamentous
fungus isolated from nature being stimulated by the presence of this
vitamin. Tatum and Beadle (1945) reported a mutant of Neurospora
which was deficient for pantothenic acid.

Specificity. As in the case of thiamine and inositol, the structure of
pantothenic acid is almost specific for activity. A hydroxypantothenic
acid synthesized by Mitchell et al. (1940) has a varying ability to replace
pantothenic acid for some organisms. The activity of this compound
for the Gebriide Mayer yeast was low as compared with pantothenic acid.

Mode of action. Pantothenic acid was found to favor the accumula-
tion of glycogen by yeasts (Williams et al, 1936), and to increase markedly
the rate of carbon dioxide evolution by the pantothenic acid-deficient
Gebriide Mayer yeast (Pratt and Williams, 1939). More recent work
(Novelli and Lipmann, 1947) has show^n that pantothenic acid is phos-
phorylated and acts as a coenzyme. This enzyme system performs vari-
ous oxidations and acetylations in the cell.

PYRIDOXINE

Pyridoxine is also known as adermin or as vitamin Be. While inositol
and pantothenic acid were first investigated as growth factors for micro-
organisms, pyridoxine was discovered as a result of animal research.
This vitamin was isolated independently by five groups of workers in
1938 and was synthesized the next year. The structural formula is given
below :

HO-
CHs-

■^ n— CH2OH

Pyridoxine

Pyridoxine is quite soluble in water and is stable to acid and alkah but is
destroyed by light.

Fungi deficient for pyridoxine. Partial or total deficiencies for this
vitamin have been reported for various species and strains of yeasts
(Eakin and Williams, 1939; Burkholder, 1943). Among these are Sac-
charomyces carlsbergensis var. mandshuricus Y-379, S. chodati Y-140, S.
oviformis, S. ludwigii, and Mycoderma valida Y-7.

Leonian and Lilly (1942) found that the omission of either thiamine or
pyridoxine alone from the medium was without effect on 9 of the 10 strains

200 PHYSIOLOGY OF THE FUNGI

of yeast tested. However, the omission of both pyridoxine and thiamine
caused a decrease in the growth of two of these strains. Apparently
these two yeasts were capable of synthesizing either thiamine or pyri-
doxine, provided that the other vitamin w^as present. This is a common
effect among fungi partially deficient for two or more vitamins. The
presence of one vitamin for which a fungus is partially deficient may
enable the fungus to synthesize other vitamins with greater ease.

Among the filamentous fungi, deficiency for pyridoxine seems to be
characteristic of certain species of Ceratostomella and a few other fungi
(Robbins and Ma, 1942o, 19426). Some species reported to be deficient
for pyridoxine, with other deficiencies given in parentheses, are Cerato-
stomella ulmi, C. ips (thiamine, biotin), C. pseudotsugae (thiamine), C.
piceaperda 240 (biotin), C. pini (thiamine, biotin), C montium (thiamine,
biotin), C. pilifera, C. multiannulata (thiamine), C. pluriannulata (thia-
mine), C. microspora (thiamine, biotin), Ophiostoma catonianum (thia-
mine), Trichophyton discoides (thiamine, inositol).

Specificity. One of the important uses of vitamin-deficient organisms
is for the purpose of vitamin assay. Certain vitamin-deficient fungi
and bacteria are used to determine the vitamin content of foodstuffs and
other natural products. For such tests to be of any value, it is necessary
to know if the organism used responds to substances other than the
vitamin itself. Snell et at. (1942) found that Streptococcus faecalis gave
much greater apparent yields of pyridoxine when used for assay than did
yeast. It was then discovered (Snell, 1942) that autoclaving pyridoxine
with the basal medium for 20 min. increased the activity of pyridoxine
forty-one times, and that this change in activity for certain organisms
was correlated with oxidation and heating with certain amino acids.

Snell (1944) then postulated that vitamers of pyridoxine were formed
by these treatments. When this problem was under investigation, these
vitamers of unknown structure were called "pseudopyridoxine," which
was later found to consist of either one or both of the following compounds :

HO-
CH3

^^— CHoOH HO— r^^— CH2OH

Pyridoxal Pyridoxamine

These two compounds were synthesized by Harris et al. (1944) and tested
by Snell.

It was concluded that this vitamin consists of three or more closely
related compounds. Saccharomyces carlsbergensis responds about equally
to the three compounds, while the reaction of certain bacteria is much
greater to pyridoxal and pyridoxamine than to pyridoxine, Ceratosto-

VITAMINS 201

mella ulmi grew at a more rapid rate with pyridoxamine than vvith pvri-
doxal or pyridoxine (Snell and Rannefelt, 1945). All three forms of this
vitamin occur in natural products. Assays for this vitamin are discussed
in Chap. 10.

Mode of action. One of the earliest clues to the action of pyridoxine
was discovered by Snell and Guirard (1943) in the interrelationship
among glycine, alanine, and pyridoxine and growth of Streptococcus
faecalis R. They found that alanine could replace pyridoxine for this
organism and that glycine caused inhibition which was overcome by
the addition of either alanine or pyridoxine. It was also found that
/3-alanine, serine, and threonine inhibited growth. It is possible that
alanine serves as a precursor for pyridoxine in this organism, or that one
function of pyridoxine is the synthesis of alanine. At any rate the action
of pyridoxine appears to be connected with either amino-acid synthesis
or amino-acid utilization, or both. Like other vitamins, pyridoxine (or
its conversion products) has been assumed to function in the cell as a
part of a coenzyme.

Pyridoxal is phosphorylated before it functions in enzyme systems.
In this it is like thiamine and pantothenic acid. Pyridoxal phosphate
is said to function as a coenzyme in the transformation of tryptophane
into indole by Escherichia coli (Wood et at., 1947). We may assume that
the function of this vitamin is the same in the fungi as in the bacteria.

p-AMINOBENZOIC ACID
p-Aminobenzoic acid has the following structure:

COOH

NH2
p-Aminobenzoic acid

Rubo and Gillespie (1940) found p-aminobenzoic acid to be a growth
factor for nine strains of Clostridium acetohutylicum. Most of the interest
in this compound centers in its antagonistic action to sulfonamides. A
discussion of this subject is presented in Chapter 11.

Fungi deficient for p-aminobenzoic acid. Robbins and Ma (1944)
reported Rhodotorula aurantica to be deficient for p-aminobenzoic acid
and thiamine. Concentrations of as low as 0.03 /xg per liter had a positive
effect on the growth of this yeast, while maximum growth was attained
in the presence of 3 /zg per liter. The intensity of the pink color developed
by this yeast was a function of the p-aminobenzoic acid content of the
medium.

202 PHYSIOLOGY OF THE FUNGI

In so far as we are aware, no filamentous fungus isolated from nature
has been shown to be deficient for p-aminobenzoic acid. Tatum and
Beadle (1942) described a mutant of Neurospora which was unable to
synthesize this vitamin. Wyss et at. (1944) found that the availability
of p-aminobenzoic acid to the deficient mutant of Neurospora crassa was
a function of the pH of the medium (see Fig. 41).

Mode of action. The functions of p-aminobenzoic acid are unknown.
We may assume, on the basis of the behavior of other vitamins, that it
functions as a coenzyme, or as a part of a coenzyme. Recent work
indicates that p-aminobenzoic acid is a constituent part of folic acid.

RIBOFLAVIN
The structure of riboflavin is given below:

CH2OH

I
HC— OH

CH2

N N

\c^ \c=o

N C

Riboflavin

Many bacteria, especially species of Lactobacillus are unable to synthe-
size riboflavin (Peterson and Peterson, 1945). So far as we are able to
determine, none of the fungi isolated from nature have been found to be
deficient for riboflavin. This vitamin is synthesized by the fungi.
Mitchell and Houlahan (1946) described a mutant of Neurospora which
required the addition of riboflavin to the medium for growth at tempera-
tures above 28°C. Between 15 and 25°C. the growth rate of the mutant
without added riboflavin was equal to that of the wild type. The rate
of growth decreased rapidly as the temperature increased from 25 to

28°C.

SUMMARY

It is assumed that all living organisms require a number of vitamins, or
growth factors, for normal growth, reproduction, and other vital proc-
esses. However, organisms differ widely in their synthetic capacities
for the various vitamins. Some fungi are self-sufficient with respect to
vitamins, being able to synthesize their vitamins from pure chemicals
of a synthetic medium. Others lack the ability to synthesize sufficient

VITAMINS 203

quantities of one or more vitamins and are called vitamin-deficient fungi.
The deficiency may be single or multiple, complete or partial. Partial
deficiency may vary from nearly complete to nearly self-suflficient and
is more pronounced during the early stages of growth.

A single deficiency for thiamine has been more commonly reported
among filamentous fungi than any other type. Biotin deficiency is like-
wise commonly found, often in combination with thiamine deficiency.
Deficiencies for inositol and pyridoxine are less common. Two filamen-
tous fungi isolated from nature are reported to be deficient for nicotinic
acid. Numerous other deficiencies have been induced in mutants by ir-
radiation. Some yeasts show complete or partial multiple deficiencies
for three to six vitamins, while relatively few filamentous fungi are
deficient for as many as three vitamins.

Absolute deficiencies are not known to be influenced by the environ-
ment, while conditioned deficiencies may be affected either by nutritional
factors or by factors of the physical environment. Among these, tem-
perature and the composition and pH of the medium seem to be the most
important.

Methods of detecting vitamin deficiencies are exact, and accurate
determination depends on the ability or inability of a fungus to grow on
a synthetic medium composed of pure chemicals, to which known amounts
of the various vitamins to be tested are added. Vegetative growth,
measured by dry weight, is apparently the most useful criterion of the
utilization of vitamins, although reproduction and other processes are
likewise affected.

Compounds having vitamin activity but differing in molecular struc-
ture are called vitamers. In general, only compounds of closely related
structure have vitamin activity.

The inhibitory effects of vitamins in excess quantities are apparently
common. They are usually evident by slight reduction in rate or maxi-
mum amount of growth and are more common with self-sufficient fungi
than with those deficient for the vitamin in question. Thiamine is more
commonly reported as a growth depressor than other vitamins. One
instance of severe inhibition due to excess inositol and temperatures near
maximum for growth is discussed.

The known effects of vitamins on the growth of fungi emphasize the
important fact that growth is a result of a number of interacting factors,
among which are the vitamins. A proper balance between the different
vitamins and with the other nutritional and environmental factors must
exist if maximum rate of growth is to take place.

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for Streptococcus lactis R., Proc. Soc. Exptl. Biol. Med. 51: 356-358, 1942.
Snell, E. E.: The vitamin activities of "pyridoxal" and "pyridoxamine," Jour.

Biol. Chem. 154: 313-314, 1944.
Snell, E. E., and B. M. Guirard: Some interrelationships of pyridoxine, alanine

and glycine in their effect on certain lactic acid bacteria, Proc. Natl. Acad. Sci.

U.S. 29: 66-73, 1943.
Snell, E. E., B. M. Guirard, and R. J. Williams: Occurrence in natural products

of a physiologically active metabolite of pyridoxine, Jour. Biol. Chem. 143 :

519-530, 1942.
Snell, E. E., and A. N. Rannefelt: The vitamin Be group. III. The vitamin

activity of pyridoxal and pyridoxamine for various organisms, Jour. Biol. Chem.

157 : 475-489, 1945.

VITAMINS 207

Stokes, J. L., J. W. Foster, and H. B. Woodward: Synthesis of pyridoxin by a

"pyridoxin-less" x-ray mutant of Neurospora sitophila, Arch. Biochem. 2:

235-245, 1943.
Stokes, J. L., and M. Gunness: Microbiological activity of synthetic biotin, its

optical isomers and related compounds, Jour. Biol. Chem. 157: 121-126, 1945.
Stokes, J. L., A. Larsen, and M. Gunness: Biotin and the synthesis of aspartic

acid by microorganisms. Jour. Biol. Chem. 167: 613-614, 1947.
Strauss, B. S.: Studies on the Be-requiring, pH-sensitive mutants of Neurospora

crassa, Arch. Biochem. 30: 292-305, 1951.
Tatum, E. L., and G. W. Beadle: Genetic control of biochemical reactions in

Neurospora: an "aminobenzoic-less" mutant, Proc. Natl. Acad. Sci. U.S. 28:

234-243, 1942.
Tatum, E. L., and G. W. Beadle: Biochemical genetics of Neurospora, Ann. Mis-
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Am. Chem. Soc. 54: 3462-3463, 1932.
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Williams, R. R., and T. D. Spies: Vitamin Bi (Thiamin) and Its Use in Medicine,

The Macmillan Company, New York, 1938.
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Arch. Biochem. 1: 143-163, 1942.
Wood, W. A., I. C. Gunsalus, and W. W. Umbreit: Function of pyridoxal phos-
phate : Resolution and purification of the tryptophanase enzyme of Escherichia

coli, Jour. Biol. Chem. 170: 313-321, 1947.
Woolley, D. W.: The nature of the antialopecia factor, Science 92: 384-385, 1940.
Wooster, R. C., and V. H. Cheldelin: Growth requirements of Penicillium digita-

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p-aminobenzoic acid to Neurospora crassa, Science 99 : 18-19, 1944.

CHAPTER 10
FUNGI AS TEST ORGANISMS

Numerous physiological problems are accessible to investigation
through the use of microorganisms. By the proper choice of deficient
organisms, it is feasible to detect minute amounts of physiologically active
compounds such as the vitamins and amino acids. Knowledge has been
gained of the way vitamins and amino acids are synthesized and destroyed
by various organisms. The amino-acid composition of proteins and the
availability of certain essential elements in soil may be determined by the
use of fungi and bacteria. These highly practical studies are based upon
a knowledge of the compounds and elements essential for the nutrition
of microorganisms. Since these are, in general, the same elements and
compounds needed by animals, there is a very close relation between
fungus and animal physiology in nutritional problems. Foodstuffs for
man and animals are the most common materials analyzed in routine
assays.

Some of the advantages which have contributed to the widespread use
of microorganisms for assay purposes are simple technique and apparatus,
sensitivity, specificity, and the short time required. Perhaps the most
important single factor is the small sample needed and the fact that little
or no purification or concentration of the active material is required.
These advantages are to be compared with chemical methods or the use
of animals for obtaining the same information. All analytical methods
have advantages and disadvantages. A knowledge of the limitations of
any method is essential for valid results.

Most microbiological assays depend upon the proportional response of
deficient test organisms to the substances for which they are deficient.
This proportional response occurs only for a limited range of concentra-
tions. The usable range of concentration depends upon the substance
being assayed, the test organism, and the basal medium. In theoiy,
any organism may be used to assay any substance for which it is deficient,
but in practice not all organisms having the same deficiency are equally
suitable. For example, Rhizohium trijolii 205 is about 100 times as sen-
sitive to biotin as Sordaria fimicola.

The following are essential to any quantitative microbiological assay:
(1) a suitable test organism; (2) the preparation of a basal medium ade-
quate in all respects, but essentially free from the substance to be assayed ;
(3) liberation, from the material to be analyzed, of the substance to be

208

FUNGI AS TEST ORGANISMS 209

assayed, in a water-soluble condition; (4) a standard sample of the sub-
stance to be analyzed ; (5) preparation of a range of concentrations of the
known and unknown substances in the basal medium ; (6) uniform inocula-
tion; (7) incubation under uniform conditions; (8) measuring the response
of the test organism; (9) construction of the standard curve from the
response of the test organism to kno\\Ti amounts of the substance under
test; (10) calculating the content of the substance contained in the sample.
The above discussion assumes the use of pure compounds in obtaining
standard curves. The utility of microbiological assay methods is not
confined to the assay of known compounds. They are of great utihty
in studies of methods of isolation of new growth factors and other active
compounds. These occur in complex natural products and, before they
are isolated, are known only by the physiological effects they produce in
living organisms. Given a deficient fungus, or other organism, it is pos-
sible to follow the efficiency of the various steps in an isolation procedure.
The isolation of many of the water-soluble vitamins has been facilitated
by the use of test fungi. The use of a biotin-deficient yeast enabled
Kogl and Tonnis (1936) to isolate biotin for the first time as a pure
compound.

GENERAL PROCEDURES

The following discussion of the steps involved in microbiological assay
may serve also as a guide to the quantitative study of the physiology of
fungi. Such studies are the surest way to gain knowledge and under-
standing of the physiology of the fungi.

Selection of test organisms. The first requirement of a test organism
is specificity for the compound under assay. A fungus which responds
to either or both moieties of thiamine is less suitable than one which
requires the intact thiamine molecule. Other considerations may out-
weigh the advantages of strict specificity, but the response of the test
organism to moieties, vitamers, and related compounds must be known.

Other considerations besides specificity enter into the selection of test
organisms. Test organisms should be easily maintained in culture,
easily handled in the laboratory, and have stable biochemical character-
istics. Rapid and uniform growth is desirable. The habit of growth is
important. A fungus which forms mucilaginous colonies which adhere
to the walls of the flasks is difficult to harvest, and yeasts which clump
are difficult to determine by turbidimetric methods.

The basal medium. Except for the compound or element under
investigation the basal medium should be complete and balanced. If a
test organism is deficient for more than one factor, all the factors except
the one under investigation should be present in optimum amounts.
Other requirements are easily available sources of carbon and nitrogen
and a medium which is adequately buffered in the optimum pH range.

210 PHYSIOLOGY OF THE FUNGI

The basal medium should be essentially free from the vitamin or other
factor under test. The response of the test organism to the basal
medium should be slight; this value is known as the blank, or control.
The size of the blank depends upon the residual concentration of the
factor in the basal medium and the amount and kind of inoculum used.
The degree to which a basal medium should be freed of the substance
under test depends upon the sensitivity of the test organism.

The best basal medium for any test organism can be determined only
after a prolonged investigation of the nutritional requirements of the
organism. This arduous task is too infrequently attempted. Fre-
quently, it is desirable to use some natural material in the medium. A
complex medium which supplies several sources of carbon and nitrogen
as well as other organic compounds may support more rapid growth than
a simple minimal medium.

The sample being analyzed may contain compounds which stimulate
or depress growth. Stimulation of growth due to the presence of acces-
sory factors, is perhaps more often encountered than growth depression.
The adequacy of the basal medium may be tested by comparing the
growth curve obtained on the sample with the standard curve. If the
response of the test organism to the sample is due solely to the factor con-
tained in the sample, the two curves will be identical. The presence of
inhibiting substances in the sample is detected when the sample curve
falls below the standard curve. Stimulating substances are revealed by
an upward drift of the sample curve.

If biologically pure compounds were available, the preparation of basal
media for assay purposes would be greatly simplified. No general
method of purification is useful for all purposes. Riboflavin is destroyed
by light, and media can be freed of this vitamin by exposure to strong
illumination. Activated charcoal (Norit or Darco) is very useful in
adsorbing residual traces of many vitamins. Recrystallization of sugars,
asparagine, and mineral salts is helpful in some instances. Casein is
extracted with hot alcohol to remove vitamins. The essential micro
elements may be removed in the ways discussed in Chap. 5. Frequently
reagents made by one manufacturer are purer in certain respects than
those of another.

Three basal media which have been used for fungi in microbiological
assays are given below.

Glucose-Asparagine

Glucose 30 e,

Asparagine 1 g.

MgS04-7HoO 0.5 g.

KH2PO4 1.5 g.

Distilled water to make 1 liter

FUNGI AS TEST ORGANISMS 211

This medium was used for thiamine assay using Phycomyces blake-
sleeanus as the test fungus (Schopfer, 1945).

Sucrose-Ammonium Tartrate-Ammonium Nitrate

Sucrose 20 g.

KH2PO4 1 g.

MgS04-7H20 0.5 g.

Ammonium tartrate 5 . 0 g.

NH4NO3 1.0 g.

NaCl 0.1 g.

CaCli 0.1 g.

B 0.01 mg.

Mo 0 . 02 mg.

Fe 0.2 mg.,

Cu 0.1 mg.

Mn 0.02 mg.

Zn 2.0 mg.

Bio tin 5 Mg

Distilled water to make 1,000 ml.

This medium was used by Horowitz and Beadle (1943) and by Beadle
(1944) for the assay of choline and inositol by biochemical mutants of
Neurospora crassa.

Glucose-Casein Hydrolysate

Glucose 25 g.

Casein hydrolysate equivalent to 2 g. casein

MgS04-7H20 0.5 g.

KH2PO4 l.Og.

Fumaric acid 1 . 32 g.

NaaCOs 1.12 g.

Fe+ + + as sulfate 0.2 mg.

Zn+ + as sulfate 0.2 mg.

Mn+ + as sulfate 0.1 mg.

Distilled water to make 1 liter

This medium was used by Leonian and Lilly (1945) for the assay of
certain vitamins. Various deficient yeasts and filamentous fungi were
used as test organisms. This medium is suitable for testing fungi for
vitamin deficiencies.

Preparing for an assay. In general, the compound being assayed
should be brought into aqueous solution before assaying. Many vita-
mins occur in a ''bound" condition and must be liberated before analysis.
The procedure used to liberate bound vitamins depends upon the vita-
min involved, as well as the nature of the substance being assayed.
Snell (1948) has listed tentative methods for the liberation of the various
vitamins. In general, acid or enzymatic hydrolysis is used. Proteins
are hydrolyzed before amino-acid assay. Acid hydrolysis is destructive

212 PHYSIOLOGY OF THE FUNGI

to certain amino acids, especially tryptophane. Alkaline hydrolysis of
proteins has been recommended for this amino acid (Greene and Black,
1944).

The concentrations of the standard compound and of the sample for
assay should be so chosen that the response of the test organism is roughly
linear. Every concentration should be run in duplicate. Control flasks
to which neither the standard compound nor the assay sample have been
added should form a part of every assay. This provides a means of
evaluating the basal medium and should never be omitted.

The type of culture vessel and the volume of the basal medium used
will depend upon the test organism. Bacteria are frequently cultured
in test tubes. These are also useful for yeasts. Uniform test tubes
which can be used in a photoelectric colorimeter allow measurement of
turbidity without transfer (Lindegren and Raut, 1947). The filamentous
fungi are usually cultured in Erlenmeyer flasks. The volume of medium
should be so chosen that the liquid is less than 1 cm. deep. All glassware
must be clean. Accuracy in measuring the basal medium and the known
and unknown solutions is essential.

Inoculation and incubation. The medium upon which the inoculum
is grown should be complete and contain an adequate but not excessive
amount of the factor under investigation. Certain fungi, especially the
yeasts, cease to be deficient for certain vitamins when continuously cul-
tured upon media free from these factors.

Spore inoculum may be used with advantage with many filamentous
fungi. Frequently it is desirable to use germinated spores for inoculum.
Phycomyces blakesleeanus spores require the Z factors for rapid germina-
tion (Robbins, 1940). If the test sample contains these factors and the
basal medium does not, early growth will be more rapid in the sample
series. It is convenient to germinate the spores of this fungus and others
by preparing a spore suspension in dilute peptone solution a few hours
before inoculation. These germinated spores grow essentially without
interruption and shorten the time of incubation. Fragmented mycelium
may also be used to advantage. A uniform amount of inoculum must be
used. This is easy to achieve when a suspension of spores or fragmented
mycelium is used. Inocula of these types provide a multitude of growing
points, which results in uniform growth. Disks of mycelium on agar
are, in general, unsatisfactory.

An obvious advantage of using a large amount of inoculum is the
shorter time required for an assay. However, there is danger of intro-
ducing with a large inoculum enough of the substance under investigation
to give abnormally high blanks. Washing the inoculum with sterile
distilled water reduces this hazard but increases the work and multiplies
the chances of contamination. A very small inoculum results in a longer
lag period, and the time required for analysis may be prolonged.

FUNGI AS TEST ORGANISMS 213

Test organisms during an assay should be cultured under uniform
conditions with respect to light and temperature. In general, the fila-
mentous fungi should not be agitated during the period of incubation.
Yeasts are frequently grown with continuous or intermittent shaking.

There are two schools of thought concerning the time of incubation
for assay. The first recommends a uniform short period of growth and
determination of the yield before the organism reaches its maximum
development. There is a saving in time in this method, but the influence
of accessory factors in the sample may make such results unreliable. A
comparison should always be made between the analytical data for a
short and a long period of incubation before choosing the length of incu-
bation period. In general, we feel that assays tend to be more reliable
when the period of incubation is long enough to allow maximum
development of the test organism.

Measuring the response. The methods used for measuring the
response of test organisms vary. The growth response of bacteria may
be measured either by titrating the acid produced or by determining the
turbidity with a suitable photoelectric colorimeter. The growth response
of yeasts may be measured as turbidity, or the cells may be weighed.
The first procedure is by far the simpler. The growth of filamentous
fungi is commonly measured by collecting the mycelium and determining
the drj^ weight (see discussion in Chap. 3).

Calculation of results. A growth curve (acidity, turbidity, or weight)
is plotted from the response of the test organism to the different concen-
trations of the standard substance. The concentration of the substance
in the sample is then calculated from the standard curve. It is necessary
to use a new standard curve for each series of assays. Unsuspected
variations in the basal medium and in technique from day to day make
this precaution necessary. In making the calculations, it is assumed that
equal amounts of the substance, whether as a pure compound or in the
sample, will cause the same amount of response by the test organism.
It is customary to report the concentrations of vitamins and micro essen-
tial elements in micrograms per gram of original sample.

As an example of the type of calculation involved in an assay, the
standard curve (Fig. 43) and protocol of a biotin assay are given below.
The substance assayed was air-dry yeast cells. Biotin was liberated
from the sample by acid hydrolysis, and the cell extract was neutralized
and made up to such volume that 1 ml. of hydrolysate was equivalent
to 50 mg. of original yeast cells. The test organism, Saccharomyces
cerevisiae, Gebriide Mayer strain, was incubated for 72 hr at 25°C.
Twenty-five milliliters of glucose-casein hydrolysate medium was used
per 250-ml. flask. The cultures were agitated 10 min. every hour. The
data for the response of the test organism to varying amounts of yeast
hydrolysate are given in Table 35.

214

PHYSIOLOGY OF THE FUNGI

Table 35. Yield of Saccharomyces cerevisiae Cells Produced when Different

Amounts of Yeast Hydrolysate Were Added to 25 Milliliters of a

Biotin-free Glucose-Casein Hydrolysate Medium

Yeast hydrolysate,

Equivalent weight
of sample, mg.

Yield, mg.

ml. per flask

Flask 1

Flask 2

0.03125
0.0625
0.125
0.25

1.5625
3.125
6.25
12.5

9.2
20.2
32.8
48.0

8.9
19.0
32.8
47.6

The amount of biotin in the original sample may then be calculated.
The amount of biotin in 6.25 mg. of the sample produced 32.8 mg. of

«40

S30

^'■^'"'^

0 0.001 0002 O004 0.006 0.008

Micrograms of biotin per flosk

Fig. 43. Standard curve for a biotin assay using Saccharomyces cerevisiae, Gebriide
Mayer strain, as the test fungus. Basal medium was glucose-casein hydrolysate,
25 ml. per 250-ml. Erlenmeyer flask. Cultures were incubated at 25°C., agitated 10
min. each hour, and harvested after 72 hr.

dry yeast cells. From the standard curve this is seen to be equivalent
to 0.0025 jug of biotin. The biotin content of the sample is therefore
equal to 0.0025 X 1,000/6.25, or 0.4 /xg of biotin per gram of sample.

VITAMIN ASSAYS

It is beyond the intent of this chapter to include detailed information
about techniques in connection with individual assays. The following
references are useful for entry into the hterature. Schopfer (1945) has
considered the philosophy underlying the use of microorganisms for
assay. Leonian and Lilly (1945) investigated the use of many test
organisms to assay the vitamin content of a single substance. This work
showed that widely different assay values for some vitamins are obtained

FVNGI AS TEST ORGANISMS 215

when different test organisms are used. The review of Snell (1948)
represents the critical judgment of an active investigator in this field.

While the filamentous fungi are frequently passed over in favor of
bacteria and yeasts, they offer certain advantages when only simple
apparatus is available, or where occasional assays are to be made. The
test organisms for the specific vitamins listed below are in part those
recommended by Snell (1948).

Thiamine. Phycomyces hlakesleeanus. This fungus responds to the
two moities of thiamine. Schopfer (1935, 1945) used a glucose-asparagine
medium and used dry weight of mycelium to measure growth. This is
an excellent organism to use in gaining experience with a microbiological
assay. Schultz et al. (1942) used Saccharomyces cerevisiae (Fleischmann's
baker's yeast) and measured the evolution of carbon dioxide, which was
proportional to the thiamine content of the sample.

Pyridoxine. Saccharomyces carlsbergensis. Snell (1945a) found that
this yeast responds about equally to pyridoxine, pyridoxal, and pyridoxa-
mine. Growth may be measured turbidimetrically or by weighing the
cells. Differential assays for these three vitamers have been devised.

p-Aminobenzoic acid. Neurospora crassa mutant. Various labora-
tories have used this organism (Tatum et al, 1946). For the effect of
pH on utilization of this vitamin see Wyss et al. (1944).

Pantothenic acid. Saccharomyces carlsbergensis. Most, if not all,
yeasts respond to the )3-alanine moiety of pantothenic acid. Atkin et al.
(1944) noted that the incorporation of Z-asparagine in the basal medium
reduced interference due to /3-alanine.

Nicotinic acid. Lactobacillus arabinosus. This organism responds
equally to nicotinic acid and nicotinamide. Growth may be measured
either by titrating the acid produced, or turbidimetrically (Krehl et al.,
1943). Zygosaccharomyces marxianus was used by Leonian and Lilly
(1945).

Inositol. Neurospora crassa mutant. This mutant was first used by
Beadle (1944) to assay inositol. It is an easy organism to handle, and
since this mutant forms few conidia, it is not a great source of contamina-
tion to a laboratoiy. Snell (1948) recommends the use of Saccharomyces
carlsbergensis for inositol assay.

Biotin. Saccharomyces cerevisiae. Various strains have been used.
Many, if not all, strains respond also to desthiobiotin (Lilly and Leonian,
1944). The existence of many biotin vitamers makes the choice of a test
organism difficult. Neurospora crassa and N. sitophila may also be used.
It is probable that some of the divergence of assay values obtained when
different test organisms are used is due to biotin complexes. Such a
complex, biocytin, has been isolated by Wright et al. (1950). The
analytical results were unchanged by acid hydrolysis when Lactobacillus

216 PHYSIOLOGY OF THE FUNGI

casei was used but were increased when L. arahinosus was the test
organism.

Riboflavin. Lactohacillus casei. Fatty acids stimulate growth.
Growth may be measured by titrating the acid formed, or turbidimetri-
cally (Roberts and Snell, 1946). It is probable that mutants of Neuro-
spora deficient for this vitamin may also be used in assay.

AMINO-ACID ASSAYS

The importance of the amino-acid composition of proteins used in
animal nutrition makes any advance in analytical methods of great
interest and value. The general techniques for amino-acid determina-
tions by microbiological procedures are the same as for other assays.
The first requirement of this type of microbiological assay is a suitable
test organism. Few fungi isolated from nature are deficient for amino
acids. For this reason bacteria have been extensively used. The follow-
ing references will give an entry into the literature on the use of bacteria
for amino-acid assay: Hutchings and Peterson (1943); Shankman (1943);
Dunn et at. (1944); Snell (1945); and Horn et at. (1950).

Some mutants of N'eurospora have been found to be deficient for amino
acids. Mutants having the following amino-acid deficiencies have been
studied: leucine, isoleucine, valine, lysine, methionine, serine, or glycine.
Only the mutant deficient for leucine appears to have been much used in
microbiological assay (Ryan and Brand, 1944; Brand et al., 1945). The
growth of a lysine-deficient mutant was completely inhibited by arginine
when the molecular ratio of arginine to lysine was 2 to 1 (Doermann,
1944).

Ryan (1948) has considered the possibility of microbiological assay
of amino acids by observing the percentage of germination of conidia
from deficient mutants in the presence of different concentrations of the
specific amino acid. An assay can be completed within a few hours by
this method. Unfortunately the inhibiting action of certain amino acids
introduces complications into the proposed method.

Mutations of Neurospora and certain other fungi have been induced
by chemicals, such as nitrite or nitrous acid, colchicine, nitrogen mustard
gas, and hydrogen peroxide, or by irradiation with ultraviolet and X rays.
These mutants are frequently characterized by inability to synthesize
various metabolites. They differ from the parent wild type in that one
or more genes have been inactivated. It is thought that each gene con-
trols a single biochemical reaction. Mutants having the same gross
deficiency may differ in the specific gene inactivated.

Horowitz (1947) studied four mutants of Neurospora which were unable
to synthesize methionine from inorganic sources of nitrogen and sulfur.
One of these mutants was able to grow in the presence of cysteine, cysta-

FUNGI AS TEST ORGANISMS 217

thionine, homocysteine, and methionine. The second was unable to
utihze cysteine but was able to utilize the other three compounds. The
third isolate utilized either homocysteine or methionine, while the fourth
isolate utilized only methionine. From these results the steps in the
synthesis of methionine and the genes inactivated may be summarized as

gene 4 gene 3 gene 2 gene 1

follows : > cysteine > cystathionine > homocysteine — -^ methi-
onine. From similar studies Srb and Horowitz (1944) concluded that

Neurospora synthesizes arginine as follows: ornithine — -^ citrulline >

arginine.

Fungus mutants have proved to be powerful tools for investigating
pathways of synthesis and utilization of vitamins, amino acids, and other
compounds, and in studies of biochemical mutations. From these studies
also comes the realization that each step in the synthesis or utilization of
a compound may be controlled or limited by a specific gene. The review
papers of Bonner (194G) and Beadle (1945, 1945a) should be consulted
for further information and literature citations.

ASSAYS FOR ESSENTIAL ELEMENTS

Microorganisms may be used to determine the presence of essential
elements. In view of the speed and accuracy of chemical and spectro-
scopic methods, it might be assumed that microorganisms would be of
little value in such applications. The value of microbiological tests
would appear to be in applications where availability as well as total
amounts are of importance. Problems of this sort frequently arise in
connection with mineral deficiencies in soil. It is recognized that the
absolute content of an essential element in a soil may not measure the
availability of that element for green plants. Microbiological and chem-
ical methods of analysis must be correlated with plant tests before they
are of much value.

The possible number of test organisms is unlimited except for the
important considerations of sensitivity, ease of handling, and time
required to make an assay. In practice, only a few organisms have been
used. There exists a wide field for investigations dealing with the cor-
relation between availability to microorganisms and availability to green
plants of certain essential elements in soil.

Copper. Mulder (1939-1940) used Aspergillus niger to determine
copper in soil. The range of concentrations in the standard series was
0.0 to 2.5 Mg Cu++ per culture; 40 ml. of medium was used in liter flasks.
One gram of sterile soil was used as the sample. The method of measur-
ing the response of A. niger to copper was very simple, inasmuch as the
number and color of the spores produced were functions of the copper
content of the medium. No spores developed on the control medium, but

218

PHYSIOLOGY OF THE FUNGI

with increasing concentrations of copper the spores were yellow, yellow-
brown, gray-brown, brown, and black.

The color of the spores produced on copper-deficient media by different
isolates of A. niger varied. Excellent correlation between the copper
content of various soils as determined by this method and the incidence
of copper deficiency in grain was found. Some of Mulder's results are
presented in Table 36.

Table 3G. The Correlation of Copper Deficiency in White Oats and the
Copper Content of the Soil as Determined by Aspergillus niger Method
All soil was from the same field. (Mulder, Antonie van Leeuwenhock 6, 1940.)

Available Copper,
Condition of Oats Mg per G. of Soil

Severely diseased 0 . 25

Less severely diseased 0.8

Healthy (from a portion of the field not showing the disease) 1.7

Healthy (copper sulfate added to the soil) 2.5

Magnesium. Smit and Mulder (1942) postulated that a microbio-
logical method would show better correlation with magnesium deficiency
in green plants than chemical methods. This was confirmed for the
Netherlands soils investigated. Azotobacter chroococcum and Aspergillus
niger were used as test organisms. Preference w^as given to the fungus
inasmuch as only 4 to 5 days w-ere required for an assay. A simple
technique was used, and visual comparison w^as sufficiently accurate to
diagnose magnesium deficiency in soils.

Potassium. Aspergillus niger was used by Niklas and Toursel (1940)
to determine available potassium and other elements in soils. These
authors weighed the mycelium produced. Rogosa (1944) has shown that
Lactobacillus casei may be used to determine small amounts of potassium.

rABLE 37. The Effect of Molybdenum Content of a Glucose-Nitrate Medium

UPON Yield of Mycelium and Sporulation of Aspergillus niger

(Mulder, Plant and Soil 1, 1948.)

Mg Na2Mo04-2H.A
in 50 ml. medium

Mg. mycelium
per culture

Sporulation

Appearance of
mycelium

0.0
0.0025
0.010
0.050

165
294

558
868

Normal

Entirely mucous
Partially mucous
Partially mucous
Normal

Molybdenum. The amount of this element needed by fungi and green
plants is greater when nitrogen is supplied as nitrate than when ammo-
nium nitrogen is furnished. This fact introduces a complication into the
microbiological assaj^ of molybdenum in that the sample must be ashed

FUNGI AS TEST ORGANISMS 219

before analysis. It is probable that amino acids and other nitrogen
sources containing reduced nitrogen would also affect the amount of
molybdenum needed. Mulder (1948) investigated the use of Aspergillus
niger as a test organism (Table 37). For further discussion and refer-
ences to the use of microorganisms in essential-element assay see Vande-
caveye (1948).

SUGARS

Yeasts and other microorganisms have been used to separate optical
isomers and complex mixtures of sugars. Pasteur (18G0) used Pemcillium
glaucum to obtain the "unnatural" isomer of tartaric acid from rfZ-tartaric
acid. Fischer and Hertz (1892) used brewer's yeast to ferment D-galac-
tose, while L-galactose in the same medium was not utilized. Auernheimer
el al. (1948) used the specific fermentative powers of Hansenula suaveolens
and Candida guilliermondi in the separation of L-arabinose and D-xylose
obtained from the hydrolysis of straw and corn cobs. H. suaveolens does
not utilize L-arabinose, while C. guilliermondi utilizes both pentoses.
Saccharomyces carlshergensis was used to demonstrate the absence of
D-glucose in the hydrolysates. These yeasts were used in conjunction
with chemical methods of analysis. Appling et al. (1947) found Sac-
charomyces carlshergensis var. mandschuricus to ferment D-galactose but
not L-galactose. Similarly, H. suaveolens utilized D-xylose but not
L-xylose.

These citations indicate the usefulness of yeasts and other organisms
in the solution of problems difficult to solve by other methods. The
value of microorganisms in such applications is due to their specificity.

TESTS FOR CERTAIN METABOLIC PRODUCTS

Fungi excrete into the media in w^hich they grow various physiologically
active substances. In the older literature these are referred to as staling
'products. Among the metabolic products are those which may stimulate
or inhibit growth and reproduction. The kind and the amount of com-
pounds excreted depend upon the particular fungus involved as well as
the composition of the medium. The effect of the metabolic products ot
one fungus upon another is simply demonstrated when fungi are gro^vn
in association. The beneficial effect of one fungus upon another was
demonstrated by Kogl and Fries (1937). Neither Nematospora gossypii
or Polyporus adustus grew when inoculated alone into a synthetic medium,
but when both fungi were inoculated together in the same flask, both
began to grow rapidly after about a week. N. gossypii is deficient for
biotin but synthesizes thiamine, while P. adustus is deficient for thiamine
but synthesizes biotin. Kogl and Fries called this artificial symbiosis.
Schopfer and Guilloud (1945) cite other examples in connection with
work on strains of Candida guilliermondi involving vitamin deficiencies.

220

PHYSIOLOGY OF THE FUNGI

By using a series of test organisms of known deficiencies, it is easy to
demonstrate that fungi excrete vitamins. It is a common experience to
find deficient fungi growing in association with contaminants. The
method is simple and consists of inoculating plates of vitamin-free medium
with two test fungi (Fig. 44). Not all fungi excrete the same amount of a

Fig. 44. Test demonstrating the excretion of biotin by Aspergillus 7'ugulosus (right),
whengrownwith Sordariafimicola (biotin-deficient) on vitamin-free glucose-asparagine
medium. Sordaria (left) made only slight growth until it approached the colony of
Aspergillus, where a zone of stimulated growth is evident.

given vitamin. This may be shown by choosing test fungi such as
Sordaria fimicola, which requires more biotin for fruiting than for growth.
Some fungi excrete enough biotin to allow grow^th of S. fimicola, while
others excrete enough biotin to allow reproduction also. Other com-
pounds besides the vitamins may be excreted and favor the growth of
other organisms. Further instances of the favorable effect of one fungus
on the sporulation of another are discussed in Chap. 14.

The metabolic products of one fungus may inhibit the growth of
another. This phenomenon may be frequently observed on contaminated
plates (Fig. 45). Fleming (1929) discovered the action of penicillin in
this way.

Many fungi apparently produce substances which inhibit the germina-
tion of their spores. Schopfer (1933) found that spores of Phy corny ces
blakesleeanus would not germinate on agar media upon which this fungus
had grown. If such a "staled" plate was autoclaved, the medium would
then allow germination and growth of P. blakesleeanus. These results

FUNGI AS TEST ORGANISMS 221

indicate that the spore-inhibiting substance was either volatile or
unstable. This inhibitory substance was not identified.

Fig. 45. Test for antibiotic production by growing two organisms in association on
the same agar plate. Helminthosporium sativum on the left and an unidentified
actinomycete on the right.

TESTING FABRIC PROTECTANTS

While the deterioration of cellulosic materials exposed to the weather
or in contact with the soil is not solely due to the action of bacteria and
fungi, these organisms are the chief agents of destruction. The problem
of deterioration of cellulosic materials has received a vast amount of
attention, especially in connection with military materiel in humid tropic
climates. Work on this problem involves the identification of the respon-
sible microorganisms, laboratory tests, and use of test fungi in evaluating
protectants.

The basis of the various methods for determining cellulolytic activity
consists in inoculating cotton duck or other test material with the fungi
under test. The degree of cellulolytic activity is determined by measur-
ing the decrease in tensile strength of the test specimen. The test
medium used is usually an inorganic salt solution having pH 6.8. It is
desirable to use a buffered medium inasmuch as cellulase is most active
around pH 7. White et at. (1948) note that many fungi which are
strongly cellulolytic under laboratory conditions cause but httle damage
in the field. They believe that, under a given set of natural environ-
mental conditions, the actual decay of fibers is caused by a relatively
few species of fungi. Among the strongly cellulolytic fungi are Mem-

222

PHYSIOLOGY OF THE FUNGI

noniella echinata (the variability in strains in laboratory tests is possibly
correlated with biotin deficiency), Chaetomium spp., especially C. glo-
bosian (Greathouse and Ames, 1945), Myrothecium verrucaria (as strong
a cellulose decomposer as yet found in laboratory tests), Trichoderma
viride, and Thielavia sepedonium.

The reduction in tensile strength of cotton duck maintained under
specified conditions is used as a measure of the destructive effects of
fungi on fabrics. The data in Table 38 are taken from White et al. (1948).

Table 38.

Assay of Fungi for Cellulolytic Activity Based upon Loss of
Tensile Strength of Cotton Duck
(White et al, Mrjcologia 40, 1948.)

Species

Aspergillus niger PQMD 25a

A. terreus PQMD 72f

Chaetomium funicolum PQMD 351.

C. globosum PQMD 32b

Fusarium oxysponim Fla C-8

Gliomastix convoluta PQMD 4c

Myrothecium verrucaria PQMD 70h
Thielavia sepedonium PQMD 47g. .

Trichoderma viride PQMD 6a

T. viride PQMD 63d

Strength retained, %

6 days

9 days

12 days

100

103

105

—

100

Growth

at end of

experiment

4
4
4
3
4
4
4
4
0

The evaluation of protective fungicides for fabrics, paper, and other
cellulosic materials consists in comparing the effects of known cellulolytic
fungi upon treated and untreated specimens of material. In addition
to causing loss of tensile strength, some fungi cause great damage by
surface growth (mildew). Abrams (1948) has reviewed the techniques
used at the Bureau of Standards for testing mildew- and rotproofing
agents. Aspergillus niger was used to determine mildew resistance, and
the effectiveness of various treatments was evaluated by visual observa-
tion. Chaetomium globosum and a species of Penicillium (USDA 66)
were used in rot-resistance tests. Of some 36 compounds tested, copper
naphthenate and pyridyl mercury compounds were most effective. The
effectiveness of the fungicides varies with the test organisms used. For
data on fungicide evaluation the reader is referred to Abrams (1948).

SUMMARY

The use of microorganisms for analytical purposes is based upon specific
biochemical characteristics of selected test organisms. Within a certain
range of concentration, the response is proportional to the amount of

FUNGI AS TEST ORGANISMS 223

test substance present in the medium. Among the substances for which
quantitative assay procedures have been developed are the vitamins,
amino acids, and essential elements. Microorganisms have also been
used to discover pathways of biochemical synthesis and degradation, to
separate isomers, and for other analytical purposes.

The essential features of a microbiological assay are (1) a suitable test
organism, (2) a suitable basal medium essentially free from the substance
under test, (3) preparation of the sample, (4) a reference standard (a
pure compound where possible), (5) two series of cultures to which a
known range of concentrations of the standard and unknown have been
added, (6) uniform inoculation, (7) incubation under uniform conditions,
(8) measuring the response of the test organisms, (9) construction of a
standard curve, and (10) calculating the results.

When microbiological assay procedures are used, it is unnecessary to
isolate the compound being assayed from the other constituents present
in the sample. The preparation of the sample for assay is usually simple
and ordinarily involves hydrolysis. Microbiological procedures usually
require a short time to complete. The amount of sample needed is small,
which is an important consideration in some problems. Microbiological
assays are invaluable, provided that suitable test organisms are available,
in devising chemical procedures for the isolation of new vitamins and
other physiologically active compounds.

Biochemical mutants of Neurospora and other fungi are particularly
useful in determining the pathways of synthesis of amino acids and other
compounds.

REFERENCES

Abrams, E.: Microbiological deterioration of organic materials, its prevention and
methods of test, Natl. Bur. Standards (U.S.), Misc. Pub. 188, 1948.

Appling, J. W., E. K. Ratcliff, and L. E. Wise: Chemical and microbiological
differentiation of enantiomorphs of galactose and xylose, Anal. Chem. 19 : 496-
497, 1947.

Atkin, L., W. L. Williams, A. S. Schultz, and C. N. Frey: Yeast microbiological
methods for determination of vitamins. Pantothenic acid, Ind. Eng. Chem.,
Anal. Ed. 16: 67-71, 1944.

AuERNHEiMER, A. H., L. J. WicKERHAM, and L. E. Schniepp: Quantitative deter-
mination of hemicellulose constituents by fermentation, Anal. Chem. 20:
876-877, 1948.

Beadle, G. W. : An inositoUess mutant strain of Neurospora and its use in bioassays,
Joiir. Biol. Chem. 156 : 683-689, 1944.

Beadle, G. W. : Biochemical genetics, Chem. Revs. 37 : 15-96, 1945.

Beadle, G. W. : Genetics and metabolism in Neurospora, Physiol. Revs. 25 : 643-663,
1945a.

Bonner, D.: Biochemical mutations in Neurospora, Cold Spring Harbor Symposia
Quant. Biol. 11 : 14-24, 1946.

Brand, E., F. J. Ryan, and E. M. Diskant: Leucine content of proteins and food-
stuffs, Jour. Am. Chem. Soc. 67: 1532-1534, 1945.

224 PHYSIOLOGY OF THE FUNGI

DoERMANN, A. II.: A lysineless mutant of Neurospora and its inhibition by arginine,
Arch. Biochem. 5 : 373-384, 1944.

Dunn, M. S., S. Shankman, M. N. Camien, W. Frankland, and L. B. Rockland:
Investigations of amino acids, peptides, and proteins. XVIII. The amino acid
requirements of Leuconostoc viesenteroides P-60, Jour. Biol. Chem. 156: 703-713,
1944.

FxscHER, E., and J. Hertz: Reduction der Schleimsaure, Ber. d. deut. chem. Ges.
25: 1247-12(51, 1892.
■^Fleming, A.: On the antibacterial action of cultures of a Penicillium, with special
reference to their use in the isolation of B. influenzae, Brit. Jour. Exptl. Path.
10: 226-236, 1929.

Greathouse, G. a., and L. M. Ames: Fabric deterioration by thirteen described
and three new species of Chaetomium, Mycologia 37 : 138-155, 1945.

Greene, R. D., and A. Black: The microbiological assay of trytophane in proteins
and foods. Jour. Biol. Chem. 155 : 1-8, 1944.

Horn, M. J., D. B. Jones, and A. E. Blum: Methods for microbiological and chemi-
cal determinations of essential amino acids in proteins and foods, f7.»S. De-pt.
Agr. Misc. Pub. 696, 1950.
*HoRowiTZ, N. H.: Methionine synthesis in Neurospora. The isolation of cysta-
thionine. Jour. Biol. Chem. 171 : 255-264, 1947.

Horowitz, N. H., and G. W. Beadle : A microbiological method for the determina-
tion of choline by use of a mutant of Neurospora, Jour. Biol. Chem. 150 : 325-333,
1943.

Horsfall, J. G.: Fungicides and Their Action, Chronica Botanica Co., Waltham,
1945.

HuTCHiNGS, B. L., and W. H. Peterson: Amino acid requirements of Lactobacillus
casei, Proc. Soc. Exptl. Biol. Med. 52 : 36-38, 1943.

Kogl, F., and N. Fries: Ueber den Einfluss von Biotin, Aneurin und Meso-Inosit
auf das Wachstum verscheidener Pilzarten, Zeit. physiol. Chem. 249: 93-110,
1937.

KoGii, F., and B. Tonnis: Ueber das Bios-Problem. Darstellung von krystallisier-
tem Biotin aus Eigelb, Zeit. physiol. Chem. 242 : 43-73, 1936.

Krehl, W. a., F. M. Strong, and C. A. Elvehjem: Determination of nicotinic acid.
Modifications in the microbiological method, Ind. Eng. Chem., Anal. Ed., 15:
471-475, 1943.

Leonian, L. H., and V. G. Lilly: The comparative value of different test organisms
in the microbiological assay of B vitamins. West Va. Agr. Expt. Sta. Bull. 319,
1945.

Lilly, V. G., and L. H. Leonian: The anti-biotin effect of desthiobiotin, Science 99 :
205-206, 1944.

LiNDEGREN, C. C, and C. Raut: The effect of the medium on apparent vitamin-
synthesizing deficiencies of microorganisms, Ann. Missouri Botan. Garden 34:
75-84, 1947.

Mulder, E. G.: On the use of micro-organisms in measuring a deficiency of copper,
magnesium and molybdenum in soils. Anionic van Leeuwenhoek 6: 99-109,
1939-1940.

Mulder, E. G. : Importance of molybdenum in the nitrogen metabolism of micro-
organisms and higher plants, Plant and Soil 1 : 94-1 19, 1948.

NiKLAS, H., and O. Toursel: Die Bodenuntersuchung mittels Aspergillus niger,
Bodenkunde und Pflxinzen erndkr. 18 : 79-107, 1940.

Pasteur, L. : Note relative au Penicillium, glaucum et a la dissymetrie moleculaire
des produits organiques naturels, Compt. rend. acad. set. 61 : 298-299, 1860.

FUNGI AS TEST ORGANISMS 225

RoBBiNS, W. J.: Effect of extracts of Phycomyces upon its development, Am. Jour.

Botany 27: 559-564, 1940.
Roberts, E. C, and E. E. Snell: An improved medium for microbiological assays

with Lactobacillus casei, Jour. Biol. Chem. 163 : 499-509, 1946.
RoGOSA, M.: Microbiological method for the quantitative determination of small

quantities of potassium, Jour. Biol. Chem. 154: 307-308, 1944.
*Ryan, F. J. : The germination of conidia from biochemical mutants of Neurospora,

Am. Jour. Botany 35: 497-503, 1948.
Ryan, F. J., and E. Brand: A method for the determination of leucine in protein

hydrolysates and in foodstuffs by the use of a Neurospora mutant, Jour. Biol.

Chem. 154: 161-175, 1944.
ScHOPFER, W. H.: Etude d'un cas de stimulation unilaterale et d'un cas d'inhibition

chez un micro-organisme, Compt. rend. soc. phys. hist. nat. Genhve. 50: 152-154,

1933.
ScHOPFER, W, H.: Recherches sur I'emploi possible d'un test vegetal pour la vitamine

Bi. Essai d'etalonnage. Bull. soc. chim. biol. 17: 1097-1109, 1935.
ScHOPFER, W. H. : Les Tests microbiologiques pour la determination des vitamines,

Experientia 1 : 1-68, 1945.
ScHOPFER, W. H., and M. Guilloud: Un cas de stimulation unilateral chez las

microorganismes explique par une action vitaminique, Verhandl. naturforsch.

Ges. Basel 61: 299-314, 1945.
ScHULTZ, A. S., L. Atkin, and C. N. Frey: Determination of vitamin Bi by yeast

fermentation method. Improvements related to the use of sulfite cleavage and

a new fermentometer, Ind. Eng. Chem., Anal. Ed. 14: 35-39, 1942.
Shankman, S.: Amino acid nutrition of Lactobacillus arabinosus, Jour. Biol. Chem.

150:305-310, 1943.
Smit, J., and E. G. Mulder: Magnesium deficiency as the cause of injury in cereals,

Mededeelingen van de Landbouwhoogeschool, Deel 46, Verhandeling 3, 1942.
Snell, E. E. : The microbiological assay of amino acids. Advances in Protein Chem. 2 :

85-118, 1945.
Snell, E. E. : The vitamin Be group. Evidence for the occurrence of pyridoxamine

and pyridoxal in natural products, Jour. Biol. Chem. 157: 491-505, 1945a.
*Snell, E. E.: Use of microorganisms for assay of vitamins, Physiol. Revs. 28: 255-

282, 1948.
Srb, a. M., and N. H. Horowitz: The ornithine cycle in Neurospora and its genetic

control, Jour. Biol. Chem. 154: 129-139, 1944.
Tatum, E. L., M. G. Ritchey, E. V. Cowdry, and L. F. Wicks: Vitamin content of

mouse epidermis during methylcholanthrene carcinogenesis. I. Biotin, choline,

inositol, p-aminobenzoic acid and pyridoxine, Jour. Biol. Chem. 163 : 675-682,

1946.
Vandeca\teye, S. C. : Biological methods of determining nutrients in soil in Diagnos-
tic Techniques for Soils and Crops (edited by H. B. Kitchen), The American

Potash Institute, Washington, 1948.
* White, W. L., R. T. Darby, G. M. Stechert, and K. Sanderson: Assay of cellu-

lolytic activity of molds isolated from fabrics and related items exposed in the

tropics, Mycologia 40 : 34-84, 1948.
Wright, L. D., E. L. Cresson, H. R. Skeggs, T. R. Wood, R. L. Peck, D. E. Wolf,

and K. Folkers: Biocytin, a naturally-occuring complex of biotin. Jour. Am.

Chem. Soc. 72: 1048, 1950.
Wyss, O., V. G. Lilly, and L. H. Leonian: The effect of pH on the availability of

p-aminobenzoic acid to Neurospora crassa, Science 99: 18-19, 1944.

CHAPTER 11
METABOLITE ANTAGONISTS

This chapter and the one following will deal with chemical compounds
which inhibit, injure, or kill fungi. Much can be learned about "normal"
physiological processes by studying the factors which interfere with them.
The ideas to be discussed here are applicable to the entire field of phys-
iology, and some of our illustrative material will deal with organisms
other than fungi. The reviews of Woolley (1944), Welch (1945), Wright
(1947), Mcllwain (1947), and Roblin (1946, 1949) are extensive and well
documented and should be consulted for additional references.

Metabolites are chemical substances which are essential for the func-
tioning and maintenance of living cells. Metabolites may be synthesized
by the organism or obtained from the medium, e.g., vitamins, amino
acids, etc.

An antimetabolite, or antagonist, is a compound which interferes with
the utilization of a normal metabolite. Wright (1947) has classified
antagonists (more specifically antivitamins) on the basis of their mode of
action: (1) those which act by virtue of destroying or inactivating a
metabolite; (2) those which combine irreversibly with enzymes (non-
competitive inhibition) ; and (3) those which combine with enzymes but
w^hich may be displaced by increased concentration of the normal metabo-
lite (competitive inhibition).

Noncompetitive enzyme inhibition is so called because an increase in
the concentration of the normal coenzyme or metabolite molecules does
not reverse the inhibition. Inhibitors of this type act by combining
with some atom or molecular group of either a coenzyme or an apoenzyme.
Among inhibitors of this type we may list the heavy metals, various
organic mercury and arsenic compounds, iodoacetate, and quinones,
which inactivate enzymes by combining with free sulfhydryl groups (see
Singer, 1945, and McElroy, 1947, for references). Among the inhibitors
which act on the iron-porphyrin enzymes are cyanide, azide, hydrogen
sulfide, and carbon monoxide. Most of the discussion to follow will deal
with competitive antagonists.

Metabolite antagonists are analogues of normal metabolites, but not
all analogues of a metabolite are necessarily antagonists. These "for-
eign" molecules, because of their close resemblance to normal metabolites,
combine with enzymes in the same manner as normal metabolites. How-

226

METABOLITE ANTAGONISTS

227

ever, these foreign molecules are not transformed by the enzyme to which
they are bound. If the antagonist is an analogue of a coenzyme, it
presumably forms a pseudoholoenzyme which is unable to function. The
close structural relation between a metabolite (p-aminobenzoic acid) and
its antagonist (sulfanilamide) is shown in Fig. 46.

6.7 A.

H H

J A !

— ►|2.3A.|-^
p-Aminobenzoate Ion

—►[2.4 A.
Sulfanilamide

Fig. 46. Interatomic distances and structural relationships of p-aminobenzoate ion
and sulfanilamide. (Courtesy of Roblin, Chem. Eng. News 27 : 3624, 1949. Published
by permission of American Chemical Society.)

In spite of the large number of compounds which have been tested for
antagonism, it is not possible to specify exactly what changes in metabolite
molecules are required to produce antagonists. A single modification of a
metabolite molecule is more hkely to produce an antagonist than two or
more changes in structure. This is to be expected, for an antagonist
must closely resemble the corresponding metabolite. Replacing a
carboxyl group with a sulfonic-acid group has been effective in many
instances.

The specific action of enzymes has been likened to the relation of a
lock and its key. Unless an enzyme and a substrate molecule are related
in this fashion, no reaction will take place. A modern diagrammatic
representation of the lock-and-key simile is shown in Fig. 47. The
mechanism of competitive inhibition may be visuahzed by referring to
this figure. Metabolite antagonists may be thought of as "wrong" keys,
which jam the lock mechanism. As long as a false key is in the lock,
it prevents the true key from entering and opening the lock.

Compounds which resemble coenzymes in structure compete for the
active surface of apoenzymes. Because of similarity in structure, an
apoenzyme-foreign molecule complex, or pseudoholoenzyme is formed.
Such a pseudoenzyme is unable to function. The reversal of enzyme
inhibition in such instances is caused by the addition of more coenzyme
molecules. The argument is the same when substrate analogues are
involved. For example, 3-fluorophenylalanine inhibits the utihzation

228

PHYSIOLOGY OF THE FUNGI

of phenylalanine (a normal metabolite) by Neurospora crassa (Mitchell
and Neimann, 1947).

The effect of an antagonist will depend upon the concentration of the
normal metabolite present in the medium and cells and upon the organ-
ism. In general, enzymes have a greater affinity for metabolites than
for antimetabolites. Since both metabolite and antagonist compete
for the same enzyme, the amount of inhibition will depend upon the
relative concentrations rather than upon the absolute amounts of these
compounds present. The amount of an inhibitor required to reduce the

nAP

Substrate

Products
free enzyme

Enzyme -substrate
complex

Fig. 47. A diagrammatic illustration of Fischer's simile that an enzyme and its
substrate are related as are a lock and its key. (Courtesy of McElroy, Quart. Rev.
Biol. 22 : 26, 1947. Published by permission of The Williams & Wilkins Company.)

amount of growth to one-half will depend upon the ratio of inhibitor and
metabolite present. In simple instances, at least, this ratio is equal to a
constant and is called the inhibition constant, or index. The amounts
of sulfadiazine and p-aminobenzoic acid required to reduce the amount
of growth of Streptococcus faecalis R to one-half the normal value gave an
inhibition index of 333 (Lampen and Jones, 1946).

The inhibition index is valid only for the particular conditions used
in an experiment and for the particular strains of the organism used. In
the case of self-sufficient organisms the use of an amount of inhibitor less
than that required for total inhibition will only decrease the rate of
growth, and thus the inhibition index will change with the time of incuba-

METABOLITE ANTAGONISTS 229

tion. This is due to the synthesis of the metabohte by the organism.
Sulfanilamide inhibits the growth of Aspergillus niger, but the fun-
gus overcomes this inhibition as the time of incubation is prolonged
(Hartelius, 194G). The concentration of a metabolite in the control
cultures should be less than the amount which allows maximum growth,
because of the nonlinear response of an organism to the metabolite at
high concentrations.

The composition of the medium is an important consideration in any
investigation of metabolite antagonism. If adequate amounts of a
natural metabolite are present, the action of an inhibitor may be over-
looked. Synthetic media should be used. The composition of the
medium used may also affect the action of an inhibitor in another way.
If metabolite A is transformed into metabolite B by an organism, the
presence of metabolite B in sufficient amount for optimum growth may be
expected to nullify any amount of an antagonist for metabolite A. An
antagonist of metabolite B, however, would exhibit normal competitive
inhibition, Shive and Macow (1946) have pointed out that, by the use
of a suitable series of inhibitors, it is possible to follow the transformations
of a given metabolite step by step. These authors designate this use of
metabolite antagonists as inhibition analysis. Rydon (1948) found
Bacterium typhosum to synthesize tiyptophane by the following steps:
anthranilic acid — > indole — > tryptophane. The 2- and 4-methylanthran-
ihc acids were potent inhibitors against anthranilic acid but not against
indole or tryptophane. Certain analogues of indole and tryptophane
were inhibitors of these metabolites.

In discussing metabolite antagonists in a general way, it should be
borne in mind that these compounds may inhibit only certain organisms,
or a particular organism only under certain conditions. For example,
desthiobiotin is a biotin antagonist for Ceratostomella pini and Lacto-
hacillus casei, while this compound replaces biotin for many strains of
Saccharomyces cerevisiae (Lilly and Leonian, 1944). Woolley (1944,
1946) is of the opinion that the established facts of inhibition and reversal
are more important than the hypotheses which are adopted to explain
these phenomena. However, the concept of competitive metabohte
antagonism has been very useful in correlating a vast amount of experi-
mental work in apparently unrelated fields.

ANTIVITAMINS

Antivitamins are known for all the water-soluble vitamins which have
been synthesized and for at least one of the fat-soluble vitamins (vitamin
K).

p-Aminobenzoic acid antagonists. When the sulfonamides were intro-
duced into medicine, it was quickly found that serum and other natural

230

PHYSIOLOGY OF THE FUNGI

products antagonized the inhibitive action of sulfanilamide on the growth
of certain bacteria. Rubbo and Gillespie (1940) discovered that p-amino-
benzoic acid was a growth factor for certain bacteria. Woods (1940)
found that p-aminobenzoic acid in low concentration overcame sulfanila-
mide inhibition. A general theory was proposed by Fildes (1940) to
explain the antagonism between metabolites and compounds having
closely related structures.

1400

8 10 12

Doys of incubotion

Fig. 48. The effect of various concentrations of sulfanilamide (amounts per flask)
upon the time of spore germination and upon the rate and amovnt of growth of
Aspergillus niger in flasks containing 55 ml. of sucrose— ammonium sulfate medium at
32°C. (Drawn from the data of Hartelius, Compt. rend. trav. lab. Carlsberg, S^r.
physiol. 24: 181, 1946.)

Sulfanilamide was first considered to be antagonized by p-amino-
benzoic acid, rather than the reverse. This was due to the discovery
of the therapeutic value of sulfanilamide before it was known that
p-aminobenzoic acid was a vitamin. The structural relation between
these compounds has already been noted. The literature dealing with
the sulfonamides is abundant, but most of it relates to bacteria and
medicine. Relatively few papers have been published on the effects
of these compounds on the growth of fungi.

Hartelius (1946) investigated the effect of sulfanilamide upon the
growth of Aspergillus niger and found that the amount of inhibition was
dependent upon the amount of inoculum used, the concentration of
sulfanilamide in the medium, and the time of incubation. The curves
in Fig. 48 illustrate the effect of time of incubation on inhibition, a factor
which is too often overlooked in experiments of this kind. The curves

METABOLITE ANTAGONISTS 231

in Fig. 48 indicate that A. niger synthesizes either p-aminobenzoic acid
or some other compound which reverses the inhibitory action of sulfa-
nilamide. When p-aminobenzoic acid was added to the medium, sulfa-
nilamide no longer inhibited the growth of yl. niger (Hartelius and Roholt,
1946). Other fungi have been shown to react like A. niger when cultured
in media containing sulfanilamide (Fourneau et al., 1936).

It has been assumed that self-sufficient fungi require the same vitamins
as the deficient species. The synthesis of a vitamin may suggest its
need but does not demonstrate it. Antivitamins (or other antimeta-
bolites) provide a way of demonstrating the need of self-sufficient fungi
for the vitamins they synthesize. Thus A. niger requires p-aminobenzoic
acid just as Rhodotorula aurantica does, but this need can be demonstrated
only in the presence of a specific reversible inhibitor such as sulfanilamide.
This technique offers a possible way of discovering new vitamins and
other metabolites. If a compound inhibits growth, it is worth while to
search for compounds which overcome this inhibition reversibly.

For most purposes sulfanilamide has been replaced by other sulfona-
mides. However, sulfanilamide appears to be the most active sulfona-
mide against fungi. For a review of the clinical aspects of the sulfona-
mides in mycoses and for literature citations, see Wolf (1947).

Stoddard (1947) has reported the sulfonamides to be of some value in
controlling the X disease of peach (a virus). Addition of p-amino-
benzoic acid lessened the effectiveness of the treatment.

It is recognized that the simple Woods-Fildes theory of competitive
inhibition is inadequate to explain completely the mechanism of sulfona-
mide therapy. In vivo the environment is much more complex than in
simple laboratory media. For further information and references to the
literature, see Sevag et al. (1945) and Mudd (1945).

Thiamine antagonists. Thiamine may be inactivated by an enzyme,
thiaminase, which is found in fish viscera (Sealock et al., 1943) and prob-
ably occurs in other organisms. Foxes which are fed raw fish may
develop a thiamine-deficiency disease (Chastek paralysis). The mode of
inactivation was further investigated by Krampitz and Woolley (1944),
who found that thiamine was destroyed by a process of enzymatic hydrol-
ysis whereby the thiazole and pyrimidine moieties were formed. Mucor
ramanniamis (thiazole-deficient) and Endomyces vernalis (pyrimidine-
deficient) were used as test organisms in the preliminary work. Another
thiamine antagonist of unknown nature has been reported to occur in
bracken fern (Weswig et al., 1946).

Pyrithiamine, an analogue of thiamine, has been used in studies of
competitive thiamine inhibition. Unfortunately, the exact structure of
this compound is not known. In papers published before 1949 it was
assumed that pyrithiamine had the structure now assigned to neopyri-

232

PHYHIOLOGY OF THE FUNGI

thiamine (Wilson and Harris, 1949). Pyrithiamine appears to differ
from neopyrithiamine in the amount of pyrimidine moiety it contains.
The formula for neopyrithiamine is given below.

N=C— NH2-HBr CH3 CH2— CH2— OH

CHs — C C — CHj

N— CH Br

Neopyrithiamine

Robbins (1941) found low concentrations of pyrithiamine to replace
thiamine for Pythiomorpha gonapodyoides (pyrimidine-deficient), while
high concentrations inhibited growth. Pyrithiamine did not replace
thiamine for Phycomyces blakesleeanus (requires both moieties) or Phyto-
phthora cinnamomi (requires intact thiamine). The inhibition of growth
of various fungi and bacteria caused by pyrithiamine was overcome by
increasing the thiamine content of the medium (Woolley and White,
1943). The inhibition index is given in Table 39. The efficiency of
pyrithiamine as a thiamine antagonist is related to the specific vitamin
requirements of the organisms tested. The inhibition index was low for
those species which require intact thiamine, intermediate for those which
require either or both moieties, and high for self-sufficient species.

Table 39.

The Efficiency of Pyrithiamine as an Inhibitor of Fungus and
Bacterial Growth
(Woolley and White, Jour. Exptl. Med. 78, 1943.)

Organism

Inhibition index

pyrithiamine

thiamine

Thiamine
requirement

Ceratastomella fimbriata

C. penicillata

Phytophthora cinnamomi

130

800

400,000

40,000

5,000,000

Intact thiamine
Intact thiamine
Intact thaimine

Chalaropsis thielavioides

Intact thaimine

Fndomyces vernalis

Pyrimidine

Mucor ramannianus

Thiazole

Saccharomyces cerevisiae

Neurospora crassa

Lactohacillus arabinosus

L. casei

Both moieties
None
None
None

Pyrithiamine was found to inhibit sporulation of Ceratostomella fimbri-
ata, Choanephora cucurhitarum, and Chaetomium convolutum (Lilly and
Barnett, 1948). This inhibition was overcome by thiamine. Pyrithia-
mine was reported to be a more efficient antagonist for diphosphothia-
mine than for thiamine when Penicillium digitatum was used as a test
organism (Sarett and Cheldelin, 1944).

METABOLITE ANTAGONISTS 233

Pyrithiamine causes a thiamine deficiency disease in mice, which may
be cured or prevented by the administration of sufficient thiamine (Wool-
ley and White, 1943). Neopyrithiamine is reported to be four times as
active as pyrithiamine for the rat (Wilson and Harris, 1949).

Biotin antagonists. Many biotin vitamers are known which are highly
specific. The efficiency of an antibiotin in some instances may depend
upon whether biotin or one of its vitamers is the competing metabolite.
The formulas of two of the compounds are given below. Compare with
the formulas of biotin and desthiobiotin given in Chap. 9.

CO CO

/ \ / \

HN NH HN NH

CHs— CH Cn(CH.2)5— SO3H H2C CH(CH2)5— COOH

Sulfonic-acid analogue of desthiobiotin Imidazolidonecaproic acid

Desthiobiotin and imidazolidonecaproic acid differ only by a methyl
group. Desthiobiotin was found to act as a biotin vitamer for Saccha,
romyces cerevisiae and other yeasts (Dittmer et al., 1944; Lilly andLeonian-
1944), while imidazolidonecaproic acid is an antibiotin for S. cerevisiae
(Dittmer and Du Vigneaud, 1944). Both compounds are antibiotins
for Lactobacillus casei. The sulfonic-acid analogue of desthiobiotin was
shown by Duschinsky and Rubin (1948) to be more active against desthio-
biotin and oxybiotin than against biotin for S. cerevisiae. The replace-
ment of a carboxyl group by a sulfonic-acid group appears to be a rather
general method of changing a metabolite into an antagonist. Further
examples of this will be cited in connection with pantothenic and amino-
acid antagonists.

Egg white contains a specific protein which combines with biotin and
thus renders this vitamin inactive. This inactivity is due to the molecu-
lar size of the avidin-biotin complex, which prevents its absorption by
organisms. Raw egg white may be used to produce experimental biotin
deficiency in animals. Avidin is no longer active after heating, and
bound biotin is released by this treatment. This specific protein has
been used to separate biotin vitamers into two groups, for avidin com-
bines only with those compounds w^hich have an intact urea ring structure.
The papers of Eakin et al. (1941) and Burk and Winzler (1943) may be
consulted for further details.

Pantothenic acid antagonists. Yeasts are the only fungi which have
been reported to be deficient for pantothenic acid, and in most instances
/3-alanine replaces the intact vitamin molecule. One of the commonly
studied pantothenic acid antagonists is the compound called pantoyl-
taurine. The formulas for pantothenic acid and pantoyltaurine are
given belo'rt

234

PHYSIOLOGY OF THE FUNGI

HO— CH2— C(CH3)2-
HO— CH2— C(CH3)2-

CHOH— CO— NH-

Pantothenic acid

-CHOH— CO— NH-

Pantoyltaurine

CH2— CHo— COOH

-CH2— CH2— SO3H

Pantoyltaurine is the sulfonic-acid analogue of pantothenic acid.
Snell (1941) studied the competitive inhibition of yeast growth by pan-
toyltaurine and found that this compound was effective when pantothenic
acid was the metabolite supplied in the medium but that pantoyltaurine
did not compete with /3-alanine. The data in Table 40 illustrate this
difference.

Table 40. The Effect of Pantoyltaurine on the Growth of Saccharomyces
cerevisiae in the Presence of Pantothenic Acid and (3-Alanine
Inoculum used, 0.02 mg., time of incubation, 16 hr. (Snell, Jour. Biol. Chem. 141,
1941.)

Calcium

pantothenate,

yug/lO ml.

Sodium salt
of pantoyl-
taurine,
Mg/10 ml.

Moist

cells,

mg/10 ml.

/3- Alanine,
Mg/10 ml.

Sodium salt
of pantoyl-
taurine,
Mg/10 ml.

Moist

cells,

mg./lO ml.

0.0

0.3

0.0

0.03

0.5

6.6

0.3

2.8

0.5

1,000

2.9

0.5

5.5

0.5

5,000

0.4

0.3

1,000

3.0

0.5

10,000

0.3

10,000

3.0

30.0

10,000

6.6

0.5

5,000

5.5

0.5

10,000

5.7

The synthesis of pantothenic acid via /3-alanine by Escherichia colt
is inhibited by cysteic acid (sulfonic-acid analogue of aspartic acid).
This inhibition is reversed by /3-alanine or pantothenic acid (Ravel and
Shive, 1946). For further information concerning other pantothenic
acid and other antagonists, the review of Roblin (1946) should be
consulted.

Pyridoxine antagonists. Some of the pyridoxine analogues studied
by Robbins and Ma (1942) inhibited the growth of Ceratostomella ulmi.
This inhibition was reversed by additional pyridoxine. The substitution
of an ethyl group for the methyl group of pyridoxine produced an antag-
onist for C. ulmi, but ethyl pyridoxine was as active for excised tomato
roots as pyridoxine itself. The above authors suggest that ethyl pyri-
doxine might be a chemotherapeutic agent for the Dutch elm disease.
The formulas of ethyl pyridoxine and desoxypyridoxine are given below :

HO-

C2H2-

/^-CHoOH

-CH2OH

HO-

/%— CH2OH

\n^

CH3—

Ethyl pyridoxine

Desoxypyridoxine

METABOLITE ANTAGONISTS

235

Martin et at. (1948) found desoxypyridoxine to be slightly more effec-
tive against pyridoxal than pyridoxine, when Saccharomyces cerevisiae
was used.

Vitamin K antagonists. There are at least two naturally occurring
compounds which have vitamin K activity. Certain synthetic analogues
are used in medicine to replace the natural vitamins. All these com-
pounds are substituted 1,4-naphthoquinones. The structural formula
for vitamin K2 is given below:

-CHs CH3 CH3

I I

-CHo— (CH=C— CH2— CH2)6— CH=C— CH3

Vitamin K2

Horsfall (1945) has reported 2-methyl-l,4-naphthoquinone to be a weak
fungicide, although this compound replaces natural vitamin K in medi-
cine. On the other hand, 2,3-dichloro-l,4-naphthoquinone (Phygon) is
a potent fungicide (Ter Horst and Felix, 1943).

— CHa

O
2-Methy 1- 1 , 4-naphtho quinone

—CI
—CI

2 , 3-Dichloro- 1 , 4-naphtho quinone

Phygon may act as a fungicide by virtue of combination of the quinone
with free amine or sulfhydryl groups. This mechanism probably inac-
tivates certain enzymes noncompetitively. On the other hand, Phygon
is structurally related to vitamin K, and a competitive type of inhibition
should also be possible. Woolley (1945) investigated the inhibitory effect
of 2,3-dichloro-l,4-naphthoquinone and 2-methyl-l,4-naphthoquinone on
the growth of Saccharomyces cerevisiae and Endomyces vernalis. The first
compound was more toxic than the second. In less than toxic concen-
trations, the second compound partially overcame the toxicity of the
first. The amount of 2,3-dichloro-l,4-naphthoquinone required to
inhibit yeast (half maximum growth) was 1.7 jug per liter, while 230 ng
of 2-methyl-l,4-naphthoquinone were required to produce the same
amount of inhibition. Some of Woolley 's data are presented in Table 41.
Many potent antimalarial drugs are 1,4-naphthoquinone derivatives
(Fieser et al., 1948).

It has been assumed in our discussion of the effects of antagonists on

236 PHYSIOLOGY OF THE FUNGI

organisms that antimetabolites are active by virtue of interfering with
various enzymatic processes. It is also interesting to note that com-
petitive inhibition has been demonstrated with isolated enzyme systems,
Schopfer and Grob (1949) found the action of urease to be inhibited by
2-chloro-l,4-naphthoquinone. Most of the activity was restored by the
addition of 2-methyl-l,4-naphthoquinone (vitamin K3).

Table 41. The Reversal of Inhibition Caused by 2,3-Dichloro-1,4-naphtho-
QuiNONE by 2-Methyl-1,4-naphthoquinone
Test fungus, Saccharomyces cerevisiae. Concentration of 2,3-dichloro-l,4-naphtho-
quinone, 0.005 jug/ml. (Woolley, Proc. Soc. Exptl. Biol. Med. 60, 1945. Published
by permission of the Society for Experimental Biology and Medicine.)

2-methyl-l,4- Turbidity
naphthoquinone, ^g/ml. (100 = no growth)

0.0 93

0.04 60

0.02 68

0.01 77

0.005 85

Other vitamin antagonists. The sulfonic-acid analogue of nicotinic
acid inhibits the growth of certain bacteria (Mcllwain, 1940). Appar-
ently this analogue has not been tested in nicotinic acid-deficient fungi.
Woolley (194Ga) has reported maize to contain a "pellagragenic" agent
which may tentatively be considered as a naturally occurring anti-
nicotinic-acid factor.

Among the recently developed insecticides, 7-hexachlorocyclohexane
is of considerable value. Kirkwood and Phillips (1946) have shown that
the growth of Saccharomyces cerevisiae is inhibited by this compound, and
that the inhibition is overcome by meso-inositol. The other isomers of
hexachlorocyclohexane were not very effective inhibitors of yeast growth ;
neither are they of much value as insecticides. These observations point
to competitive inhibition as a possible mechanism of insecticidal action
of this compound.

AMINO-ACID ANTAGONISTS

Organisms must either synthesize or obtain from exogenous sources
the different amino acids they require for the synthesis of protein. Anti-
metabolites which antagonize the synthesis or utilization of essential
amino acids would have a profound effect upon growth or other functions
of organisms. The role of amino acids is not confined to the synthesis
of proteins but extends to the synthesis of other essential metabolites.
An amino-acid antagonist may act in two ways, (1) by inhibiting protein
synthesis and (2) by inhibiting the synthesis of essential metabolites
which are derived from amino acids, either directly or indirectly. If
an amino a^id functions in more than one way, the action of an amino-

METABOLITE ANTAGONISTS

237

acid antagonist may be overcome, at least in part, by the action of second-
ary metabolites as well as the primary metabolite. The toxic effect of
3-acetylpyridine on rats is reversed by either nicotinic acid amide or
tryptophane (Woolley, 1945a).

Analogues. Mitchell and Niemann (1947) found that the halogenated
derivatives of phenylalanine and tyrosine competitively inhibit growth
of the wild strain of Neurospora crassa (Table 42). The most effective
of these inhibitors was 3-fiuoro-DL-phenylalanine. The structural form-
ulas for this analogue and the natural metabolite are shown below:

^^-CHo— CHNH2— COOH

CHo— CHNH2— COOH

Phenylalanine

3-Fluorophenylalanine

Table 42. Inhibition of Growth of Neurospora crassa by Some Halogenated

Alpha-amino Acids
Basal medium contained 30 mg. of DL-phenylalanine or 20 mg. L-tyrosine per liter
depending upon the antagonist tested. (Mitchell and Niemann, Jour. Am. Chem.
Sac. 69, 1947. Published by permission of the American Chemical Society.)

Compound

Mg./ml. for 50%
inhibition

Moles inhibitor
Moles amino acid

3-Fluoro-DL-phenylalanine

3-Fluoro-DL-tyrosine

3-Fluoro-L-tyrosine

3-Fluoro-D-tyrosine

0.04
0.23
0.15
0.41

1.2

10.5

6.8

18.5

The other halogen derivitives (chloro, bromo, and iodo) were less effective
inhibitors. 3-Fluorophenylalanine was shown to be an effective inhibitor
for various other fungi and bacteria.

The effect of j8-2-thienylalanine on the growth of a strain Saccharomyces
cerevisiae and certain bacteria has been studied by Ferger and Du
Vigneaud (1948). The formula for this thiophene analogue of phenyl-
alanine is given below:

HC C— CHo— CHNH2— COOH

i3-2-Thienylalanine

Only the l isomer is active in competing with phenylalanine. The replace-
ment of divalent sulfur ( — S — ) by a vinylene group ( — CH=CH — ), or
vice versa, often leads to the production of an antimetabolite. As
another example, the effect of replacing sulfur in cysteine by radicals

238

PHYSIOLOGY OF THE FUNGI

containing the vinylene group may be cited. Dittmer et al. (1948) found
methallylglycine, allylglycine, and crotylglycine to inhibit the growth of
Saccharomyces cerevisiae and Escherichia coli. The effects of these three
antimetabohtes on the growth of yeast are shown in Fig. 49.

50 100 150 200 250 300

Micrograms of unsoturoted amino acids per 7.5 ml.

Fig. 49. The inhibition of growth of Saccharomyces cerevisiae, strain 139, by DL-allyl-
glycine, DL-methallylglycine, and DL-crotylglycine. (Courtesy of Dittmer, Goering,
Goodman, and Cristol, Jour. Am. Chem. Soc. 70: 2501, 1948. Published by permis-
sion of the American Chemical Society.)

Natural amino acids. Antagonism among the amino acids is not
limited to competitive inhibition between naturally occurring amino
acids and their analogues. Robbins and McVeigh (1946) found hydroxy-
proline to inhibit the growth of several dermatophytes: Tricho-phyton
mentagrophytes, T. gypseum (granular form), T. purpureum, Epidermo-
phyton fiocculosum, and Microsporum canis. This inhibition was over-
come by proline. The relationship of these two naturally occurring
amino acids is shown below:

H2C-
H.C

-CH2
CH— COOH

HOHC-

H2C

-CHo

in-

COOH

H
Proline

H
Hyd ro xy proline

Low concentrations of hydroxyproline stimulated growth of Tricho-
phyton purpureum, while higher concentrations inhibited growth. Addi-
tion of hydroxyproline to a glucose-asparagine medium increased the
growth of Polyporus squamosiis. Hydroxyproline was without effect
on the growth of 19 other species of fungi. Whether amino-acid antago-
nisms may limit the nitrogen utilization of natural mixtures of these

METABOLITE ANTAGONISTS 239

compounds is unknown, but the possibility of inhibition should be kept
in mind when only a few amino acids are used in a medium. The effect
of any single compound upon a fungus may be modified by the other
constituents of the medium.

Harteiius (1946a) found glutamic and aspartic acids, glutamine, and
asparagine to inhibit the growth of a strain of yeast when suboptimumal
amounts of /^-alanine were used in the medium. These amino acids did
not inhibit growth when pantothenic acid was used. In fact, these com-
pounds stimulated growth under these conditions. The inhibitory effect
in the presence of /3-alanine was overcome by increasing the concentration
of this provitamin. To obtain maximum growth in the presence of 50
mg. of glutamic acid per flask (55 ml.), twenty times as much j9-alanine
was required as when glutamic acid was omitted from the medium.
Harteiius attributed this effect to the combination of /3-alanine and
glutamic acid to form an inactive dipeptide.

Among the naturally occurring amino acids, L-canavanine is found
free in jack beans. Canavanine is an analogue of arginine; the formulas
are shown below.

HjN— C(=NH)— NH— CH2— CH2— CH,— CH(NH2)— COOH

Arginine

H2N— C(=NH)— NH— O— CH2— CH2— CHCNH,)— COOH

Canavanine

Horowitz and Srb (1948) studied the effect of canavanine on three
wild-type strains of Neurospora and found one strain to be inhibited
completely by concentrations greater than 1.25 mg. per liter; another
strain was only partially inhibited, while the third strain was tolerant.
Genetic analysis indicated that tolerance and susceptibility segregated
by alternative forms of a single gene. L-Arginine was effective in over-
coming canavanine toxicity, while L-lysine was less effective. Three
molecules of arginine overcame one molecule of canavanine in the strain
of Neurospora most sensitive to this inhibitor. A similar competitive
inhibition between canavanine and arginine in various bacteria has also
been observed (Volcani and Snell, 1948).

Other metabolite antagonists. Woolley (1944a) found benzimidazole
to inhibit the growth of Saccharomyces cerevisiae and Endomyces vernalis.
This inhibition was overcome by adenine and guanine. The structural
relationship between benzimidazole and adenine is shown below:

N=C— NH2

HC C— NH

\
CH

— C— N
Benzimidazole Adenine

240 PHYSIOLOGY OF THE FUNGI

DEVELOPMENT OF FASTNESS

An organism which has become tolerant, or resistant, to an inhibitor
(analogue, drug, antibiotic, etc.) after exposure is said to be fast, or more
specifically pyrithiamine-fast, sulfanilamide-fast, or penicillin-fast, as
the case may be. Fastness is a very common phenomenon, although it
appears to have been but little studied in fungi. It is an important factor
which limits the use of many antibiotics and the sulfonamides in medicine.
This phase of fungus physiology deserves more attention than it has
received. It is conceivable that the prolonged use of a single fungicide
to control a fungus pathogen could lead to the development, or selection,
of a strain which would be relatively tolerant to the effect of the fungicide.
Such findings do not appear to have been reported from field studies, but
this possibility should be kept in mind.

Fungi do become fast to various antagonists. WooUey (19446), by
repeatedly subculturing Endomyces vernalis in a medium containing
pyrithiamine, developed a strain which withstood twenty-five times the
concentration of pyrithiamine which served to reduce the growth of
the parent strain to half the maximum. In this instance, fastness was
correlated with the ability of the pyrithiamine-fast strain to cleave the
inhibitor molecule into its cyclic moieties. Thus, the development of
pyrithiamine fastness may be ascribed to the formation of an adaptive
enzyme which destroyed the antagonist. Escherichia coU, which is not
inhibited by pyrithiamine, also hydrolyzed this compound. These
results indicate that adaptive enzymes may play a role in the develop-
ment of fastness.

In addition to resistance or fastness which develops in organisms cul-
tured in the presence of an inhibitor, it has been found recently that
various bacteria not only develop resistance but may develop strains
which are actually dependent upon the presence of the "inhibitor" before
they can grow. Yegian et al. (1949) have found that culturing Myco-
bacterium tuberculosis in the presence of streptomycin gave rise to strains
which were fast to this antibiotic and also produced strains which cannot
grow unless streptomycin is present in the medium.

SUMMARY

The normal utilization of a metabolite may be prevented or inhibited
in three ways: (1) destruction or removal in an unavailable combination
of a metabolite; the enzymatic hydrolysis of thiamine and the combina-
tion of biotin with avidin are representative examples of this mode of
inactivation ; (2) the noncompetitive inhibition of various enzymes by
such compounds as iodoacetate, cyanide, and azide; (3) competitive
inhibition due to metabolite antagonists. This type of inhibition is

METABOLITE ANTAGONISTS 241

overcome by increasing the concentration of the normal metabohte.
Antagonists are known which inhibit the functioning of vitamins, amino
acids, and other metabohtes.

It is postulated that a metabolite and its antagonists compete for the
active surface of specific enzymes. The ratio of inhibitor to metabolite
required to reduce growth to one-half its normal value is called the inhibi-
tion index. Effective inhibitors have small inhibition indexes. The
same compound may act as an antagonist for some fungi and as a metabo-
lite for others; e.g., desthiobiotin. The medium used for investigating
inhibition is important, for the presence of a normal metabolite, or a
secondary metabolite derived from it, may prevent inhibition, A given
compound may be considered as an antagonist, but it is only an antagonist
for certain species, and then only under certain conditions. Organisms
may acquire a tolerance or resistance to inhibitory agents and become
fast. In extreme instances they become dependent upon the inhibitor,
which then acts as a kind of growth factor.

The competitive nature of many inhibitions is firmly established. In
most instances there is a close structural relation between a metabolite
and its antagonists. The theories which have been advanced to explain
these phenomena have been useful in correlating the results of research
and for increasing our insight into metabolic processes,

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242 PHYSIOLOGY OF THE FUNGI

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CHAPTER 12
THE ACTION OF FUNGICIDES

The never-ending warfare which man must wage against parasitic fungi
in order to protect his crops has been ably chronicled by Large (1940).
The saprophytic species which decay wood and other cellulosic materials
cause great economic loss, although these species perform a necessary
and indispensable role in maintaining the carbon cycle in nature. It is to
man's interest and profit that the deterioration of textiles and lumber be
prevented or delayed and that his crops be protected from pathogenic
fungi. This is done by the use of fungicides, which either kill or inhibit
the action of fungi.

By definition, an agent which kills fungi is a fungicide. A fungistatic
agent merely causes inhibition. The same agent is commonly capable of
producing both actions. A discussion of the terms fungicidal and fungi-
static is given by McCallan and Wellman (1942). These authors point
out that the fungistatic activity of an agent is broader than its fungicidal
activity.

Both physical and chemical agents may be fungicidal and fungistatic.
Of the physical agents, heat and ultraviolet radiation are probably most
commonly used, while many chemical compounds are ''toxic" to fungi.
Whether an agent is fungicidal or fungistatic is primarily a matter of
degree of intensity and duration of exposure. We may assume that these
agents, whether chemical or physical, act directly upon certain specific
enzymes or enzyme systems. If the action is less severe and may be
reversed, the result is fungistasis, while if it is irreversible, the action is
fungicidal. Since most of the agents employed by man are chemical
compounds, much of the following discussion will be limited to the
mechanism of action of these compounds.

Chemical fungicides may be applied as eradicants or as protectants. A
protectant is applied to the plant or other material before the inoculum
arrives at the infection court and often functions only after the fungus
spore germinates. An eradieant kills the fungus already present on or in
the substrate material.

The lethal action of a chemical depends upon both the concentration
of the active compound or ion and the time of exposure. Species of
fungi exhibit great variation in ability to resist the action of certain
fungicides. Many fungi are killed by exposure to a few parts per million

245

246 PHYSIOLOGY OF THE FUNGI

of cupric ion, while a few species have been reported to grow in a saturated
solution of copper sulfate (Starkey and Waksman, 1943). There is no
useful universal fungicide.

The intelligent choice of a fungicide depends upon a number of factors,
the major ones being the species of fungus to be controlled and the nature
of the material to be protected. The solubility of the fungicide is of great
importance. For most efficient preservation of wood or protection as a
spray, a fungicide must have a low solubility in order that the protection
may extend over a long period of time. For surface sterilization a highly
soluble fungicide is used. When a fungicide is to be used on a living
plant (or other organism), the relative sensitivity of the host and of the
fungus to the fungicide must be considered. Host sensitivity limits the
use of many potent fungicides. A useful fungicide must be more toxic
to the fungus than to the host. For example, copper fungicides are quite
toxic to cabbage, cucumber, and pea seed, while beet, eggplant, pepper,
and spinach seed are relatively tolerant to copper.

Although there is an enormous accumulation of literature on fungicides,
their composition, application, limitations, and economic value (see
Frear, 1948, and Horsfall, 1945), relatively little has been published on
the mechanism of fungicidal action. This is a practical as well as an
academic question, for the intelligent use of known fungicides and the
search for new and better ones are based upon a knowledge of how they
act.

In the past the most important inorganic fungicides have contained
compounds of copper, mercury, or sulfur. In the future, however, excel-
lent fungicides may be made from other toxic elements. For example,
cadmium is of potential interest, but the present supply is limited. In
controlling fungi and other pests, there is always the danger that they
will become tolerant, or fast, to a given toxicant. This means that the
more susceptible individuals are killed and that a greater amount of a
given fungicide is required to control the more tolerant population which
is then built up. It is desirable from several viewpoints to have satis-
factory reserve fungicides in the armory of the plant pathologist.

COPPER

The first copper salts to be used as fungicides were the sulfate and
acetate (Prevost, 1807). These salts are soluble, and even in low con-
centration they are too toxic for many uses. Since all the copper is
available at once, these salts are toxic to plants, especially to young parts.
These soluble salts have a further disadvantage when used as a spray, for
a heavy dew or rain will easily wash them off. However, these salts,
especially copper sulfate, were successfully used for treating seed grain to
destroy surface contaminants. This treatment was devised by Prevost
to control bunt.

ACTION OF FUNGIDICES

247

The next advance in copper fungicides was not until 1885, whei
Millardet published the formula for making the famous fungicide
Bordeaux mixture. Millardet recommended that 8 kg. of copper sulfate
pentahydrate (bluestone) be dissolved in 100 liters of water. This solu-
tion was then mixed with 15 kg. of quicklime slaked in 30 liters of water.
The chemistry of Bordeaux mixture is more complicated than was
assumed at first. Instead of cupric hydroxide, a series of basic sulfates
are formed, the composition being dependent upon the ratio of coppei
sulfate and calcium hydroxide used (Frear, 1948). Bordeaux mixture is
a copper compound or compounds of low solubility. According to
Goldsworthy and Green (1936), Bordeaux mixture in equilibrium with
water yields a solution containing about 4 p. p.m. of copper. However,
McCallan and Wilcoxon (1936) found that well-washed 4-4-50 Bordeaux
mixture was soluble only to the extent of furnishing 1 p. p.m. of copper.
After this material was thoroughly dried, as in a spray film, the solubihty
in terms of copper decreased to 0.2 to 0.3 p. p.m. McCallan and Wilcoxon
have reported a comparison between the amounts of Bordeaux mixture
and copper sulfate required to inhibit the germination of 90 per cent of
the spores of a few species. These data are given in Table 43.

Table 43. The Relative Efficiency of Bordeaux Mixture and Copper Sulfate

IN Inhibiting Spore Germination
(McCallan and Wilcoxon, Contribs. Boyce Thompsori Inst. 6, 1936.)

Cu, mg. /liter, for LD 90

Species

Bordeaux
mixture

Copper

sulfate

Uromyces caryophyllinus

Sclerotinia fructicola

180
120
390
500
2,400

1.74
1 20

Botridis paeoniae

2 23

Glomerella cingulata

Alternaria solani

1.40
6.72

If Bordeaux mixture or other copper spray or dust of low solubility
furnishes less than 1 p. p.m. of copper to the solutions with which it is in
equilibrium, it is obvious that the concentration of copper is too low for
any great amount of toxicity. We must also take into account the rate
^ of solubility of the ''insoluble" copper compounds, for if the rate of
solution is slow, the maximum concentration may not be attained in
time to prevent infection. The only hypothesis which would account
for the lethal action of copper compounds of such low solubility would be
that of cumulative action. A germinating spore in a saturated solution
of the copper compound in equilibrium with the solid copper compound
would remove copper from the solution. This process would cause more

248 PHYSIOLOGY OF THE FUNGI

of the copper compound to dissolve until the spore was no longer able to
take more copper from the solution. This theory is attractive because
of its simplicity, but there seems to be no very good evidence for it
(McCallan, 1929).

In practice, Bordeaux mixture and other "insoluble" copper sprays
act as if they were more soluble than is indicated by chemical tests.
However, in practice the fungicide is exposed to the action of the atmos-
phere, the host plant, and the fungus spores. This is a more complicated
situation than that found in the chemical determination of solubility.
Barker and Gimingham (1911) found that intact leaves increased the
soluble copper from Bordeaux mixture to some extent but were of the
opinion that the host plant had only a slight influence on the solubility
of such sprays. However, if the leaves were injured, they were quite
effective in bringing copper into solution. The possibility that the spores
exert a solvent action on "insoluble" copper compounds has long been
considered by plant pathologists. The spores of at least some species
do exert a solvent action on Bordeaux mixture. McCallan and Wilcoxon
(1936) showed that the amount of copper brought into solution by the
soluble materials washed from or excreted by 100 million spores of some
species varied as follows: Uromyces caryophyllimis, 1.01 mg.; Sclerotinia
fructicola, 0.76 mg.; Neurospora sitophila, 0.12 mg.; Botrytis paeoniae,
0.10 mg.; Glomerella cingulata, 0.046 mg. ; Aspergillus niger, 0.023 mg.;
and Alternaria solani, 0.013 mg. Enough spores of Neurospora sitophila
were collected so that the nature of the soluble materials from the spores
could be identified chemically. Malic acid was isolated and identified.
The presence of amino acids also was detected. Both malic acid (or
malates) and various amino acids dissolve "insoluble" copper compounds
under nevitral or alkaline conditions with the formation of soluble complex
copper compounds. McCallan and Wilcoxon showed that sodium
cuprimalate and a copper-glycine compound were about as toxic as copper
sulfate. On the other hand, Goldsworthy and Green (1936) were of the
opinion that spore secretions played a minor role in increasing the solu-
bility of Bordeaux mixture, but the evidence of McCallan and Wilcoxon
seems quite conclusive.

Basic copper carbonate (malachite, Cu(OH)2-CuC03) and cuprous
oxide (CuiO) are used in treating seeds. Since the seed covered with
these materials is planted in soil which contains a variety of protein
degradation products, it is easy to understand how these substances are
made sufficiently soluble to be fungicidal. Marten and Leach (1944)
studied the effect of various nitrogenous compounds upon the solubility
of cuprous oxide. Gelatin and peptone were less efficient in dissolving
cuprous oxide than were glycine, aspartic acid, asparagine, or cystine.
Ammonium hydroxide was also a solvent for cuprous oxide. With all

ACTION OF FUNGICIDES 249

these "solvents" the solutions were blue in color, which indicates that
the copper was oxidized to the cupric state. Marten and Leach investi-
gated the toxicity of the copper-glycine compound to Pythnim debaryanum.
It was noted that an excess of glycine protected the fungus from the
action of the copper. Some 200 times as much copper was required to
inhibit growth when glycine was present in the medium as when it was
absent. Thus, it seems that whether a given amount of copper is toxic
or not depends upon the nature and amount of certain constituents in
the medium or substrate.

One may ask, By what mechanism does the copper ion cause fungistasis,
or how does the copper kill? The common explanation of the toxic
action of the heavj'^ metals (copper, mercury, and silver) is based upon the
property of these ions of precipitating or denaturing proteins. Enzymes
are proteins, and it would be expected that the heavy metals would
inactivate these catalysts. However, not all enzymes are equally inac-
tivated by low concentrations of heavy-metal ions. The enzymes which
require free sulfhydryl groups for activity appear to be especially suscepti-
ble to inactivation by ions of heavy metals. It is probable that copper
causes fungistasis by combining with the sulfhydryl groups of certain
enzymes. At this stage, the action of copper is reversible. Goldsworthy
and Green (1936) found that spores of Sderotinia fructicola which had
been treated with insufficient copper to kill made normal growth when
sown on copper-free medium. As long as an inhibition is reversible, the
process is one of fungistasis. Death of the spore results when irreversible
changes occur.

There is reason to believe that the injurious effect of copper fungicides
upon the host plant is due to the same mechanism that operates in fungus
spores. Foster (1947) attributed the sensitivity to copper of certain
seeds to their content of sulfhydryl enzymes.

mercury'

While a number of inorganic salts of mercury have been used as anti-
septics, only two have had wide application as fungicides. Mercuric
chloride (corrosive sublimate, bichloride of mercuiy, HgCl2) is a soluble,
highly poisonous compound. It is commonly used for surface steriliza-
tion in a concentration of 1/1,000. Mercuric chloride is occasionally
used as a special-purpose fungicide.

Mercurous chloride (calomel, HgCl or Hg2Cl2) is essentially insoluble
in water, sufficiently so to be used in medicine. Calomel slowly decom-
poses into mercury and mercuric chloride. This decomposition is accel-
erated by sunlight, which may account for the successful use of calomel
to control dollar spot, brown patch, and other turf diseases.

The organic mercury compounds have won wide acceptance in the

250 PHYSIOLOGY OF THE FUNGI

treating of seed to control the attack of fungi which cause damping-off
and of certain seed-borne pathogens. The organic mercurials are free
from many of the objections inherent in the inorganic compounds of
mercury. In general, they combine less avidly with proteins, are more
selective in their action, and are far less toxic to animal life. As used for
seed protection, they are commonly diluted with an inert carrier. Most
if not all such organic mercury compounds are sold under trade names,
but the active components are required by law to be stated on the label.
Among these organic mercury compounds are ethylmercury phosphate
(Semesan Jr. and New Improved Ceresan), ethylmercury chloride
(Ceresan) and hydroxymercurichlorophenol (Semesan).

The organic mercury compounds used as sprays and for treating seeds
are in general related to mercuric chloride in the following way:

C2H6— Hg— CI CI— Hg~Cl

Ethylmercury chloride Mercuric chloride

The ethyl group has replaced a chlorine atom in mercuric chloride.
The type formula for compounds like this may be written as R — Hg — X,
where R may be any alkyl or aryl (or other) group and X represents any
anion, I~, Cl~, 0H~, NOs", P04~. The anion greatly modifies the solu-
bility of the compound in water. In general, this type of organic mer-
cury compound is volatile, and this property may be assumed to aid in
penetration. Other organic mercury fungicides are derivatives of alkjd
and aryl mercuric hydroxides. These compounds can react with organic
acids to form salts. The relation of these compounds to mercuric hydrox-
ide is shown below:

CeHs— Hg— OH HO— Hg— OH

Phenylmercury hydroxide Mercuric hydroxide

Compounds of this type are used to protect cellulose and leather products.

Parker-Rhodes (1942) investigated the toxicity of the following mer-
cury compounds to Macrosporium sarcinaeforme and Botrytis allii: mer-
curic acetate, mercuric chloride, methylmercury nitrate, and tolylmer-
curic nitrate. All these compounds were toxic to M. sarcinaeforme, and
all except methylmercury nitrate were toxic to B. allii. Perhaps methyl-
mercury nitrate is not soluble enough in fat for the spores of this fungus
to absorb a toxic amount of the compound. Dillon- Weston and Booer
(1935) found that vapor of ethylmercury iodide was toxic to TiUetia spores
in the laboratory but afforded no control in the field.

The soluble inorganic mercury salts are protein precipitants, and this
property may explain in part their mode of action when used in high
concentrations. These salts are frequently fungistatic or bacteriostatic,
since the very firmness of the union between the mercuric ion and the cell
membrane may form a barrier to further penetration. The first action

ACTION OF FUNGICIDES 251

of mercuric ion is to cause stasis, which may be reversed by treating the
cells with reagents which have a high affinity for mercury. McCalla
(1940) demonstrated that cells of Escherichia coli which had been treated
with mercuric chloride could be revived by hydrogen sulfide. If stasis
due to mercury is not overcome within a certain time, irreversible changes
occur and death of the cells results.

Organic mercury compounds are not protein precipitants, and this is
one of their advantages as disinfectants and fungicides. Fildes (1940)
ascribed the action of mercury compounds to combination of mercuric
ion with the sulfhydryl group of essential metabolites and enzymes.
Others have shown that organic mercury compounds act similarly.
According to this view, enzyme inhibition is the basis of the action of
mercury compounds. Fildes found that the action of mercury was
antagonized by compounds which contained free sulfhydryl groups
(thioacetate, cysteine, glutathione). Neither cystine ( — S — S — ) nor
methionine ( — S — ) was effective in overcoming mercury toxicity.

Organic mercury compounds appear to act by the same mechanism as
the mercuric ion. p-Chloromercuribenzoate was found to inhibit the
action of various sulfhydryl enzymes which take part in carbohydrate
metabolism, e.g. succinic acid oxidase, yeast carboxylase, malate oxidase,
and ketoglutarate oxidase. This organic mercury compound also inhib-
ited the action of c?-amino acid oxidase, transaminase, Z-glutamate oxidase,
and other enzymes (Barron and Singer, 1945; Singer and Barron, 1945).
In many instances the inhibitory action of p-chloromercuribenzoate on
these enzymes could be reversed by glutathione, cysteine, or hydrogen
sulfide.

Cook et at. (1946) found phenylmercuric nitrate to depress the respira-
tion of Saccharomyces cerevisiae. This depression in rate of respiration
was overcome by various compounds having a free — SH group; e.g.,
cysteine and homocysteine, while cystine and methionine were without
effect. The work of these investigators and of others makes it highly
probable that mercury compounds are toxic because they inactivate
certain essential enzyme systems. The enzyme inhibitions discussed
above are examples of noncompetitive inhibition. These inhibitions are
reversible, as in the case of competitive inhibition, but the reversing
agents are nonspecific, or not limited to a single metabolite.

SULFUR

Of the nonmetallic elements, sulfur and certain of its compounds are
widely used as protective and eradicant fungicides. The toxicity of the
nonmetallic elements is dependent upon the state of oxidation. In many
instances, compounds in the higher states of oxidation are the least toxic.
For example, sulfur in the form of the free element (S) and of sulfide

252

PHYSIOLOGY OF THE FUNGI

(S=) forms excellent fungicides, while sulfites (SOa^) are only slightly-
toxic and sulfates (SO 4=) are nontoxic.

According to Large (1940), elemental sulfur has been used to control
powdery mildew for slightly over 100 years. The effectiveness of sulfur
increases as the particle size diminishes. Finely divided sulfur adheres
to plant surfaces much better than larger particles. In addition, the
distance between particles tends to be decreased when fine particles are
used, and the infection court is thereby better protected. The odds are
increased that a fungus spore falling upon a treated leaf will be within the
range of action of a particle of sulfur. An example of the effect of particle
size of sulfur on toxicity is given in Table 44. The greater toxicity of

Table 44. The Relation between the Particle Size and Toxicity of a Sulfur
Dust to the Conidia of Sclerotinia americana
(Wilcoxon and McCallan, Phytopathology 20, 1930.)

Treatment

Mean diameter of
sulfur particles, tx

Germination, %

Control

285

142

97.6

Ground roU sulfur

62.8

Ground roll sulfur

47.2
29.1
20.7

the finely divided sulfur is due to the fact that sulfur enters the spore in
the form of vapor. The amount of vapor formed from a given amount of
sulfur in a given time depends upon the area of the exposed surface, as
well as upon temperature. Therefore, the fineness of the sulfur particles
governs the effective concentration of sulfur vapor and its effectiveness
as a fungicide.

McCallan (1946) estimated the yearly consumption of sulfur in the
United States alone to be 142 million pounds. Of this amount 110
million pounds is used as sulfur dust, 5 million pounds as wettable sulfur,
and 27 million pounds as lime-sulfur. Approximately 62 per cent of
this is used primarily for the control of apple scab alone.

Since elemental sulfur is insoluble, its action upon fungi cannot be
attributed to the sulfur in this form. Two general theories have been
proposed to explain the action of sulfur. One theory holds that the
action is due to oxidized sulfur, such as SO2 or SO3 (which form sulfurous
and sulfuric acid, respectively, with water) or pentathionic acid, H2S5O6.
According to the second theory, the reduced form of sulfur, H2S, is the
active toxic agent. Both these theories are supported by published
experimental evidence. All these compounds are toxic to fungi under
certain conditions, if in high enough concentrations. However, to

ACTION OF FUNGICIDES 253

account satisfactorily for the toxic properties of sulfur, it must be demon-
strated that the toxic agent is produced under the conditions which
prevail in the field in quantities sufficient to account for the observed
effects. Any evaluation of these hypotheses must take into account all
the variables involved. The caution of Wilcoxon and McCallan (1930)
is pertinent:

In making comparisons of the toxicity of chemical substances to fungus spores,
there are two requisites for obtaining accurate results which, though quite
obvious, have not always received the consideration they deserve, (a) The
substance whose toxicity is to be measured must be available in a pure state
and of known concentration, and (6) the technique employed must be capable of
distinguishing between the toxicity of the substances it is desired to compare.

It is agreed that elemental sulfur is not the toxic agent and that sulfur is
transformed into the toxic agent. There are three possible agencies for
such transformations: the atmosphere, the plant on which the sulfur is
dusted, and the fungus spores or mycelium. Sulfur acts at a distance,
and since sulfur is volatile at room temperature, this property offers an
explanation. Sulfur vapor is a gas, and in this state it should be more
easily transformed into the toxicant.

Sulfur is slowly oxidized by the oxygen of the atmosphere to form sulfur
dioxide, but the rate at which this reaction occurs at ordinary tempera-
tures makes it impossible for this reaction to account for all the toxic
properties of sulfur, even though sulfur dioxide is toxic to fungus spores
(McCallan and Weedon, 1940).

Young (1922) set forth the hypothesis that pentathionic acid is the
toxic agent formed from sulfur. It is agreed, even by those who do not
support Young's hypothesis, that this acid is formed on the surface of
sulfur dust. A considerable number of papers were published during
the next decade which gave support to this view (Liming, 1932). Wil-
coxon and McCallan (1930) investigated this theory thoroughly and
concluded that pure pentathionic acid had no toxic properties for the
spores of Sderotinia americana, Botrytis sp., Macrosporium sarcinaeforme,
and Uromijces caryopJujUinus. If sufficient pentathionic acid was used
to reduce the pH to about 4, spore germination was inhibited. Solutions
of sulfuric acid having the same pH were equally toxic. Neutral salts
of both acids were nontoxic. Roach and Glynne (1928) likewise found
pentathionic and sulfuric acids to have the same toxicity when tested
against the winter sporangia of Synchytrium endohioticum. Wilcoxon
and McCallan (1930) performed a decisive experiment when they washed
one lot of sulfur dust with alkali to remove pentathionic acid and com-
pared this pentathionate-free dust with the original sample, which con-
tained a trace of this acid. No difference in toxicity of the washed and
control samples of this sulfur dust was found.

254

PHYSIOLOGY OF THE FUNGI

There is now general agreement that hydrogen sulfide is the common
toxic compound produced from sulfur. Not only is hydrogen sulfide
toxic to fungus spores, but the mechanism for its production is also pres-
ent. It is known that hydrogen sulfide is produced from sulfur both by
the treated plant and by the fungus spores,

McCallan and Wilcoxon (1931) made qualitative tests for the ability
of the spores of 17 species of fungi to produce hydrogen sulfide from sulfur.

All produced this substance, but in
varying amounts and at varying
rates. They showed that the spores
need not be in direct contact with
solid sulfur to produce hydrogen
sulfide. Figure 50 illustrates the
method used by these investigators
to demonstrate this phenomenon.

These authors investigated the
toxicity of hydrogen sulfide to the
spores of eight species of fungi.
These experiments were performed
in a flowing stream of air which con-
tained known amounts of hydrogen
sulfide, and the concentration in the
water droplet in which the spores
were suspended was calculated from
Henry's law. These precautions are
necessary because hydrogen sulfide is
unstable. Neglect of this fact by

LEAD ACETATE
PAPER WHITE

LEAD ACETATE
PAPER BLACKENED

COLLODION SAC
SPORE SUSPENSION
SULPHUR PASTE

Fig. 50. The production of hydrogen
sulfide by Sclerotinia spores separated
from sulfur by a collodion membrane.
Note that the production of hydrogen
sulfide takes place on the spore side of
the membrane and not on the sulfur
side. (Courtesy of McCallan and
WUcoxon, Contribs. Boyce Thompson
Inst. 3: 26, 1931.)

earlier investigators led to an underestimation of the toxicity of hydrogen
sulfide. These results of McCallan and Wilcoxon are presented in Fig.
51. From these curves it is seen that spores of Venturia inaequalis,
Uromyces caryophyllinus, and Puccinia antirrhini are inhibited by very
low concentrations of hydrogen sulfide, while the spores of Botrytis sp.
and Glomerella cingulata are scarcely affected by ten times as much
hydrogen sulfide. By increasing the hydrogen sulfide concentration to
60 p. p.m., complete inhibition of germination of the spores of these two
species was obtained. The spores of these eight fungi were shown to
produce varying amounts of hydrogen sulfide per unit weight of spores.
Whether hydrogen sulfide produced by spores would prove toxic would
therefore depend upon the ability of the particular spores to produce
hydrogen sulfide and the sensitivity of the spores to this substance. The
correlation is shown in Table 45.

The actions of sulfur and hydrogen sulfide are parallel, and it may be
concluded that sulfur is toxic to the spores of certain species by virtue

ACTION OF FUNGICIDES

255

of absorption of sulfur vapor and its reduction to hydrogen sulfide within
the spore. Thus, the spores of susceptible species destroy themselves.
It is not thought that the hydrogen sulfide evolved from leaves or other
spores is absorbed in lethal quantities under natural conditions.

0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

Concentration -milligrams per liter

Fig. 51. Toxicity of hydrogen sulfide to urediospores of Uromyces caryophyllinus
and Puccinia antirrhini and to conidia of Venturia inaequalis, Sclerotinia americana,
Macrosporiuni sarcinaeforme, Pestalotia stellata, Glonierella cingulata, and Botrytis sp.
(Courtesy of McCallan and Wilcoxon, Contribs. Boyce Thompson Inst. 3: 31, 1931.)

Liquid lime-sulfur is a common spray material and is prepared by
boiling sulfur and calcium hydroxide together. The chief active ingre-
dient is calcium polysulfide. After deposition on leaves the calcium
polysulfide is quickly decomposed, yielding sulfur and calcium sulfide,

Table 45. Comparison between the Toxicity and the Production of Hydrogen
Sulfide, Expressed in Units Equal to the Amounts of Hydrogen Sulfide
Required to Reduce Germination 50 Per Cent
(McCallan and Wilcoxon, Contribs. Boyce Thompson Inst. 3, 1931.)

Species

Mg. H2S
required to
reduce ger-
mination of

1,000,000
spores 50%

Mg. H2S

produced by

1,000,000

spores in

12 hr.

Production of H2S

expressed in units

equal to the amount

of H2S required to

reduce germination

50%

Venturia inaeaualis

0.001
0.002
0.006
0.013
0.043
0.049
0.532
0.665

0.002

0.019

0.13

0.039

0.013

0.001

0.027

0.002

2.0

Uromyces caryophyllinus

Puccinia antirrhini

9.5
2.2

Sclerotinia americana

Macrosporiuni sarcinaeforme

Pestalotia stellata

Glonierella cingulata

3.0
0.30
0.02
0.05

Botrytis sp

0.003

256 PHYSIOLOGY OF THE FUNGI

which in turn may decompose by hydrolysis to yield hydrogen sulfide
and calcium hydroxide. At the same time some of the calcium poly-
sulfide is oxidized to calcium thiosulfate and sulfur. See Frear (1948)
for a discussion of the chemistry involved.

It is known that lime-sulfur exerts an eradicant action on some fungi,
including Venturia inaequalis, when first applied. After a few days
this spray exerts only a protective action like that of elemental sulfur,
which probably depends on the elemental sulfur set free by the decom-
position of various constituents comprising lime-sulfur. The eradicant
action, then, depends upon either the calcium polysulfide or calcium
sulfide. We may consider that sulfide ion (S=) is the toxic agent. The
alkalinity of the spray may aid in penetration into the mycelium already
present.

Lime-sulfur solution may be treated with ferrous sulfate or aluminum
sulfate in the spray tank to produce colloidal sulfur and hydrogen sulfide.
Aluminum sulfate, AI2 (804)3, hydrolyzes to form aluminum hydroxide
and sulfuric acid. A lime-sulfur spray so treated has only a protective
action. It has lost its eradicant value. We may assume, therefore, that
the decomposition of lime-sulfur in spray tanks when treated with acid
(aluminum sulfate) and the decomposition on the leaf follow a somewhat
similar pattern. This scheme of producing colloidal sulfur has the draw-
back that the added iron, when ferrous sulfate is used, is toxic to vegeta-
tion, and dangerous amounts of hydrogen sulfide are evolved.

We may assume that hydrogen sulfide exerts its toxic action on fungus
spores by inactivating certain enzymes. Hydrogen sulfide is known to
inactivate many enzymes, including catalase, cytochrome oxidase, dopa
oxidase, lactase, and others. Generally, hydrogen sulfide and cyanide
inhibit the same enzymes. It is thought that these metalloenzymes are
inhibited by sulfide or cyanide because these agents react with iron or
copper to form highly insoluble or little-ionized compounds or complexes.

ORGANIC FUNGICIDES

The newer fungicides, with few exceptions, are either organic or organo-
metallic compounds. The organic mercury compounds were considered
with the inorganic compounds of mercury, since the mechanism of action
appears to be the same in both types of compounds. Many of the
organic fungicides exhibit greater specificity than the inorganic fungicides.
The possibilities of modification in the structure of organic compounds
are almost unlimited. The study of organic fungicides, therefore, offers
the opportunity of correlating structure with type and intensity of fungi-
cidal action.

Aldehydes. The first organic fungicide to attain wide acceptance was
formaldehyde. At one time this compound was used for the surface
sterilization of grain and potato tubers, but at present formaldehyde is

ACTION OF FUNGICIDES

257

little used. Formaldehyde reacts with free amino groups, and it is
probable that its fungicidal action depends upon this property. Some
other aldehydes also have fungicidal properties (Uppal, 1926).

Quinones. While there are two series of quinones (ortho, or 1,2, and
para, or 1,4), we shall consider only the 1,4-quinones as fungicides. The
simplest quinone is p-benzoquinone. Quinones are cyclic compounds
which possess a characteristic pair of double bonds. Such a configuration
of double bonds is called quinoid and is possessed by many dyes, some
of which are fungicides. If a considerable series of toxic compounds
possess a common functional group or groups, it may be assumed that
these groups are involved in fungicidal activity. According to Horsfall
(1945), 1,4-benzoquinone has a slight toxicity to fungi. The four hydro-
gens in 1,4-benzoquinone can be replaced by chlorine to form chloranil
(Spergon), which greatly increases the fungicidal properties. The struc-
tural formulas for these compounds are given below:

O O

— H

-H

CI—

Cl-

— CI

-CI

o o

1,4-Benzoquinone Chloranil (Spergon)

Spergon has been used as a seed protectant.

Substituted naphthoquinones are more important fungicides than,
the benzoquinones. Among these, 2,3-dichloro-l,4-naphthoquinone
(Phygon) is reported to be five to eight times as effective as Spergon (Ter
Horst and Felix, 1943). Some of the naphthoquinones synthesized by
plants are fungicides. Juglone, 5-hydroxy-l,4-naphthoquinone, is found
in walnut hulls and is secreted by walnut roots. The isomeric 2-hydroxy-
1,4-naphthoquinone (lawsone) is found in henna leaves. Juglone is
reported to be as toxic to fungus spores as Bordeaux mixture. Juglone
controls black spot of roses as well as sulfur does (Gries, 1943, 1943a).
It is also toxic to many plants. Little et al. (1948) isolated 2-methoxy-
1,4-naphthoquinone from Impatiens balsamina. This compound was an
active fungicide which exhibited no phytotoxicity toward tomato and
bean plants. The formulas of two naphthoquinone fungicides are given
below:

O O

-OCH3

2-Methoxy-l,4-naphthoquinone

OH O

Juglone

258

PHYSIOLOGY OF THE FUNGI

The fungicidal action of substituted quinones may be due in part to
their property of reacting with free amino groups of proteins (Theis,
1945). Substituted naphthoquinones as antagonists of vitamin K were
discussed in Chap. 11. Most of the available evidence indicates that
the principal mechanism of quinone toxicity lies in its noncompetitive
inhibition of sulfhydiyl enzymes.

It has been suggested that the mechanism of inhibition is dependent
upon the structure of the substituted naphthoquinones. Colwell and
McCall (1946) found the fungistatic and fungicidal concentrations of
2-methyl-l,4-naphthoquinones to be the same when Aspergillus niger
and an unidentified fungus were used as test organisms. Addition of
sodium thioglycolate or cysteine antagonized the toxic action of this
naphthoquinone. These authors postulate that only naphthoquinones
unsubstituted in position 3 react wdth sulfhydryl groups, for 2-methyl-3-
methoxy-l,4-naphthoquinone was not antagonized by thioglycolate or
cysteine. The reaction between certain naphthoquinones and sulfhydiyl-
containing compounds can be demonstrated in vitro.

The amounts of various substituted 1,4-naphthoquinones required to
cause a 50 per cent inhibition of isolated yeast carboxylase and similar
reduction in the germination of Monilinia fructicola spores w^ere roughly
parallel (Foote et al., 1949). Carboxylase is a sulfhydryl enzyme. It is
probable that other sulfhydryl enzymes are also inhibited by naphtho-
quinones. For further information on the mechanism of quinone inhibi-
tion, see Geiger (1946).

Dyes. Various dyes are fungistatic compounds. Malachite green
and crystal violet are used to control various fungus infections of the
skin. Both these dyes have a benzoquinoid structure, as is shown below:

^ ^

-N(CH3)2

(CH3)2N='

Malachite green

Leonian (1930) made a study of the toxicity of malachite green to many
species and strains of Phytophthora and found only three species {P.
hydrophila, P. melongenae, and P. sp.) able to grow in the presence of
1 p. p.m. of malachite green. Other species were more sensitive to this
dye. P. colocasiae and P. richardiae failed to grow in nutrient solutions
containing 1 part of malachite green in 16 million parts of medium.
Leonian (1932) investigated the growth-inhibiting properties of malachite

ACTION OF FUNGICIDES

259

(CH3)2N

N(CH3)2

Crystal violet

green and crystal violet upon 26 species and isolates of Trichophyton.
Malachite green proved greatly superior to crystal violet. Over half
the isolates tested failed to grow in the presence of 1 part of malachite
green to 50,000 parts of medium, and many failed to grow in the presence
of 1 p.p.m. of this dye. Crystal violet allowed some growth in all isolates
tested at a concentration of 1 part in 50,000 parts of medium. Placing
the inoculum in direct contact with the medium containing the dye was
more lethal than placing the agar inoculum plug with the mycelium upon
the surface of the test medium. Some other dyes such as methylene
blue are also toxic to fungi. Both malachite green and methylene blue
inhibit carboxylase (Horsfall, 1945).

Dithiocarbamates and related compounds. Barratt and Horsfall
(1947) have reported extensive investigations on the homologues and
analogues of disodium ethylenebisdithiocarbamate (Nabam). In gen-
eral, these compounds are formed when primary and secondary amines
react with carbon disulfide. The formula for Nabam is given below:

S H

H S

Na— S— C— N— CH2— CH2— N— C— S— Na
Disodium ethylenebisdithiocarbamate (Nabam)

The zinc (Ziram) and ferric (Ferbam) salts of dimethyldithiocarbamate
are effective fungicides for the control of certain fungus pathogens. The
formula for dimethyldithiocarbamate is given below:

II
(CH3)2— N— C— SH
Dimethyldithiocarbamate

The oxidation product of dimethyldithiocarbamate is tetramethylthluram
disulfide (Thiram), which has some value as a seed protectant.

The dithiocarbamate fungicides, such as Nabam, yield hydrogen sulfide
on hydrolysis. This reaction takes place spontaneously in the presence
of moisture. The mechanism of hydrogen sulfide toxicity has already
been discussed. The second mechanism which has been proposed
involves the formation of insoluble mercaptides of certain essential metals.

2G0 PHYSIOLOGY OF THE FUNGI

In addition, Nabam on decomposition yields an unidentified toxic gaseous
compound, which is neither hydrogen sulfide nor sulfur dioxide (Rich
and Horsfall, 1950).

Specific organic reagents for metals. The essential nature of certain
micro elements for fungus growth and the role of these elements in
enzymes were discussed in Chaps. 4 and 5. The chemistry of these
specific organic reagents is treated by Yoe and Sarver (1941). These
reagents form insoluble or slightly ionized compounds with metals.
Zentmeyer (1944) tested various organic analytical reagents and found
8-hydroxyquinoline (Oxine) and ammonium nitrosophenylhydroxylamine
(Cupferron) to be fungistatic. 8-Hydroxyciuinoline inhibited the growth
of Fusarium oxysporum var. lycopersici, Ceratostomella ulmi, and a species
of Penicillium. The effectiveness of 8-hydroxyquinoline in forming
chelate salts increases as the pH values increase. Below pH 3.5 complex
formation does not take place with zinc, copper, iron, and manganese.
Zinc ion reacts with 8-hydroxyquinoline as shown below:

+ H+

I I r

OH O— Zn

8-Hydroxyquinoline Zinc complex of 8-hydroxyquinoline

The fungistatic effect of 8-hydroxyquinoline on Fusarium oxysporum
var. lycopersici and Ceratostomella ulmi was overcome by increasing the
zinc content of the medium. In the presence of 8-hydroxyquinoline
there was competition between this compound and one or more enzyme
systems for the zinc present in the medium. Whether or not an organic
compound such as 8-hydroxyquinoline will act as a fungistatic agent
depends upon the concentration of the reagent, the amount of fungus
mycelium, and the concentration of the metallic ion for which the two
sj^stems compete. One would expect that such fungicides, in common
with all others, would be more effective when the mass of the fungus is
small.

Other organic fungicides. Many other types of organic compounds
are fungicides, and an intensive search for new ones is in progress. Brief
mention of some of these developments is made below. Geiger (1948)
reports various unsaturated ketones to be active against Aspergillus
niger, Trichoderma koningii, Cryptococcus neoformans, and Trichophyton
mentagrophytes. The mode of action resembles that of the naphtho-
quinones in that sulfhydryl enzymes, including succinic acid dehydro-
genase, triose phosphate dehydrogenase, and urease, are inhibited. The
fungistatic activity of ethylenic and acetylenic compounds has been

ACTION OF FUNGICIDES 261

tested on Fusarium graminearum, Penicillium digitatum, and Botrytis
aim (McGowan et al., 1948). The fungicidal action of substituted
pyrazoles was tested on spores of Alter naria oleracea and Sclerotinia
americana in the laboratory, and for the control of apple scab, cedar-apple
rust, and late blight of potato and tomato. Some of these compounds
show promise, although the mechanism of action is not known (McNew
and Sandholm, (1949). For a survey of the newer fungicides see Well-
man (1948).

EVALUATING FUNGICIDES

The preliminary tests of fungicidal activity are made in the laboratory
in order to eliminate inactive compounds or to compare the activities
of different compounds under identical conditions. Evaluation in the
greenhouse and field is the final test of a new fungicide. This discus-
sion will be limited to a general consideration of laboratory testing of
fungicides.

Fungus spores rather than mycelium are used in most laboratory tests
because it is the function of a protectant fungicide to kill or inhibit spore
germination. Three basic types of procedures may be used in laboratory
tests (McCallan, 1947): (1) Spores are suspended in solutions or suspen-
sions of the fungicide under test, and the inhibition of germination is
noted as a function of time of exposure and concentration of the fungicide.
This is a modification of the Rideal- Walker method of evaluating anti-
septics. (2) The compound to be tested is incorporated in a suitable
solid or liquid medium, which is then inoculated with spores of the test
fungi. The amount of inhibition of germination or growth is determined.
(3) Glass slides are covered uniformly with the fungicide, and after dry-
ing, the spores are sown on the treated slides. The inoculated plates
are then placed in constant-humidity chambers and the percentage of
germination determined after 20 to 24 hr. ; or the effectiveness of a fungi-
cide may be studied as a function of time of exposure.

The second and third methods appear to be the most useful. Fleury
(1948) studied the fungistatic action of thiourea on Aspergillus niger
by adding this substance to a liquid basal medium. Thiourea was a
much more potent inhibitor w^hen nitrate nitrogen was used than when
ammonium or organic nitrogen was present in the medium. Agar
medium has been used by Leben and Keitt (1949) to assay the amount of
toxicant on leaf surfaces. A suspension of spores of Glomerella cingulata
was prepared in warm (38 to 40°C.) agar medium. Five milliliters of
this seeded medium was added to Petri dishes which contained 15 ml. of
solidified agar medium. After the seeded agar had solidified, leaf disks
of uniform size were cut from sprayed leaves and placed on the agar. The
amount of toxicant present on the leaf surface was determined by measur-

262 PHYSIOLOGY OF THE FUNGI

ing the diameter of the zone of inhibition. Disks of blotting paper to
which fungicides have been added may be used to determine their potency.
Thornberry (1950) has suggested the use of filter-paper disks for the
evaluation of fungicides and bactericides. Filter-paper disks appear
to be more suitable than blotting paper. In this method seven filter-
paper disks are uniformly spaced on a Petri dish, and 0.09 ml. of the
toxicant in aqueous solution is added per disk. The zone of inhibition
is a measure of the effectiveness of the fungicide.

The glass-slide method appears to simulate more closely the conditions
under which the spores of plant pathogenic fungi germinate in nature.
The Committee on the Standardization of Fungicidal Tests of the Ameri-
can Phytopathological Society has considered this method important
enough to publish a detailed and documented summary (1943), to which
the student is referred for further information and references. This
committee recommended the use of spores of the following species for
this test: Alternaria solani, Glomerella cingulata, Macrosporium sarcinae-
forme, Sclerotinia fructicola, Penicillium expansum, and Rhizopus nigri-
cans. For accurate work, at least two of these test fungi should be used.
The effectiveness of a fungicide is determined by calculating the percent-
age of inhibition of spore germination. The methods of evaluating data
obtained in fungicide tests are discussed by Horsfall (1945).

SUMMARY

A fungicide is an agent capable of killing some fungi. Fungicides may
be either water-soluble or nearly insoluble. The action of fungicides
of the first class is immediate; that of the second class is delayed. Eradi-
cant fungicides are of the first class, w^hile protective fungicides are of
the second. Fungistasis is the complete or partial inhibition of one or
more life processes of a fungus. This inhibition is reversible. The same
chemical compound may cause fungistasis or may be a fungicide, depend-
ing upon the concentration and time of exposure. The same substance
may be a fungicide for one species, cause fungistasis of a second, and be
without effect upon a third. Fungistasis precedes fungicidal action.

Before a fungicide can act upon a fungus, the toxicant must get into
the fungus cells, or at least reach the protoplasmic membrane. While
other factors undoubtedly enter into the mechanism of fungicidal action,
the principal point of attack appears to be enzyme systems. The heavy-
metal fungicides appear to act by inhibiting various sulfhydryl enzymes.
Fungus spores transform sulfur into hydrogen sulfide, which inhibits the
metalloenzymes. Organic fungicides, so far as is known, are also enzyme
inhibitors.

In the past, fungicides containing copper, mercury, and sulfur have
been the most useful. Recently, organic fungicides have become impor-

ACTION OF FUNGICIDES 263

tant and promise to be used even more extensively in the future. Organic
fungicides are generally more specific than inorganic fungicides. Satis-
factory fungicides for the control of certain diseases are still undiscovered.

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264 PHYSIOLOGY OF THE FUNGI

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Ann. Applied Biol. 35: 25-36, 1948.
McNew, G. L., and N. K. Sandholm: The fungicidal activity of substituted pyra-

zoles and related compounds. Phytopathology 39 : 721-751, 1949.
Marten, E. A., and J. G. Leach: Some factors influencing the solubility of cuprous

oxide in relation to its toxicity as a fungicide, Phytopathology 34 : 459-470, 1944.
Parker-Rhodes, A. F. : Studies on the mechanism of fungicidal action. IV. Mer-
cury, Ann. Applied Biol. 29: 404-411, 1942.
Provost, B. : Memoire sur la cause immediate de la carie ou charbon des bl^s, et de

plusieurs autres maladies des plantes, et sur preservatifs de la carie, 1807.

Trans, by G. W. Keitt, Phytopathological Classics No. 6, American Phyto-

pathological Society, Menasha, 1939.
Rich, S., and J. G. Horsfall: Gaseous toxicants from organic sulfur compounds,

Am. Jour. Botany 37: 643-650, 1950.
Roach, W. A., and M. D. Glynne: The toxicity of certain sulphur compounds to

Synchytrium endobioticum, the fungus causing wart disease of potatoes, Ann.

Applied Biol. 15: 168-190, 1928.
Singer, T. P., and E. S. G. Barron: Studies on biological oxidations. XX. Sulf-

hydryl enzymes in fat and protein metabolism. Jour. Biol. Chem. 157: 241-253,

1945.
Starkey, R. L., and S. A. Waksman: Fungi tolerant to extreme acidity and high

concentrations of copper sulfate, Jour. Bad. 45: 509-519, 1943.

ACTION OF FUNGICIDES 265

Ter Horst, W. p., and E. L. Felix: 2,3-Dichloro-l,4-naphthoquinone, a potent

fungicide, Ind. Eng. Chem. 35: 1255-1259, 1943.
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Thornberry, H. H. : a paper-disk plate method for the quantitative evaluation of

fungicides and bactericides. Phytopathology 40: 419-429, 1950.
Uppal, B. N.: Toxicity of organic compounds to the spores of Phytophthora colocasiae

Rac, Jour. Agr. Research 32 : 1069-1097, 1926.
Wellman, R. H.: Synthetic chemicals for agriculture. II. Fungicides, nematocides,

rodenticides and weed killers, Chem. hids. 63 : 223-229, 1948.
WiLCOXON, F., and S. A. E. McCallan: The fungicidal action of sulphur. I. The

alleged role of pentathionic acid, Phytopathology 20: 391-417, 1930.
YoE, J. H., and L. A. Sarver: Organic Analytical Reagents, John Wiley & Sons

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100 : 294-295, 1944.

CHAPTER 13
METABOLIC PRODUCTS

The most important product of fungus metabolism is carbon dioxide,
and the most important function of the fungi in the economy of nature
is the destruction of plant and animal remains. The use of fungi for
food antedates written history. The use of fvmgi for the preparation
of bread and wine developed as a household art. From the time of
Pasteur, the study of fermentation has led to an ever-increasing knowl-
edge and understanding of the activities of microorganisms. The pro-
duction of antibiotics and vitamins, alcohol and organic acids, and the
potential utilization of waste agricultural products are current fields of
research and industrial activity. For extensive treatment of these
subjects the reader is referred to Prescott and Dunn (1949) and Foster
(1949).

DECOMPOSITION OF ORGANIC MATERIALS

Brefeld (1908) called fungi "Organismen der Verwesung" and con-
sidered them to be indispensable agents in maintaining the essential-
element balance of nature. Saprophytic fungi and bacteria prevent the
accumulation of plant and animal debris and return the elements that
compose these materials to the storehouse of nature, where they are
reused by new generations of plants and animals. In this role, sapro-
phytic fungi are designated as "vegetable vultures" by Rolfe and Rolfe
(1926), for they act as scavengers in the plant world.

Green plants assimilate carbon in the form of carbon dioxide. Waks-
man (1938) has assembled the data with regard to the amount of carbon
in the biosphere. It is estimated that the atmosphere contains 600
billion tons of carbon in the form of carbon dioxide, and plants are esti-
mated to remove 16 billion tons yearly. Thus, the carbon content of the
atmosphere is sufficient for about 40 years, if no carbon dioxide were
returned to the air.

The complete destruction of plant and animal remains by fungi and
bacteria requires a long time, although some plant constituents, such as
soluble sugars and other carbohydrates, are quickly utilized. Presum-
ably the fungi are the most important organisms in this process. Other
plant constituents, such as the waxes and lignin, are attacked more
slowly. The more resistant constituents are slowly modified to form

266

METABOLIC PRODUCTS 267

humus. Some of the carbon and other essential elements is converted
into bacterial and fungus protoplasm, which after death is subject to
decay. In the end, humus is converted into carbon dioxide, water, and
other simple compounds, which are used again. The importance of
humus as a soil constituent is ably discussed by Waksman (1938). In
addition to the carbon cycle, the fungi also play an important part in the
cycles involving the release and utilization of the other essential elements.

FUNGI AS FOOD

Many curious details about the early use of fungi as food have been
collected from classical and other sources by Buller (1914) and by Rolfe
and Rolfe (192G). The mushrooms were no doubt among the first fungi
used as food by man. Yeast became part of his diet when the arts of
brewing and baking were discovered. The widespread use of fermented
beverages, under certain dietetic circumstances, has an important bearing
on nutrition and health. J. S. Wallerstein (1939) has discussed primitive
brewing practices and the geographical distribution of the art. The beer
of the Middle Ages Avas turbid, owing to its content of suspended yeast
cells (Thaysen, 1943).

The nutritive value of any food depends upon its composition and
digestibility and the assimilability of its hydrolytic products. The early
writers, in the absence of precise information, were of the opinion that
fungi had little value as food. The nutritive value of fungi, of yeast in
particular, will be discussed from the standpoint of protein content and
value, vitamins, fats, and minerals.

Assuming good digestibility, the value of fungus protein is determined
by its amino-acid composition. Rose (1938), in a long series of careful
experiments, has determined which amino acids are essential for man and
animals. Some nine or ten amino acids were found to be essential (Table
20). If the protein part of a diet is deficient in a single essential amino
acid, nitrogen is lost from the body, or inefficient utilization of protein
results. More of a poor protein must be consumed in order to increase
the intake of essential amino acids to satisfactory levels. The amino-
acid composition of yeast and some other proteins is given in Table 46.
Yeast protein compares favorably w4th casein or meat with respect to
essential amino acids.

Less complete data are available for the amino-acid composition of
fleshy fungi. According to Lintzel (1941), the proteins of Psalliota
campestris, Cantherella cibarius. Boletus edulis, and Morchella esculenta
are about equal to animal protein. From 100 to 200 gr. (dry weight) of
these mushrooms was required to maintain the nitrogen balance in a man
weighing 70 kg. Fitzpatrick et al. (1946) found the tryptophane content
of P. campestris to be 5 mg. per 100 g.

268

PHYSIOLOGY OF THE FUNGI

Table 46. Approximate Amino-acid Composition (in Per Cent) of Some Plant
AND Animal Proteins Calculated to 16 Per Cent Nitrogen
(Block and Boiling, Arch. Biochem. 7, 1945. Published by permission of Academic
Press, Inc.)

Amino acid

Arginine

Histidine

Lysine

Tyrosine

Tryptophane .
Phenylalanine

Cystine

Methionine . . .
Threonine. . . .

Leucine

Isoleucine ....
Valine

Yeasts *

Meat

Casein

Corn
gluten

Max.

Min.

5.3

3.1

7.7

4.1

3.1

2.3

2.9

2.5

1.6

8.1

6.7

7.2

7.5

0.8

3.7

3.4

6.4

6.7

1.5

1.2

1.3

1.2

0.7

4.6

2.9

4.9

5.2

6.4

1.1

0.9

1.3

0.4

1.1

2.8

2.6

3.3

3.5

4.0

6.0

5.1

5.4

3.9

4.1

8.5

6.1

7.7

12.1

24.0

6.2

5.5

5.2

6.5

5.0

5.9

4.6

5.7

7.0

5.0

Polished
rice

7.2
1.5
3.2
5.6
1.3
6.7
1.4
3.4
4.1
9.0
5.3
6.3

* Eight strains analyzed.

The value of fungus protein in nutrition can be assessed only in relation
to the amino-acid composition of the remainder of the diet. If the
dietary proteins are low in certain essential amino acids, the supplemen-
tary value of yeast (or other) protein may be great. The cereal grains,
which furnish the bulk of protein for the population of the world, are
generally low in one or more essential amino acids. Usually cereal
protein is low in lysine or tryptophane or both. Sure (1946, 1947) studied
the effect on the growth of rats of adding 1, 3, and 5 per cent of dried
yeast to diets which contained cereals as the sole source of protein. The
most marked effect of yeast occurred on a maize diet. At the end of a
10-week experimental period the rats receiving only cereal weighed
27.3 g., while the rats which received an additional 1 per cent yeast
weighed 50.5 g. Rats which received the cereal plus 3 and 5 per cent
yeast weighed 91.8 and 109.9 g., respectively. The effect of yeast was
not so great when wheat or rice supplied the protein in the diet. In
general, the most promising use of yeast protein in human nutrition is
as a supplement rather than as a sole source of protein.

Yeasts are efficient in absorbing and concentrating the vitamins present
in the media in which they grow (Gorcica and Levine, 1942). The
relative value of yeast as a source of vitamins depends upon the vitamin
content of the other constituents of the diet. The prevalence of vitamin
deficiency diseases (beriberi, pellagra, and others) is evidence that the
vitamin content of many diets is inadequate.

METABOLIC PRODUCTS

269

A dramatic demonstration of the value of yeast as a source of vitamins
is reported by Bray (1928), onetime medical officer, Nauru, Central
Pacific. The mandating government prohibited the brewing of toddy
(palm wine) and allowed the sale of refined sugar. The results of these
dietary changes were appalling. Soon, 40 per cent of the infants born
in 1 year perished of infantile beriberi (thiamine deficiency) before reach-
ing the age of 6 months. The restoration of toddy and enforced con-
sumption of the dregs, i.e., the yeast, reduced the incidence of beriberi
to one death in 16 months. Truly, Bray was right in calling toddy the
elixir of life of the Nauruans. Piatt and Webb (1945) have noted that a
simple maize diet which was inadequate with respect to riboflavin and
nicotinic acid was made adequate in these respects by converting a por-
tion of the dietary maize into maize beer.

The vitamin content of yeasts depends upon the species or strain and
the conditions of cultivation. Some representative data are presented
in Table 47.

Table 47. Vitamin Content of Seven Food Yeasts
Results in milligrams per 100 g. of dry yeast. (Von Loesecke, Jour. Am. Dietet.
Assoc. 22, 1946. Published by permission of the American Dietetic Association.)

Species

Torula utilis

Saccharomyces cerevisiae*

S. cerevisiae

S. cerevisiae f

S. cerevisiae^

S. cerevisiae f

S. cerevisiaeX

Thiamine

1.7
17.0
20.5
17.5
17.5
16.0

3.0

Riboflavin

4.7
8.0
7.6
4.2
4.5
3.6
7.5

Nicotinic
acid

19.0
25.0
29.0
48.0
37,0
32.0
38.0

Pantothenic
acid

86.0

112.0
122.0
86.0
72.0
74.0
13.5

* Six per cent salt added.
t Debittered brewer's yeast.
t Primary yeast.

The production of fats by fungi is discussed elsewhere in this chapter.
The usual fatty acids, including palmitic and oleic acids, are found in fat
synthesized by fungi. Apparently few studies have been made on the
value of fungi as sources of fat and essential minerals in human nutrition.

CULTIVATION OF FUNGI FOR FOOD

The ants were perhaps the first to cultivate fungi as a source of food
(see Leaoii, 1940, for discussion and references). Fungi have been used
for centuries in the Orient as food for man. The Chinese grow Hirneola
polytricha and the Japanese grow Armillaria shii-take on oak saplings.
The mushroom cultivated almost exclusively in the Occident is Agaricus

270 PHYSIOLOGY OF THE FUNGI

(Psalliota) campestris. The method of cultivating this species on com-
posted horse manure was developed near Paris before 1700. For informa-
tion on mushroom growing the reader is referred to Duggar (1915).

While attempts to replace composted horse manure by other substrates
have been made, none appears to be entirely satisfactory. Humfield
(1948) has suggested that Psalliota campestris be grown in large fermentors
and the mycelium rather than the fruit bodies be used for food. Aspara-
gus butt juice, a waste agricultural product, is a suitable medium. The
chemical composition of mycelium and that of the fruit bodies is similar
and the flavor comparable. This approach perhaps offers a way to culti-
vate other desirable species, including the morels and the truffles. Nord
(1948) has suggested that the mycelium of Fusarium lini be used for food.

The use of yeasts to convert low-grade carbohydrates, such as wood
sugar and molasses, into food has interesting possibilities. It is necessary
to fortify these carbohydrates with other nutrients for the cultivation of
yeast. Phosphates, a source of potassium, and nitrogen, in the form of
urea, ammonia, or ammonium salts, are added. The function of yeast is
to convert inorganic nitrogen into protein. Animals are unable to
assimilate ammonia or urea directly but require nitrogen in the form of
protein or amino acids. Inorganic nitrogen may be converted into
proteins by green plants or by certain microorganisms. The use of urea,
a derivative of ammonia, as cattle fodder is an example of the synthesis
of protein by the microflora of the rumen.

The possibility of using wood waste for yeast propagation was investi-
gated in Germany during the First World War. In 1944 it is reported
that 9,000 tons of food yeast were produced in Germany. Fermentable
carbohydrates are obtained from wood as a by-product of sulfite paper
manufacture, or by direct hydrolysis. Before sulfite liquor or wood
hydrolysate is used for yeast culture, it is treated with calcium carbonate
to adjust the pH and precipitate impurities. After the addition of
nutrients the solution is heavily inoculated with the desired strain of
yeast. Aeration is necessary for high yields of yeast. The weight of
yeast produced amounts to about half the weight of sugar utilized. Such
yeast is approximately 50 per cent protein (Harris et al., 1948). The
economics of fodder-yeast production from sulfite liquor have been studied
by Schleef (1948). The use of by-product molasses for the production
of food and fodder yeasts should offer fewer technical difficulties than the

use of wood sugar.

FAT PRODUCTION

Serious efforts to utilize fungi for the synthesis of fats were made in
Germany during the First World War and continued thereafter. The
technical problems encountered proved difficult, but some success was
achieved by 1942 (Hesse, 1949). The controHing factor in fat production

METABOLIC PRODUCTS

271

appears to be the carbon-nitrogen ratio. As long as an adequate supply
of nitrogen is present, little fat is synthesized. If the carbohydrate sup-
ply is high when the nitrogen is exhausted, assimilable fat is synthesized.
Linder (1922) termed these two phases 'protein generation and Jat genera-
tion. Fat-laden cells of many fungi appear to be incapable of cell division.
Fat formation takes place only in the presence of an abundant supply
of oxygen. The relation between sugar concentration and amount of fat
synthesized by Penicillium javanicum is illustrated in Fig. 52.

C7>

2500

2000

Myce

//um"''

1500

Per

cent fat

innn

3»

200

300 400

Groms of glucose per liter

500

Fig. 52. The effect of the concentration of glucose on the amount of mycelium and
amount of fat synthesized by PenicilUum javanimm cultured in 75 ml. of medium for
12 days. (Drawn from the data of Lockwood, Catholic Univ. of America Biol. Ser.
13, p. 8, 1933. Published by permission of the Catholic University of America.)

Among the fungi investigated for fat synthesis are Endomyces vernalis,
Oidium lactis, Tonda utilis, Rhodotorula ghdinis, and species of Aspergil-
lus, Penicillium, Mucor, and Fusarium. From a practical standpoint,
only fungi which are capable of synthesizing fat in submerged culture
are of potential value. E. vernalis and 0. lactis do not produce fat effici-
ently in submerged culture. The fat content of various filamentous fungi
was determined by Preuss et al. (1934) and Ward et al. (1935). The use
of E. vernalis for fat and protein synthesis has been reviewed by Raaf
(1941). Starkey (1946) studied fat production by an unidentified soil
yeast, which under favorable conditions contained from 50 to 63 per cent

272 PHYSIOLOGY OF THE FUNGI

crude lipide. A list of species of Penicillium and Aspergillus which syn-
thesize considerable fat is given in Table 48.

Table 48. The Crude Fat Content op Dried Mycelium of Various Species op
PeniciUiuin and Aspergillus as Determined by Extraction with Ether
(Ward et al., Ind. Eng. Chem. 27, 1935. Published by permission of the American
Chemical Society.)

Species Crude Fat, %

Penicillium flavo-cinereum 28.5

P. piscarum 26-28

P. oxalicum 24.4

P. roqueforti 22.9

P. javanicum 22 . 2

Aspergillus flavus 16.0

Various theories of the mechanism of fat synthesis have been published
and are reviewed by Foster (1949) and Hesse (1949). Most of these
consider acetaldehyde or acetate to be the product of intermediary metab-
olism used in fat synthesis. This emphasizes the importance of pyruvic
acid in fungus metabolism. Various investigators have shown that
acetaldehyde may be converted into fat by yeasts. The glycerol required
for fat synthesis is thought to arise from the reduction and hydrolysis of
dihydroxyacetone phosphate or 3-phosphogly eerie aldehyde (scheme VI,

Chap. 7).

PRODUCTION OF VITAMINS

Only a few species of fungi and bacteria produce vitamins in large
enough amounts to be of interest in industry. Biological synthesis must
compete with chemical synthesis on a cost basis. The recovery of vita-
mins as a by-product of commercial processes or the use of waste materials
as the basis of a cheap medium may make biological synthesis attractive.

Riboflavin is produced so abundantly by Candida guilliermondi under
certain cultural conditions that it crystallizes in the medium (Burkholder,
1943). Among the factors found to influence the amount of riboflavin
synthesized, the sources of carbon and nitrogen and aeration are impor-
tant. Various investigators have found the concentration of iron in the
medium to have a profound influence on the amount of riboflavin syn-
thesized by various organisms. Iron concentrations in excess of 10 ^g
per liter decreased the amount of riboflavin synthesized by C. guillier-
mondi and C. fiareri (Tanner et al., 1945; Tanner and Van Lanen, 1947).
The optimum iron concentration for riboflavin synthesis by Clostridium,
acetobutylicum is said to be 1 mg. per liter. Hickey (1945) has suggested
the use of 2,2'-bipyridine to inactivate excessive concentrations of iron
in industrial fermentations. By maintaining the iron concentration
between 40 and 60 ng per liter, Levine et al. (1949) found the maximum
yields of riboflavin produced by C. guilliermondi and C. fiareri to be 175
and 567 ng per ml., respectively. Pilot-plant yields were somewhat less.

METABOLIC PRODUCTS 273

Eremothecium ashbyi was shown to produce as much as 157 mg. per
liter of riboflavin when cultivated on glucose-peptone medium (Renaud
andLachaux, 1945). Aeration was necessary. Foster (1947) has recom-
mended a molasses medium for the commercial production of riboflavin
by E. ashbyi. The closely related species, Ashbya gossypii, also synthe-
sizes riboflavin in large amounts (Tanner et al., 1949).

Peltier and Borchers (1947) determined the amount of riboflavin
produced by 240 isolates of soil fungi when grown on wheat bran. Forty-
five isolates produced 2 mg. or more of riboflavin per 100 g. of dry mold
bran. An unidentified species of Aspergillus produced 5.8 mg. of ribo-
flavin per 100 g. of substrate. Species of Fusarium and Aspergillus were
outstanding producers of riboflavin.

The commercial microbiological synthesis of riboflavin depends upon
the use of either E. ashbyi or C. acetobutyliciim (Tanner et al., 1949).

Vitamin B12 was isolated in crystalline form from liver and shown to
contain cobalt (Rickes et al., 1948; Smith, 1948). It is the only vitamin
so far discovered which contains a metal as an integral part of the mole-
cule. Streptomyces griseus and other microorganisms synthesize this
vitamin. Sheep and cattle pastured on cobalt-deficient soils (Florida,
Australia, New Zealand) develop a deficiency disease. Ingested cobalt
is more effective than injected cobalt in overcoming this condition. It
may be assumed that cobalt is used in the synthesis of vitamin B12 by the
action of the microorganisms of the rumen and intestine. Vitamin B12
appears to be the anti-pernicious-anemia factor (West, 1948). Whether
it is the animal protein factor is undecided. The cow-manure factor may
be vitamin B12 (Lillie et al., 1948).

Until the structure of vitamin B12 is determined and methods of syn-
thesis developed, certain natural products will remain the only source
of this vitamin. The only organic moiety of vitamin B12 so far disclosed
is l-a-D-ribofuranosido-5,6-dimethylbenzimidazole (Brink et al., 1950).
Vitamin B12 is obtained as a by-product from various industrial processes,
especially streptomycin production. It is evident that the medium must
contain cobalt; within limits, the amount of vitamin B12 synthesized by
Streptomyces griseus is a function of the cobalt content of the medium.
Maximum synthesis was observed when the medium contained 1 to 2 mg.
of cobalt per liter (Hendlin and Ruger, 1950).

None of the other vitamins appears to be synthesized by fungi in
amounts which would make the latter attractive sources for the isolation
of pure vitamins. The value of these vitamins in fungi used for food was
discussed previously. Yeast can be fortified with thiamine so that it
may serve as a therapeutic agent. By adding synthetic thiamine to an
aerated yeast culture, yeast was produced which contained 6 mg. of
thiamine per g. (Van Lanen et al., 1942).

274 PHYSIOLOGY OF THE FUNGI

ENZYME PRODUCTION

The industrial production and use of enzymes from microorganisms
in the Occident is fairly recent, although the use of fungi as amylolytic
agents by the peoples of the Orient for the preparation of koji and other
foods is an old art. For this purpose, mixed cultures of species of Asper-
gillus and Rhizopus are grown upon the rice or soybean substrates, the
enzymes being used without separation. The pioneering work of Taka-
mine (1914) on the amylases of A. oryzae was especially important.

The ability of fungi to produce amylase is widely distributed, but only
a few species are used commercially for this purpose. The amount of
amylase produced varies with the species or isolate and the cultural condi-
tions. Le Mense et al. (1947) screened 359 isolates of Penicillium and
Aspergillus and found 42 isolates to produce amylase in submerged
culture. The activity of the species of Penicillium ranged from 0.1 to
0.6 enzyme unit per milliliter of culture medium. One isolate of A.
niger (NRRL 337) was found to be especially adapted for the production
of amylase in submerged culture. The production of amylase was highly
dependent upon the composition of the medium. Corn meal was espe-
cially valuable in increasing amylase production when added to basal
media composed of corn steep liquor, dried tankage, soybean meal, or
thin stillage. Amylase production was stimulated by the addition of 10
to 20 mg. of sodium chloride per liter of culture medium. Addition of a
mixture of chlorinated phenols (Dowcide G) inhibited sporulation and
increased amylase production (Erb et al., 1948).

Others have found different isolates of the same species to produce
varying amounts of amylase. Hao et al. (1943) studied the production
of amylase by 27 isolates of various species of fungi when grown upon
wheat bran. A. oryzae, Rhizopus delemar, and R. oryzae produced the
largest amounts of amylase. A. oryzae was the fungus of choice because
of ease of handling.

In practice, fungus amylases are produced and utilized in three general
ways. (1) In the amylo process, starch is solubilized by autoclaving with
a trace of a mineral acid, and the mash is inoculated with a species of
Rhizopus, which produces amylase abundantly, and a species of yeast.
The function of the Rhizopus species is to convert the starch into ferment-
able sugars, from which the yeast produces alcohol. For a description
of this process see Owen (1933). (2) The fungus may be grown upon a
solid substrate such as bran and the resulting moldy mass (mold bran)
dried (Underkofler et al., 1946). The fresh material may be used without
drying (Roberts et al., 1944). (3) Fungus amylases may be produced in
submerged aerated cultures much as antibiotics are produced. The
culture medium may be used directly to replace malt as a saccharifying
agent.

METABOLIC PRODUCTS 275

Fungus amylases are used to replace malt amylase for the saccharifica-
tion of starch, Myrback (1948) is of the opinion that amylase from A.
niger is an a-amylase, but it differs from a-amylase of malt in that it has a
higher capacity for saccharification. For a comparison of fungus and
malt amylase and the economic considerations involved, see Underkofler
et al. (194G). The yield of alcohol is said to be slightly higher when
fungus amylase is used in place of malt for saccharification.

Fungi are the source of other enzymes of commercial interest, including
pectinase and sucrase. Pectinase is used in the clarification of fruit
juices. For a survey of the commercial production of fungus enzymes
seeL. Wallerstein (1939).

ALCOHOLIC FERMENTATION

Yeasts are used almost exclusively for the commercial production of
fermentation alcohol, but alcoholic fermentation is not restricted to these
fungi. Pasteur (1872) observed that Penicillium glaucum, Aspergillus
glaucus, and Mucor raceniosus produced alcohol under anaerobic condi-
tions. Further information on alcohol production by filamentous fungi
may be found in the monograph of Raistrick et al. (1931), who determined
complete carbon balances for 96 species of Aspergillus, 75 species of
Penicillium, 8 species of Citromyces (Penicillium) , 23 species of Fusarium
and 36 miscellaneous species. The original report should be consulted
for details and the quantitative methods used. All the 23 species of
Fusarium studied produced alcohol. From this and other reports in the
literature, it must be concluded that this property is common among
species of this genus. Many species of Aspergillus and Penicillium pro-
duced alcohol, as did species of other genera. Only a few of the species
studied failed to produce detectable amounts of alcohol. The apparatus
used in these studies is shown in Fig. 53.

The concentration of alcohol which inhibits the growth of fungi varies
with the species or strain. In general, yeasts are more tolerant of alcohol
than the filamentous fungi. The upper limit for most yeasts is about 12
per cent alcohol, although some strains are more tolerant. The suscepti-
bility to alcohol limits the alcohol concentration of naturally fermentt>?
beverages. The rate of fermentation decreases as the concentration (k
alcohol increases.

Not all isolates of a species are equally efficient in producing alcohcu
For example, eight isolates of Fusarium lini produced varying amountfe
of alcohol on the same medium. The more virulent pathogens on flax
produced the most alcohol (Letcher and Willaman, 1926). A correlation
between sporulation and alcohol production by Aspergillus flavus was
noted by Yuill (1928). In general, sporulating cultures produced less
alcohol than nonsporulating cultures.

The most important condition w^hich governs alcoholic fermentation

276

PHYSIOLOGY OF THE FUNGI

^ ^

r^HI

sterilization

Cone.

Cotton wool

mh:

Fig. 53. Apparatus for studying the metabolic products of fungi and other micro-
organisms. The apparatus consists of five units: A gasholder, P; a train for the puri-
fication and sterilization of air or other gases, A-E\ the culture flask, F; a train for the
quantitative absorption of carbon dioxide, H-M; an aspirator, Q, for the collection
of gaseous products of metabolism other than carbon dioxide. (Redrawn from Birkin-
shaw and Raistrick, Trans. Roy. Soc. (London), Ser. B, 220: 14, 1931. Published by
permission of the Royal Society.)

is the supply of oxygen. The relation between fermentation and anaero-
bic conditions was recognized by Pasteur, who summarized his extensive
investigations on fermentation as "la vie sans air." The essential feature
of fermentation is anaerobic dissimilation of carbohydrates. Growth and
fermentation are competitive processes, for fungi require oxygen for
growth. In practice it is advantageous to carry out fermentations in the

METABOLIC PRODUCTS 277

presence of some air, especially at the start. This allows some increase
in the number of cells and reduces the amount of inoculum required. The
amount of oxygen available to submerged mycelium or cells, unless
vigorous aeration is used, is insufficient to inhibit alcoholic fermentation
by certain species.

Alcoholic fermentation has been studied since the time of Lavoisier.
Few fields of study have been so valuable in increasing our understanding
of the life processes of microorganisms. Harden (1932) has concisely
reviewed the early work and theories on fermentation. The idea that
yeasts as living fungi were the proximate cause of fermentation did not
gain acceptance for many decades. The eminent Wohler (1839) ridiculed
this idea in a lively skit, in which he declared that he had followed the
entire process microscopically. Briefly, he states that the responsible
organism developed from an egg and had the shape of a Beindorf distilling
flask; "... diese Infusorien fressen Zucker, entleeren aus dem Darm-
kanal Weingeist, und aus den Harnorganen, Kohlensaure."

The enzymatic nature of alcohol fermentation was established by
Buchner (1897). The enzymatic transformations involved in fermenta-
tion were discussed in Chap. 7. Further information and references may
be found in Summer and Somers (1947), Tauber (1949), Prescott and
Dunn (1949), Meyerhof (1944, 1949), Nord and Mull (1945), and Foster
(1949).

The larger part of the world-wide fermentation industry is devoted
to the production of ethyl alcohol. During the war year of 1945 some
600 million gallons of 95 per cent ethyl alcohol was produced in the United
States alone. Less than one-third this amount was produced in 1948.
Of this amount 64 per cent was produced by fermentation (Lee, 1949).
While any source of fermentable sugars may be used for the production
of alcohol, the more common raw materials include molasses, starch from
various sources, hydrolyzed cellulose or wood sugar, and fruit juices. It
is beyond the scope of this text to discuss the commercial production of
industrial and beverage alcohol. For information on these subjects see
Prescott and Dunn (1949).

ORGANIC ACIDS

Many fungi synthesize organic acids, which accumulate in the medium.
These acids include oxalic, citric, succinic, fumaric, malic, lactic, itaconic,
kojic, gluconic, and others. Commonly, a species may synthesize a
variety of related acids. The isolates of a given species may differ widely
in synthetic capacity. To obtain maximum yields, it is necessary to
control nutritional and environmental factors closely. The optimum
conditions for one isolate may differ from those of another isolate of the

278 PHYSIOLOGY OF THE FUNGI

same species. Acid production by fungi is discussed in detail by Foster
(1949), Prescott and Dunn (1949), and Wallvcr (1949).

The meaning of the term fermentation has been expanded by most
authors to include aerobic as well as anaerobic processes. The produc-
tion of most organic acids and antibiotics Ijy fungi takes place in the
presence of oxygen, and these processes are not fermentations in the
restricted (anaerobic) sense of the term. Indeed, adequate aeration is
one of the salient features of such processes. Aeration may be achieved
by cultivating the fungi on the surface of shallow layers of medium in
pans or trays ; or the fungi may be cultivated in closed tanks, which may
contain as much as 15,000 gal. of medium. Aeration is provided by
mechanical stirring and blowing in sterile air under pressure.

The organic acids discussed in this chapter are derived from carbo-
hydrates present in the medium. In general, media highly unbalanced
with respect to carbohydrates are used. The balanced medium devel-
oped by Steinberg for the cultivation of Aspergillus niger (Chap. 2) has
a carbon-to-nitrogen ratio of 29 to 1, while the medium recommended by
Currie (1917) for the production of citric acid by ^. niger has a carbon-to-
nitrogen ratio of 72 to 1. A fungus first utilizes the nutrients in the
unbalanced medium for the production of mycelium (growth phase).
The excess carbohydrate which remains when the nitrogen is exhausted
is dissimilated ("fermentation" phase). Advantage is taken of such
preformed mycelium, for if the original medium is replaced by fresh
medium, the mycelium continues to dissimilate carbohydrate. The
replacement medium is frequently more unbalanced than the growth
medium. For example, Karow and Waksman (1947) used for A. wentii
a growth medium with a carbon-to-nitrogen ratio of 135 to 1, while the
replacement medium had a carbon-to-nitrogen ratio of 270 to 1.

Economic amounts of organic acids may accumulate in the medium
because the normal use of these compounds for the synthesis of mycelium
is prevented by the imposed experimental conditions. If the nitrogen
supply is exhausted, no more protoplasm can be formed. The mycelium
then dissimilates sugars enzymatically. Enough nutrients are supplied
in replacement media to repair and maintain the enzyme systems of the
fungus in a vigorously functioning state. The enzymes, other than those
concerned with certain phases of carbohydrate dissimilation, are largely
idle because of the lack of suitable substrates.

A fungus commonly produces several organic acids at the same time.
Citric and oxalic acids are produced by many isolates of A . niger, and the
relative amounts of these acids may be varied by controlling the pH of
the medium. In general, a highly acid medium (pH 2.0 to 3.0) favors the
synthesis of citric acid, while less acid media favor the production o/
oxalic acid.

METABOLIC PRODUCTS

279

Citric acid. Wehmer was the first to recognize the commercial possi-
bilities of citric acid synthesis by two species of Citromyces {Penicillium).
Selected isolates of Aspergillus niger appear to be used in industry,
although the propertj^ of producing citric acid is common to many fungi.
The following fungi have been suggested for commercial citric acid pro-
duction (Von Loesecke, 1945): Citromyces pfejferianus, C. glaber, C.
citricus, Aspergillus carhonarius, A. glaucus, A. clavatus, A. cinnamomeus,
A.fumaricus, A. awamori, A. aureus, Penicillium arenarium, P. olivaceum,
P. divaricatum, P. sanguifluus, P. glaucum, Mucor pyriformis.

The production of citric acid in the United States increased from about
5 million to 26 million pounds between 1935 and 1945 (Von Loesecke.
1945). Presumably most of this was "fermentation" citric acid. At
present it is beheved that most citric acid is produced by surface cultures.

Citric acid is formed from many sources of carbon. Sucrose is said
to be the best carbon source for the production of citric acid. There is
less agreement upon the value of other sugars. Different investigators
have found glucose, fructose, and maltose to vary from good to poor. In
part, this is to be attributed to the use of different isolates and different
experimental conditions. Beet molasses is used in industry. The
suitability of this substrate is said to vary with the source and year of
production (Bernhauer and Knobloch, 1941). The evaluation of carbon
sources is complicated by the metallic elements they contain, especially
iron and manganese. Methods of treating beet and cane molasses to
remove inhibiting impurities are described by Perlman et al. (1946),
Gerhardt et al. (1946), and Karow and Waksman (1947). The inhibiting
effect of metallic ions on the production of citric acid from sugars is illus-
trated by the data in Table 49.

Table 49. The Effect of Removing Metallic Contaminants from Three
Sugars, by the Process of Cationic Exchange, on the Prodlction of
Citric Acid by Aspergillus niger, Wisconsin Strain 62
(Perlman et al., Arch. Biochem. 11, 1943. Published by permission of Academic
Press, Inc.)

Sugar used

Treatment

Yield*

of citric acid, %

Sucrose from cane

Not treated

21.4

Treated

64.0

Sucrose from beet

Not treated

11.3

Treated

66.8

Glucose

Not treated

20.5

Treated

60.0

* Theoretical yield 123 per cent.

The production of citric acid in submerged culture was tried at an early
date and abandoned in favor of surface culture. However, recent litera-

280 PHYSIOLOGY OF THE FUNGI

ture indicates that submerged culture may be the preferred process in
the future. Average yields of 72 g. of anhydrous citric acid per 100 g.
of sucrose in the medium have been obtained in the laboratory (Shu
and Johnson, 1948).

The formula for citric acid is given below:

CH2— COOH

I
HO— CH— COOH

CH2— COOH

Citric acid

Any theory of citric acid formation must take into account the following
facts: Citric acid, a branched-chain compound, is synthesized from carbon
sources containing from two to seven carbon atoms. Yields of citric
acid may approach 90 per cent of the sugar used (Wells et al., 1936).
The amount of carbon dioxide evolved is low, which suggests either
reutilization or a mechanism of producing the necessary intermediates
without the production of carbon dioxide. Reutilization of carbon
dioxide seems the more probable, for Foster et al. (1941) showed Asper-
giUus niger to utilize radioactive carbon dioxide in the synthesis of citric
acid. The more probable pathway of synthesis is via the Krebs cycle
(Chap. 7) and the supplementary formation of oxalacetic from pyruvic
acid and carbon dioxide (Wood-Werkman reaction).

Gluconic acid. A considerable number of fungi produce gluconic acid.
These include Aspergillus niger (various isolates), A. fuscus, A. cinna-
momeus, A. oryzae, Penicillium glabrum, P. glaucum, P. purpurogenum
var. ruhrisclerotium, P. chrysogenum, P. crustaceum, and Fumago vagans.
Most investigators have used selected isolates of A. niger for the produc-
tion of gluconic acid. Details of laboratory and semi-pilot-plant investi-
gations may be found in the papers of Wells et al. (1937), Gastrock et al.
(1938), and Forges et al. (1941).

Many factors influence the formation of gluconic acid. Isolates of
A. niger differ in ability to synthesize this acid. Not all isolates produce
the maximum amount of acid under identical conditions. Adequate
aeration is necessary for the enzymatic conversion of glucose to gluconic
acid. Gluconic acid is produced most abundantly when the pH of the
medium is kept near 5. Calcium carbonate is used for neutralizing the
gluconic acid formed. This is advantageous, for calcium gluconate is
used in medicine as a source of readily assimilable calcium. Frecipitation
of calcium gluconate during formation may be prevented by the addition
of boric acid or borax to the culture medium in amounts vaiying up to
2,000 p. p.m. (Moyer et al., 1940). Boron compounds are added after
the growth of mycelium is essentially complete. The mycelium may be
used as many as thirteen times by removing the spent medium and adding

METABOLIC PRODUCTS 281

fresh medium with a high glucose content but low in other nutrients
(Forges et al, 1940, 1941).

The production of gluconic acid appears to be a direct oxidation of
glucose. The enzyme responsible for this transformation is called glucose
aerodehydrogenase. This enzyme, when free from catalase, catalyzes a
reaction between glucose and oxygen. Gluconic acid and hydrogen
peroxide are the products formed. Glucose aerodehydrogenase was first
isolated from Penicillium chrysogenum and was called notatin, or penicil-
lin B, at first. Its antibiotic activity is due to liberation of hydrogen
peroxide. For recent papers on this enzyme see Keilin and Hartree
(1948, 1948a).

Lactic acid. Various lactic acid bacteria are used in the commercial
production of lactic acid. These bacteria require a complex natural
medium, which makes the purification of lactic acid laborious. Many
species of Phycomycetes produce lactic acid, and species of Rhizopus are
noteworthy in this respect. The following fungi produce lactic acid:
Rhizopus arrhizus, R. chinensis, R. elegans, R. japonicus, R. nodosus, R.
oryzae, R. pseudodiinensis, R. salehrosus, R. shanghaiensis, R. stolonifer,
R. tritici, Mucor rouxii, Monilia tamari, and Blastocladia pringsheimii.
Most of these fungi appear to synthesize c?-lactic acid, although R.
chinensis synthesizes Wactic acid (Saito, 1911).

The use of R. oryzae for production of lactic acid has been intensively
investigated (Lockwood et al., 1936; Ward et at., 1936, 1938). Glucose
appears to be the best sugar. Nitrate nitrogen is not used by this fungus.
Calcium carbonate is used in the medium to neutralize lactic acid as it is
formed. Yields increase when the cultures are aerated. As much as
75 per cent of the glucose utilized is converted into lactic acid. The
presence of added zinc increases mycelial growth but depresses the yield
of lactic acid.

The mechanism of lactic acid production by fungi is ably discussed by
Foster (1949). Under anaerobic conditions, ethyl alcohol, carbon
dioxide, and lactic acid are produced in equimolecular amounts. The
amount of lactic acid produced under aerobic conditions increases, while
the amount of alcohol decreases (Waksman and Foster, 1939). The
most probable intermediate for the production of lactic acid is pyruvic
acid.

Itaconic acid. Aspergillus itaconicus was the first fungus reported to
synthesize itaconic acid. The structural formula below shows that this
unsaturated acid is related to succinic acid.

CH2=C— COOH

HoC— COOH
Itaconic acid

282 PHYSIOLOGY OF THE FUNGI

The fungi which have been tested for itaconic acid production are mainly
selected isolates of A. terreus. Relatively few isolates produce sufficient
itaconic acid to have commercial possibilities (Calam et al., 1939; Moyer
and Coghill, 1945).

Various attempts have been made to produce mutants of A. terreus
by irradiating conidia with ultraviolet light (Raper et al., 1945). Less
success attended these efforts than comparable treatment of conidia of
Penicillium chrysogenum for obtaining mutants with enhanced penicillin
production.

Among the factors which affect the production of itaconic acid by
isolates of .4. terreus are the composition of the medium, hydrogen-ion
concentration, temperature, and aeration. Glucose and ammonium
nitrate appear to be the best sources of carbon and nitrogen. The pH
range in which itaconic acid accumulates is narrow and low, 1.9 to 2.3.
The aluminum ion is toxic to A. terreus, but aluminum trays may be used
if the concentration of magnesium ion in the medium is high. As much
as 4.75 g. of magnesium sulfate heptahydrate per liter of medium may be
used. It is probable that this high concentration of magnesium ion also
enables the fungus to withstand low pH values (Lockwood and Ward,
1945).

Fumaric acid. This unsaturated, four-carbon, dicarboxylic acid is
produced by many fungi, although only a relatively few species synthe-
size large amounts. With few exceptions, the fungi which synthesize
fumaric acid in significant amounts are Phycomycetes. The formula for
fumaric acid is given below:

HOOC— CH

II
HC— COOH

Fumaric acid

The factors which affect the production of fumaric acid by Rhizopus
nigricans were studied by Foster and Waksman (1939). The concentra-
tion of zinc was found to be especially important. Optimum production
of fumaric acid occurred in cultures receiving less zinc than that required
for optimum growth. Not all isolates of R. nigricans synthesized fumaric
acid in equal amounts or under the same conditions. One isolate studied
by Foster and Waksman (1939a) produced fumaric acid anaerobically
and aerobically, whereas another produced fumaric acid aerobically only.

Various proposals have been made to explain the mechanism of fumaric
acid formation. Anaerobic synthesis is thought to involve the formation
of oxalacetic acid from pyruvic acid and carbon dioxide (Foster and
Davis, 1948). The follo^^•ing steps would convert oxalacetic acid to
fumaric acid: oxalacetate — ^ malate — > fumarate. It is probable that
fumaric acid is produced aerobically from acetic acid as follows: 2 (ace-

METABOLIC PRODUCTS 283

cate) —> succinate —> fumarate (Thunberg-Wieland condensation). R.
nigricans produces high yields of fumaric acid from both ethyl alcohol
and acetic acid, which is evidence in favor of this scheme of formation
(Foster and Waksman, 1939).

Other organic acids. Apparently, the first organic acid to be dis-
covered as a product of fungus metabolism was oxalic acid. Many fungi
in nature contain calcium oxalate crystals. This was noted as early as
1887 by De Bary. Many species of Aspergillus and Penicillium produce
large amounts of oxalic acid, especially if enough alkali is present in the
medium to convert the acid into an oxalate. Many species of Aspergillus
which produce oxalate in the presence of a neutralizing agent also produce
citric acid in acid media (Currie, 1917). For a recent discussion of oxalic
acid production by fungi see Foster (1949).

Various species of Aspergillus, including .4. oryzae, A. flavus, A. nidu-
lans, A. giganteus, and some other fungi produce kojic acid. Kojic acid
is a cyclic compound, a pyrone, and has been shown to have antibiotic
properties (Morton et al., 1945).

ESTERS

Among the esters reported to be formed by fungi are ethyl acetate,
methyl cinnamate, methyl p-methoxycinnamate, and isobutyl acetate.
Various reports are in the literature concerning a "banana-oil" odor
being produced by fungi, but apparently amyl acetate has not been
isolated and identified as a product of fungus metabolism. Ethyl acetate
is produced by Penicillium digitatum (Birkinshaw et al., 1931) and by
Endoconidiophora moniliformis (Gordon, 1950).

ANTIBIOTICS AND DRUGS

The inhibition of one organism by another is called antagonism. The
phenomenon has been known since the time of Pasteur, and the subject
has been reviewed by Waksman (1947) in a book containing over 1,000
references. Antagonism occurs in nature as well as in the laboratory
and is of such common occurence that it is frequently overlooked. Exam-
ples are easily found by examining contaminated plates for clear areas
around the contaminants. Antagonism may be due to competition for
nutrients or to toxic substances. This discussion will deal wdth the toxic
substances produced by fungi which inhibit fungi and bacteria.

General discussion. Fungi and other organisms produce a variety
of toxic substances, which include enzymes, alkaloids, toxins, simple and
complex organic compounds, and inorganic compounds. Organic com-
pounds produced by fungi and other organisms, especially bacteria and
actinomycetes, which inhibit the life processes of microorganisms are
called antibiotics. Waksman (1947) would restrict the term antibiotic

284 PHYSIOLOGY OF THE FUNGI

to organic compounds produced by microorganisms which inhibit the
functioning of other microorganisms. General usage of the term anti-
biotic is, however, wider than this and appHes the term to those organic
compounds of fairly simple structure produced by organisms which inhibit
microorganisms. These substances are referred to more specifically as
antibacterial, antifungal, or antiviral substances.

There are no universal antibacterial or antifungal substances. Anti-
biotics are specific in action. Penicillin, for example, is active against

Fig. 54. Method of assay for antibiotics. A, control culture of Penicillium notatum
on agar medium; radial series of plugs cut at 6 days. B, agar-plug assay plate show-
ing zones of inhibition of Staphylococcus developed after agar blocks removed from A
have been incubated for 16 hr. at 37°C. (Courtesy of Raper, Alexander, and Coghill,
Jour. Bad. 48: 644, 1944. Published by permission of The Williams & Wilkins
Company.)

many Gram-positive bacteria and only a relatively few Gram-negative
organisms.

The occurrence of antibiotics is probably far more widespread than
suspected at present. The reason for this lies in the way in which anti-
biotics are discovered. Antibiotics are detected by their inhibiting action
on living organisms. A susceptible test organism is essential for the
detection of an antibiotic. For obvious reasons, human pathogenic
bacteria are most used for screening tests. If one desires to obtain anti-
fungal substances active against pathogenic fungi, these fungi should be
used as test organisms.

The same principle underlies all methods for detecting antibiotic action.
The test organisms are brought into contact with the products elaborated
by the organism suspected of producing an antibiotic. This may be done
by growing two organisms on the same Petri dish. A clear zone between

METABOLIC PRODUCTS 285

the colonies indicates inhibition (Fig. 45). A second method consists
in growing an organism on agar and cutting radially a series of agar plugs
and placing these agar disks, which contain the antibiotic, on agar plates
sown uniformly with the test organism (Raper et al., 1944). This method
is illustrated in Fig. 54. Other methods of detecting antibiotics have
been summarized by Waksman (1947).

Fig. 55. The antibiotic effect of Streptomyces sp. on two plant pathogenic fungi,
Monilinia fructicola, on the left, and Helminthosporiuin sativum, on the right.

The production of antibiotic substances by fungi is common. In a
screening test of over 400 species, which included over 300 wood-inhabit-
ing fungi and 22 dermatophytes, somewhat over 200 species produced
substances active against Staphylococcus aureus and Escherichia coli
(Robbins et al., 1945). A large number of Basidiomycetes and other
fungi have been tested for the presence of antibiotics by Wilkins and
Harris (1944). The actinomycetes are the source of many useful anti-
biotics including streptomycin, Chloromycetin, aureomycin, terramycin,
and other unidentified compounds (Waksman, 1947). With the excep-
tion of Phytophthora erythroseptica none of the Phycomycetes appear to
have been reported as producing antibacterial substances. For a survey
of Fungi Imperfecti in the role of producing antibacterial substances
(against Staphylococcus aureus) and antifungal substances (against
Botrytis allii), see Brian and Hemming (1947). The inhibiting effect of
Streptomyces sp. on two plant pathogenic fungi is shown in Fig. 55.

Many soil organisms produce antibiotics. Whether these organisms
produce antibiotics in sufficient amounts to inhibit plant pathogens under
natural conditions in the soil is not certain. It is known, however, that

286

PHYSIOLOGY OF THE FUNGI

the incidence of certain diseases may be decreased by adding certain
bacteria, actinomycetes, and fungi to soil. For references, see Grossbard
(1948), Henry (1931), Waksman (1937), and Anwar (1949).

The influence of various soil-inhabiting organisms in decreasing infec-
tion of barley by Helminthos'porium sativum has been reported by Anwar
(1949). Figure 56 illustrates some of these results. It is by no means
certain that these effects were due to the antifungal substances produced
by the antagonistic organisms.

Fig. 56. The effects of certain soil organisms on the pathonogcnicit}^ of Helmintho-
s'porium sativum on barley. Seedlings grown at 80°F. in steamed soil infested with:
A, no organisms; B, H. sativum and Bacillus subtilis: C, H. sativum and Penicillium sp. ;
D, H. sativum and Trichoderma lignorum: E, H. sativum. (Courtesy of Anwar,
Phytopathology 39: 1011, 1949.)

The situation in soil is very complicated. Basic antibiotics such as
streptomycin are adsorbed on clay; acidic antibiotics like clavacin are
apparently held less firmly. Gottlieb and Siminoff (1950) are of the
opinion that competition is more of a factor than antibiotic action as the
cause of one organism inhibiting another in the soil. Thus, either As-per-
gillus clavatus or Streptomyces griseus inhibits the growth of Bacillus
suhtilis in soil. No difference was noted between a strain of S. griseus
which produced streptomycin and one which did not.

Schatz and Hazen (1948) reported that 124 of the 243 soil Actiyiomyces

METABOLIC PRODUCTS

287

tested were antagonistic to four test human pathogens, Candida albicans,
Cryptococciis neoformans, Trichophyton gypseum, and T. rubrum.

Table 50. Antibiotics Produced by Soil-inhabiting Actinomycetes and Fungi
(Brian, Cheni. Industry 1949. Published by permission of the Society of Chemical

Industry.)

Organism

Streptomyces griseus

Nocardia gardneri
Actinomyces lavendulae

Proactinomyces cyaneus

Streptomyces venezuelae

Aspergillus jlavus

A. terreus

Fusarium orthoceras

Penicillium brevi-compactum

P. chrysogenum

P. griseofulvum
P. janczewiskii
P. patulum

Trichoderma viride

Antibiotic

Grisein

Actidione
Streptomycin

Proactinomycin
Streptothricin

Litmocidin

Chloromycetin

Aspergillic acid

Citrinin

Enniatin B

Mycophenolic acid

Penicillin

Griseofulvin
Griseofulvin
Patulin

Gliotoxin
Viridin

Properties

Antibacterial (Gram positive and
negative) ; antirickettsial ; not
antifungal

Not antibacterial; antifungal

Antibacterial (Gram positive and
negative and acid fast)

Antibacterial (Gram positive)

Antibacterial (Gram positive) ;
antifungal

Antibacterial (Gram positive and
negative)

Antibacterial (Gram positive and
negative); not antifungal; anti-
rickettsial

Antibacterial (Gram positive and
negative); antifungal

Antibacterial (Gram positive and
acid fast) ; not antifungal

Antibacterial (Gram positive and
negative); antifungal

Antibacterial (Gram positive) ; not
antifungal

Not antibacterial; antifungal

Antibacterial (Gram positive and
negative) ; antifungal

Antibacterial (Gram positive and
negative and acid fast) ; anti-
fungal

Not antibacterial; antifungal

A list of antibiotics produced by some soil-inhabiting actinomycetes
and fungi is given in Table 50. Note that some organisms produce more
than one antibiotic and that the same antibiotic substance may be pro-
duced by more than one species. Organisms differ in susceptibility to
antibiotics. This range of effectiveness is frequently called the antibioHc
spectrum. Thus, Penicillum luteum-purpurogenum is some 12 thousand
times as sensitive to gliotoxin as to streptomycin. Not all fungi are
equally inhibited by the same concentration of an antibiotic; some 11

288 PHYSIOLOGY OF THE FUNGI

times as much clavacin is required to inhibit the growth of Aspergillus
clavatus as Trichophyton mentagrophytes (Reilly et al., 1945).

Fungi produce substances which are capable of inactivating certain
plant viruses. The Basidiomycetes are especially noteworthy in this
respect (Utech and Johnson, 1950). Extracts of Trichotheciiim roseum
reduce infectivity of southern bean mosaic, tobacco mosaic, and tobacco
necrosis viruses (Gupta and Price, 1950). These authors believe that
this reduced infectivity is due to increased resistance of the host. There
is no evidence which indicates that any of the known antibiotics are
involved in the destruction of plant viruses. However, antibiotics are
known which are effective against virus diseases in man.

Preliminary studies indicate that certain antibiotics may be used to
control fungi which cause plant diseases. Actidione has been reported
by Vaughn et al. (1949) to control powdery mildew on beans and roses.
Actidione was toxic to young rose leaves at a concentration of 2.5 p.p.m.
but less toxic to bean plants. Laboratory tests indicated that actidione
is a fungistatic substance for a considerable number of plant pathogenic
fungi, including Sclerotinia fructicola, Cladosporium cucumerinum, and
Colletotrichum lagenarium. Further data on the effect of actidione on
plant pathogenic fungi are reported by Whiffin (1950).

The protective action of an antibiotic obtained from an unidentified
species of Streptomyces against Venturia inaequalis on apple has been
reported by Leben and Keitt (1949). This antibiotic has been named
antimycin.

Penicillin has been used successfully, to a limited extent, in controlling
necrosis of giant cactus, caused by Erwinia carnegieana (Boyle, 1949).
Injections of penicillin into the necrotic tissue apparently diffused through
the plant tissues for some distance, killing the bacteria. This is one of
the few cases in which an antibiotic has been used successfully in thera-
peutic treatment of plant disease.

The principal use of antibiotics is to control disease in man and animals.
Only a relatively few antibiotics are useful for this purpose. In addition
to killing or inhibiting pathogenic organisms, an antibiotic, to be useful
in medicine, must be relatively nontoxic to the host. Some of the older
and more useful antibiotics used in medicine will be discussed in greater
detail on the following pages.

Penicillin. This antibiotic drug is produced in industry by selected
isolates or mutants of Penicillium chrysogcnum and P. notatum. The
original isolate of Fleming produced from 2 to 4 units of penicillin per
milliliter of culture filtrate. P. chrysogenum Q-17Q has produced in
excess of 1,000 units per milliliter. The synthesis of penicillin is not
limited to species of the P. chrysogenum-notatum group but includes cer-
tain species of Aspergillus belonging to the A. flavus group. A few fungi

METABOLIC PRODUCTS

289

belonging to other genera also produce penicillin. Sterile culture condi-
tions must be maintained at all times, as penicillin is rapidly destroyed
by the enzyme, penicillinase, which is excreted by many bacteria. Peni-
cillin is extracted from the "fermentation" liquid, or penicillin beer,
either by extraction at pH 2.0 to 2.5 with water-immiscible solvents such

R—

— CO— NH— CH-
0=0—

-CH C— (CH3)2

-N CH— COOH

Type formula of the penicillins

as amyl acetate, or by adsorption on activated carbon. The extraction
must be carried out quickly from acid solutions, owing to the instability
of penicillin under these conditions. Penicillin forms crystalline salts
with the alkali metals. The sodium salt is usually produced.

Table 51. Names and American and British-type Designations of Four

Naturally Occurring Penicillins

Name

Type

American

British

Benzylpenicillin
A^-Pentenylpenicillin
p-HydroxybenzylpenicillLn
n-Heptylpenicillin

G
F
X
K

CHs- CHo— CH=CH— CHo—

HOC6H4CH2—

(j-H.3(0-H.'>)5 — C-H.'> —

III

Penicillin, as produced by P. chrysogenum, is a mixture of related com-
pounds. The ratios among the various penicillins depends upon the
isolate and conditions used. By the use of suitable precursors the yield
of the desired compound, penicillin G, is greatly increased. This is
desirable because penicillin G salts crystallize well and are most useful
in medicine. The type formula for the penicillins is shown below. The
precursors used for the production of penicillin G are related to phenyl-
acetic acid. Many other penicillins are known, some of which are pro-
duced only in the presence of precursors which do not occur in nature
(Behrens, 1949).

The growth of the penicillin industry in the United States is shown by
the production figures in Table 52.

Penicillin is chiefly active against Gram-positive bacteria. A few
important Gram-negative pathogens, including Neisseria gonorrhoeae and
Treponema pallidum, are controlled by penicillin. Penicillin is not active
against acid-fast bacteria or fungi. Many bacteria may become resist-
ant, or fast, to penicillin. Whether natural selection or mutation or both
are involved in this phenomenon is uncertain. The morphology and

290 PHYSIOLOGY OF THE FUNGI

physiology of penicillin-fast bacteria vao^y be abnormal (Bellamy and
Klimek 1948). Penicillin is most active against young cells, in that it
inhibits the process of cell division. For papers on the mechanism of
penicillin action see Cavallito et al. (1945), Chain and Diithie (1945);
Bailey and Cavallito (1948) ; and a series of papers by Pratt and Dufrenoy
(1949).

Table 52. The Production of Penicillin in the United States for the Years

1943 TO 1948
A unit of penicillin is 0.6 ^g. (Coghill and Koch, Chem. Eng. Neivs 23, 1045; Lee,
Ind. Eng. Chem. 41, 1949. Published by permission of the American Chemical
Society.)

Year Billions of Units

1943

1944

1,633

1945

6,852

1946

25,809

1947

41,426

1948

95,855

Further details may be found in the following selected references. For
a concise authoritative account of all phases of penicillin, see Foster
(1949). The medical aspects of penicillin therapy are discussed by
Fleming (1949). The chemistry of penicillin is covered in the monograph
edited by Clarke et al. (1949). The early history of penicillin is pre-
sented by Chain and Florey (1944) and Waksman (1947).

Streptomycin. This antibiotic was discovered in Waksman's labora-
tory in 1943, and three years later, commercial production of this drug
began. Streptomycin is synthesized by some isolates of Streptomyces
griseus. The techniques used in industry resemble those used for the
production of penicillin in submerged aerated culture. Streptomycin
is adsorbed on activated carbon as the first step in isolation and purifica-
tion. In contrast to penicillin, streptomycin is a basic compound. The
production of streptomycin in the United States increased from 1,175
billion units in 1946 to 37,710 billion units in 1948 (Lee, 1949). The
chemistry of streptomycin is reviewed by Lemieux and Wolfrom (1948).

Streptomycin is mainly active against Gram-negative bacteria and
certain acid-fast organisms, including Mycohacterium tuberculosis. This
drug controls many pathogens which are unaffected by penicillin. Organ-
isms exposed to streptomycin frequently become fast. Indeed, some
bacteria have been reported to become dependent upon the drug.

The composition of the medium influences streptomycin production.
Soybean meal appears to be a suitable source of nitrogen for commercial
production (Rake and Donovick, 1946). The influence of carbon and
nitrogen sources in synthetic media has been studied by Dulaney (1948,
1949). These results may be summarized as follows: Glucose and man-

METABOLIC PRODUCTS 291

nose are the best hexoses; maltose is the best disaccharide ; starch and
its degradation product dextrin are good carbon sources. Streptomyces
griseus does not utihze nitrate nitrogen. L-ProHne is the most favorable
single amino acid, but addition of this amino acid to other sources of
nitrogen does not increase yields (see also Thornberry and Anderson,
1948). A popular account of the development of streptomycin is to be
found in Epstein and Williams (1946).

Aureomycin. This antibiotic is produced by Streptomyces aureofaciens.
Aureomycin has been prepared in pure crystalline form (Duggar, 1948).
In addition to being effective against many Gram-positive and Gram-
negative bacteria, aureomycin is also active against certain rickettsial
and viral agents. Preliminary reports on the use of aureomycin for
treating Rocky Mountain spotted fever have been favorable (Schoenbach
et al., 1948). Aureomycin appears to be of great clinical value in treating
lymphogranuloma venereum and granuloma inguinale, two virus diseases
of man (Wright et al, 1948).

Chloromycetin. This antibiotic was discovered independently in two
laboratories (Ehrlich et al., 1947, and Carter et al., 1948). It is produced
by Strepto77iyces venezuelae and is the first useful antibiotic to be produced
synthetically on a commercial scale. Chloromycetin is active against
certain Gram-positive and Gram-negative bacteria, acid-fast bacteria,
and Rickettsia (Smith et al., 1948). It has been reported that this anti-
biotic has been used successfully to treat epidemic typhus (Payne and
Knaudt, 1948).

Chloromycetin contains non-ionic chlorine and a nitro group, two
unusual features in compounds of biological origin. The formula is given
below :

O H NHCOCHClo

N— <f >^C— C— CH2OH

O HO H

Chloromycetin (Chloramphenicol)

Ergot. Among the alkaloids produced by fungi, only those obtained
from the sclerotia of Claviceps purpurea appear to be used in medicine.
Seven related alkaloids have been isolated from ergot. These alkaloids
have different pharmacological properties. The most useful alkaloid,
ergonovine, is isolated from the others before use. Certain undesirable
effects of the other alkaloids are avoided by this procedure. Ergonovine,
and formerly a mixture of ergot alkaloids, is used to stimulate uterine
contraction. At present there is no satisfactory synthetic substitute for
ergonovine (Mass, 1950).

In 1941, 571,000 lb. and in 1944, 85,000 lb. of ergot sclerotia were
imported into the United States. If the sclerotia are consumed in large

292 PHYSIOLOGY OF THE FUNGI

enough quantities by man or animals, they cause ergotism, a disease also

known as St. Anthony's fire.

C. purpurea has been cultured under laboratory conditions but forms

neither sclerotia nor alkaloids under these conditions (Michener and

Snell, 1950). Apparently alkaloids are formed only in the sclerotia.

Ergotamine, when added to mycelial cultures of C. purpurea, was largely

destroyed.

TOXINS

Numerous toxic substances are produced by fungi in nature, and their
effects on man and animals are varied. The most severe toxins are
produced by some of the Agaricaceae, particularly by species of Amanita.
It is not known whether the toxins are present in the mycelium of these
species as well as in the fruit bodies. A few of the outstanding examples
of fungus toxins will be discussed briefly.

Amanita toxin (phalloidin) is stable to heat and drying and to the action
of digestive juices. The great majority of the deaths due to mushroom
poisoning are caused by Amanita phalloides, A. virosa, and A. verna,
which contain amanita toxin. The action of this toxin is slow, the symp-
toms being delayed for 6 to 15 hr. after the mushrooms are eaten. By
this time the toxin has been absorbed, and the patient seldom responds
to treatment. No antidote for this toxin is known. The mortality rate
is high, varying from 60 to 100 per cent (Fischer, 1918). In addition to
the three species of Amanita mentioned above, the same or a similar toxin
is present in A. spreta, A. porphyria, A. strohiliformis, A. radicata, and
A. chlorinosma. Hygrophorus conicus and Pholiota autumnalis produce
similar symptoms and may contain this toxin (Krieger, 1936).

About 1 g. of pure crystalline toxin can be extracted from 40 kg. of
A. phalloides fruit bodies. The toxic dose for white mice is 50 jug; death
results in from 1 to 2 days. Chemically, phalloidin is a polypeptide
containing six amino-acid residues. Wieland and Witkop (1940) report
that phalloidin, on hydrolysis wdth sulfuric acid, yields 1 mole each of
Z-Q!-oxytryptophane and cysteine, and 2 moles each of ^-hydroxy proline b
(not found in protein digests) and /-alanine. Kuhn et al. (1939) found
methionine in addition to cysteine (ratio 1 to 5) in phalloidin. Among
the antibiotics, gramicidin and tyrocidine are polypeptides which contain
"unnatural" amino acids and are toxic when injected into experimental
animals.

Muscarin is the principal toxic agent present in A. muscaria. It is a
quick-acting toxin, producing symptoms within 1 to 6 hr. after being
consumed. The patient usually responds well to treatment and recovery
is rapid, although death may occur. Atropin is an antidote for muscarin
which is closely related to choline. Muscarin has also been demonstrated
in A. pantherina, Russula emetica. Boletus luridus, and B. satanas. A

METABOLIC PRODUCTS 293

similar or the same toxin is present in Clitocyhe illudens, Inocyhe infelix,
I. infida, Lactarius torminosus, and B. miniato-olivaceus var. sensibilis
(Krieger, 1936; Wolf and Wolf, 1947).

HelveUic acid is known to be present only in Gyromitra esculenta. Ap-
parently there is considerable variability in the reaction of individuals
who eat this fungus. Many people have eaten it with no ill effects,
although a number of cases of poisoning and even a few deaths have been
reported. Helvellic acid is soluble in hot water. Its toxic action is due
to its blood-dissolving power.

While a number of other toxic substances have been detected in the
fleshy fungi, the exact identity of most of them is not known. For
example, species of Paneolus may cause temporary paralysis or intoxica-
tion similar to alcoholic intoxication. Some species of Amanita have
been reported to contain other toxins in addition to those discussed above
(Fischer, 1918).

The alkaloids produced by Claviceps purpurea and C. paspali are toxic
to man and animals if consumed in large enough quantities. The specific
alkaloids produced were mentioned previously. The production of
toxins is also common in the genus Fusarium. Gihberella zeae, the cause
of scab of small grains, produces toxic substances which are poisonous
to livestock fed on scabby grain (Christensen and Kernkamp, 1936).
Numerous other fungi which cause plant diseases are known to produce
toxic substances which kill the host or modify its activity (see Chap. 17).

PIGMENTS

Colored compounds produced by fungi and other organisms are called
pigments. In the fungi, some pigments accumulate in the mycelium and
spores, while others diffuse into the culture medium. The pigments
produced by a fungus are in part determined by genetic factors and in
part by the environment. Mycelium, fruit bodies, and spores may be
pigmented, or in some species the pigment is confined to the spores.

Among the fleshy fungi, brown is one of the most common colors of
fruit bodies, with yellow, orange, and red being somewhat less common.
Often a number of pigments are obviously present. Few fungi are green.
Yet, Chlorosplenium aeruginosum produces a green pigment, sylindein
(Wolf and Wolf, 1947), which stains the wood in which it grows. Blue-
stain fungi (CeratostomeUa spp.) excrete blue pigments into wood. Some
species of Boletus produce a blue or bluish-green pigment when bruised or
wounded. Tricholoma personatum and Laccaria amythestina are among
the mushrooms producing purple or violet pigments. It is said that the
red-orange pigment of the fruit bodies of Echinodontium tinctorium, the
Indian paint fungus, was used by the Indians as make-up. Few of the

294 PHYSIOLOGY OF THE FUNGI

larger fruit bodies of the fungi are entirely black, although this is a com-
mon color for perithecia, pycnidia, and spores.

Among the nutritional factors which modify the production of pigments
by fungi in culture, the micro essential elements, the carbon and nitrogen
sources, the initial pH of the medium, and the temperature are important.
Perhaps the first of these factors to be studied was the effect of iron, cop-
per, zinc, and other micro elements upon the spore color of Aspergillus
niger. Copper seems to play an outstanding role in the production of
dark spores by this fungus (Mulder, 1939), but low concentrations of
other micro essential elements also affect spore color of this fungus. The
influence of iron, copper, and zinc on the pigmentation of mycelium and
spores, and the production of soluble pigments by certain species was
studied by Metz (1930).

The investigation of the chemical structure of fungus pigments has
formed an essential part of a comprehensive study of the products of
fungus metabolism at the University of London. The citations in this
paragraph will give the reader entry into this excellent work. Many
fungi produce anthraquinone pigments. Helminthosporium gramineum
stores in its mycelium two pigments (helminthosporin and hydroxyisohel-
minthosporin), which may account for 30 per cent of the dry weight of
the mycelium. Helminthosporin is 2-methyl-4,5,8-trihydroxyanthra-
quinone (Charles et at., 1933). H. cynodontis and H. euchlaenae form
cynodontin, l,4,5,8-tetrahydroxy-2-methylanthraquinone, which is closely
related to helminthosporin (Raistrick et at., 1933). Some 12 anthra-
quinone pigments are produced by fungi (Howard and Raistrick, 1949).
Xanthone pigments are produced by H. ravenelUi and H. turcicum (Rai-
strick et at., 1936). The production of anthraquinone pigments is not
restricted to species of Helminthosporium, for Penicillium islandicum
synthesizes chrysophanic acid, 4,5-dihydroxy-2-methylanthraquinone
(Howard and Raistrick, 1950). In general, the production of these and
other pigments is modified by the cultural conditions used. The produc-
tion of helminthosporin by H. gramineum was increased when nitrate or
organic sources of nitrogen were used. Ammonium nitrogen was not
favorable for pigment production. More pigment was produced when
the initial pH was 8 than in more acid media.

Many of the water-soluble pigments produced by fungi are indicators.
P. phoeniceum and P. ruhrum produce such an indicator pigment, phoe-
nicine (2,2'-dihydroxy-4,4'-ditoluquinone). The color changes of this
indicator are from yellow to red in the pH range of 1.8 to 3.4 and from
red to violet in the range 5.4 to 6.4. As much as 2 g. of this pigment is
produced by P. ruhrum per liter of medium (Curtin et al., 1940).

The functions of fungus pigments are not well understood. It is
known that certain of these pigments are enzyme inhibitors. Others, like

METABOLIC PRODUCTS 295

citrinin, are antibiotics. The physiological activity of solanione, a purple
pigment produced by Fusarium solani, decreases growth and the efficiency
of fat formation by F. lini (Weiss and Nord, 1949). Solanione is a
1,4-naphthoquinone, and this activity is in accord with the effects of other
compounds of this series. F. graminearum synthesizes an orange-red
pigment, rubofusarin, Avhich is a xanthone (Ashley et at., 1937). Rubo-
fusarin was found to stimulate growth and to inhibit the enzymatic
dehydrogenation of isopropyl alcohol by F. lini (Sciarini et al., 1943). It
is suggestive that pigment production frequently occurs near the time of
maximum development of mycelium. Perhaps pigments influence
sporulation in some way.

Carotene is produced by Mucor hiemalis and Phycomyces blakesleeanus,
and probably by Mucor mucedo, Pilairia anomala, and Dicranophora fulva
(Schopfer, 1935). The amount of carotene produced by Phycomyces
blakesleeanus was increased by increasing the concentration of asparagine
in the medium. The plus strain of this fungus synthesized more carotene
than the minus strain. Carotene occurs in nature as three isomeric
compounds, all of w'hich may be converted into vitamin A. The carotene
found in M. hiemalis and P. blakesleeanus is ;S-carotene. Emerson and
Fox (1940) found 7-carotene to be associated with the male gametangia
of a certain species of Allomyces. Apparently carotene is also common
among the yellow or orange Ascomycetes and Basidiomycetes.

SUMMARY

The saprophytic fungi play an important, if not indispensable, part
in the degradation and decay of plant and animal residues. The most
important product of fungus metabolism in nature is carbon dioxide.
Humus is in part a result of the activities of soil-inhabiting fungi.

Various species of fungi have been used for the preparation of food and
beverages. Fungi may be used to increase the world's supply of food.
Yeasts and other fungi are able to convert w-aste carbohydrate and
inorganic nitrogen compounds into protein, fats, and vitamins. Yeast
protein, because of its content of essential amino acids, has value as a
protein supplement.

Alcoholic fermentation is not restricted to the yeasts, although these
fungi are used almost exclusively in industrj^ for this purpose. The
production of alcohol requires anaerobic or partially anaerobic conditions.

Fungi may be used to produce organic acids, of which citric acid is the
most important commercially. In general, a medium high in carbo-
hydrate and low in nitrogen favors the production of organic acids, which
are synthesized in quantity only after growth is essentially complete.

In nature one organism may be antagonistic to another because of the
competition for nutrients or because of the production of antibiotics.

296 PHYSIOLOGY OF THE FUNGI

Relatively feAV of the antibiotics known to be produced meet the pre-
requisite of being nontoxic to man, but some of those which do are enor-
mously important.

Ergonovine, one of the ergot alkaloids obtained from the sclerotia of
Claviceps purpurea, is a useful drug for which no synthetic substitute is
available.

A number of fleshy fungi produce toxins, some of which are deadly to
man if consumed in sufficient quantity. The toxins vary in chemical
nature, in severity, and in the symptoms they produce.

Pigments are assumed to serve a definite function in fungi, at least in
some instances. Some pigments are known to be antibiotics; others such
as carotene are provitamins; but in general the functions of the fungus
pigments remain unknown.

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CHAPTER 14
FACTORS INFLUENCING SPORULATION OF FUNGI

The life of the individual fungus is usually short and of uncertain dura-
tion. The continuance of the species (in most instances) depends upon
the production and dissemination of sexual or asexual spores. The
importance of spore production in the spread of epiphytotics is sufficient
reason to study the factors which control, modify, or inhibit this stage of
development in the life of the fungi. Some of the most difficult problems
which arise in the study of the life processes of the fungi are to be found
in the events and conditions which control the production of spores.

In nature, we find many examples of the influence of certain environ-
mental and nutritional factors upon reproduction of the fungi. A num-
ber of parasitic fungi produce the perfect stage only in the spring, on or in
dead host tissue. This is true of Venturia inaequalis, Clavtceps purpurea,
Gnomonia ulmi, Monilinia jructicola, Coccomyces hiemalis, Guignardia
bidwelln, and others. Is the production of the sexual stage dependent
upon the pretreatment of cold, or freezing and thawing? Is it dependent
upon a favorable temperature and perhaps favorable intensity and dura-
tion of light? Or is it a matter of the proper nutrients which are made
available only after decay of the host tissues? These are difficult ques-
tions to answer, for it is likely that the production of the sexual stage
depends upon the proper balance of a number of factors. Similarly, we
may speculate about the stimuli involved in the formation of the peri-
thecia by the Erysiphales. Most of these obligate parasites form fruit
bodies late in the growing season. Perhaps, at least in some cases, this
is a reaction to cooler weather ; or perhaps the formation of perithecia is a
result of a decreasing or changing food supply as the host nears maturity.
Other physical factors are probably involved, since we know that the
abundance of perithecia varies from year to year. It is also of physio-
logical interest that many parasitic fungi produce conidia only while the
mycelium is actively attacking the living host.

A critical investigation of the factors influencing reproduction requires
that the fungi be brought into the laboratory or greenhouse where external
factors can be controlled. Only one variable should be studied at a time,
and all other influencing factors must be controlled. It is, therefore, of
great advantage in physiological studies to be able to grow a fungus in
pure culture on synthetic or semisynthetic media. However, it n\ust be

304

SPORULATION 305

pointed out that the responses of a fungus in nature cannot always be
duplicated in the laboratory.

Snyder and Hansen (1947) have given a brief and clear statement
regarding the advantages of culturing fungi on natural media and under
natural environmental conditions. These conditions are important, if
one desires to obtain reproduction of a fungvis which does not sporulate
readily in culture. However, if one desires to study critically the indi-
vidual nutritional and environmental requirements and their effects upon
reproduction of a fungus which sporulates abundantly on the usual
cultural media, it is often necessary to subject the fungus to unfavorable
conditions. Thus, only by preventing sporulation, by varying but one
factor at a time, may we discover the need for that factor.

Riker and Riker (193G) have listed 11 methods which have been suc-
cessfully employed to induce sporulation of different fungi in culture.
Since the writing of their manual much has been learned about this phase
of fungus physiology. A revised list of the conditions known to influence
sporulation of fungi is presented in the summary of this chapter.

Kauffman (1929) called attention to the views of Klebs, who held that
living cells are influenced during their lifetime in three ways: (1) by the
specific structure; (2) by the internal conditions; and (3) by the external
conditions. Kauffman equated the first of these to heredity and the last
two to environment. The external environment comprises the various
physical and chemical factors, such as temperature, light, composition
of the medium, and the like. Kauffman used the term internal environ-
ment to designate the complicated influences and reactions between cells
wdthin the organism. The physical and chemical effects of the external
environment may be transmitted through the cells and become evident at
some distance from the point of the stimulus.

The meaning of these statements may be clearer if we consider the
effect of various external environmental factors upon fruiting. It is well
known that various external stimuli may initiate the reactions which lead
to reproduction. These stimuli must act through the internal environ-
ment. Most of the discussion that follows will be concerned with the
external environment and the resulting development of the fvmgus.
Some external factors may so modify the internal milieu as to favor
sporulation, w^hile others may inhibit or prevent sporulation.

Not all fungi respond in the same way to the external factors such as
light, temperature, or nutrition. Each species produces spores when the
internal environment is suitable, but the external factors do not operate
upon the internal environment of all fungi alike. Thus, there is no univer-
sal set of external conditions which lead to fructification in all fungi. The
external conditions favorable for sporulation must be studied for each
species. This does not imply, however, that no two fungi react alike or

306 PHYSIOLOGY OF THE FUNGI

that certain helpful generalizations concerning sporulation cannot be
drawn. It does, however, imply that the only sure way of understanding
the conditions governing reproduction in a specific fungus lies in the
experimental approach.

Again it must be emphasized that all the physical and chemical condi-
tions may be at the optima, but no reproduction can occur without the
presence of favorable genetic factors. Too often we may fail to realize
the genetic requirements. The appropriate steps should be taken to
determine whether the fungi under study are homothallic or heterothallic.
It may be difficult, indeed, to determine whether failure of a fungus to
reproduce in culture is due to unfavorable environmental conditions or
to unfavorable genetic factors. There is much yet to be learned regard-
ing the physiology of reproduction, but each new investigation is certain
to add to our knowledge of this interesting and important phase of fungus
physiology.

Vegetative growth must precede reproduction. The length of the
vegetative phase varies from organism to organism, and the same organ-
ism may remain in the vegetative phase for a longer or shorter period of
time depending upon the external environment. One of the functions
of the vegetative phase is concerned with the building up of protoplasm
and the storage of energy reserves. Reproduction is a process that draws
heavily on the reserve food. The spore is usually well stocked with these
materials. Asexual reproduction differs less from vegetative growth than
does sexual reproduction. We shall find that the conditions limiting-
sexual reproduction are usually more narrow than conditions which allow
asexual reproduction and growth.

Klebs (1900) summarized his views on reproduction in the fungi in the
form of four laws or principles as follows: (1) Growth and reproduction
are life processes, which, in all organisms, depend upon different condi-
tions. In the lower organisms the external conditions mainly determine
whether growth or reproduction takes place. (2) Reproduction in the
lower organisms does not occur as long as characteristic external condi-
tions are favorable for growth. The conditions which are favorable for
reproduction are always more or less unfavorable for growth. (3) The
processes of growth and reproduction differ, in that growth may take
place under a wider range of environmental conditions than reproduction.
Growth may take place, therefore, under conditions which inhibit repro-
duction. (4) Vegetative growth appears to be mostly a preliminary step
for reproduction in that it creates a suitable internal environment for it.
To a certain degree it is not growth in itself but the prolonged period of
assimilation accompanying growth that is decisive for reproduction.

These generalizations were published in 1900 and were based upon
Klebs's own work, as well as that of others. Many more fungi have been

SPORULATION

307

studied during the past 50 years, and some new factors have been brought
to light. It would not be surprising if some modifications in these con-
clusions would be necessaiy in the light of 50 years of research. We shall
find, however, that, in the main, many of these "laws" are still valid.

ENVIRONMENTAL FACTORS

Temperature. Temperature was recognized by Bisby (1943) as an
important natural factor governing the geographical distribution of the
fungi. The temperature must be favorable not only for growth but also
for the production and germination of the spores, if the fungus is to
survive. Certain fungi are limited by high temperatures. Among these
are Plasmodiophora brassicae, Colletotrichum lindemuthianum, Urocystis
cepulae, and certain Phycomycetes. On the other hand, certain genera
of the Gasteromycetes, such as Podaxis, Battarrea, Chlamydopus, and
Phellorina, are confined to the hot arid regions of southwestern United
States, northern Africa, central Australia, and western India. Between
these extremes we may observe many examples where seasonal tempera-
ture limits or favors reproduction.

Klebs (1900) pointed out that the temperature range which allowed
sporulation was more narrow than the range for growth. In general, the
temperature limits for sexual reproduction are narrower than the limits
for asexual reproduction. Some of Klebs's data are presented in
Table 53.

Table 53. Minimum and Maximum Temperatures (in Degrees Centigrade) for

Growth axd Sporulation of Various Fungi

(Klebs, Jahrb. iriss. Botan. 35, 1900.)

Fungus

Aspergillus repens. . .
Sporidinia grandis. . .
Piloholus rnicrosporus
Saprolegnia mixta. . . .

Growth

Min.

7-8
1-2
2-4
0-1

Max.

37-38
31-32
33-34
36-37

Asexual spores

Min.

8-9

5-6?

10-12

1-2

Max.

35-36
29-30
28-30
32-33

Sexual spores

Min.

5-6

1-2

Max.

33-34

27-28

26-27

It will be noted that the upper temperature which allowed the produc-
tion of oospores by Saprolegnia mixta is a full 10°C. less than the upper
temperature limit at which growth took place. Coons (1916) found the
temperature limits for the growth of Plenodomus fuscomacidans to be
0 to 33°C., while pycnidia formed between 6 and 30°C. Perithecia failed
to form in cultures of Ceratostomella fimbriata kept at 18°C. for 60 days
(Barnett and Lilly, 1947a). Cultures of this fungus on the same medium

308

PHYSIOLOGY OF THE FUNGI

produced abundant perithecia and ascospores at 25°C. within 11 days.
Conidia were formed at 18°C.

The most noteworthy effect of culturing a fungus at temperatures
below the optimum is the decrease in the rate of growth. It has been
found by various investigators that there is an optimum temperature for
sporulation as well as for growth. The two optima may be different.
Figure 57 shows the effect of temperature on the time required to produce
conidia by Aspergillus repens.

2 6

Q, 4

o
o

•

•\

•

— .-

10 15 20 25 30

Temperature in degrees centigrode

Fig. 57. The influence of temperature on the time required to produce conidia by
Aspergillus repens. (Drawn from data of Klebs, Jarhh. wiss. Botan. 35 : 137, 1900.)

A temperature of 28°C. was optimum for sporulation of Piricularia
oryzae (Henry and Andersen, 1948). Higher and lower temperatures of
incubation decreased the numbers of spores produced. At 32°C. the
number of spores was only 10 to 15 per cent of that produced at the
optimum temperature. Reducing the temperature of incubation to
24°C. reduced the numbers of spores to about 80 per cent of the maxi-
mum. Thus, a small temperature increase above the optimum has a
much greater effect upon the number of spores produced than a small
decrease in temperature below the optimum (Fig. 58).

In nature, fungi are exposed to fluctuating temperatures. Whether a
fluctuating temperature is more favorable in inducing sporulation than a
constant temperature appears to have been studied but little. Jones
(1946) concluded that temperature was the important controlling factor
in the production of resistant sporangia of Allomyces arhiiscula in culture,
and he beheved that "the total amount of temperature" to which the
cultures were subjected was more important than the maximum, mini-

SPORULATWN

309

mum, or degrees of fluctuation, IMathur et al. (1950) reported that 15
to 20°C. favors conidium formation by Colletotrichum lindemuthianum in
culture. Sporulation was less at 25°C. and ceased at 30°C. Mrak and
Bonar (1938) found that temperature influenced the relative size of asci
and spores of Debaryomyces. The ascus was much larger than the spore
cluster at 4°C., but the spores nearly filled the ascus at 25°C.

7 9 11

Incubation period (days)

Fig. 58. The effects of temperature and time of incubation on sporulation of Piri-
rularia oryzae on rice-polish agar. (Courtesy of Henry and Andersen, Phytopathology
38 : 272, 1948.)

An interesting selective effect of temperature upon type of asexual
sporulation is found in Choanephora cucurbitarum (Barnett and Lilly,
1950). This fungus produces two types of asexual spores, those produced
in typical sporangia and conidia borne in heads. Only the conidia are
found commonly in nature, while both types are abundant in culture.
When the fungus was grown in Petri dishes at 25°C., 87 per cent of the
reproductive structures were conidial heads, while 13 per cent were
sporangia (Table 54). When the temperature was increased to 30°C.,
this proportion was nearly reversed. At 31°C. many sporangia but no
conidia were formed. No sporulation occurred at 34°C., but mycelial
growth was abundant. Temperature also affected, either directly or
indirectly, the size of the sporangia. Those produced at 25°C. averaged
60 to 90 n in diameter, while those formed at 30 or 31°C. were much larger,
averaging approximately 145 fi. It seems likely that this effect is indirect,
being a reflection of the relative number of conidia, which are formed first
under favorable conditions. We may assume that the production of
abundant conidia uses much of the food materials which might also go

310

PHYSIOLOGY OF THE FUNGI

into the formation of sporangia. Under conditions unfavorable to
conidium production, yet favorable to sporangium formation, both the
size and abundance of sporangia are increased. The effect of temperature
was also evident when pumpkin flowers artificially inoculated with C.
cucurbitarum were brought into the laboratory and placed at 30°C.
Under these conditions both conidia and sporangia were produced.

Table

54. The Effect of Temperature upon Asexual Reproduction op

Choanephora cucurbitarum
(Barnett and Lilly, Phytopathology 40: 83, 1950.)

Temperature during
sporulation, °C.

Conidial heads
per culture

Sporangia
per culture

Average size of
sporangia, fx

25
30
31
34

2,000

150

300

1,300

1,200

60-90
148
145

Other critical temperature studies are needed, particularly those
designed to show the interrelated effects of temperature with other
environmental or nutritional factors and to determine the effects of tem-
perature upon the "internal environment" of the fungi. The tempera-
ture of incubation affects zygospore formation by Phycomyces blakes-
leeanus indirectly through the amount of acid formed in the medium
(Robbins and Schmitt, 1945).

Light. Light has been a neglected and often ignored factor in many
studies of sporulation. Too often we place fungi in the laboratory or
refrigerator according to our own convenience, not to their needs, and
expect them to reproduce as they would in nature. Under natural con-
ditions many fungi fruit only when exposed to light, often to the direct
rays of the sun, for a part of the time. Numerous observations have been
reported regarding the need for light, but too few of these reports give
data as to the intensity, duration, or quality of the light required to
initiate sporulation. We should not conclude that intensity and duration
are without effect.

A review of the early work on the influence of light on the growth and
fruiting of the fungi is presented by Coons (1916). Brefeld (1877) found
that some species of Coprinus failed to fruit in the dark. A culture of
Coprimis exposed to light for 2 or 3 hr. was then able to fruit in the normal
manner when removed from the light. He also found that higher tem-
peratures replaced, in part, the beneficial effect of light for some species.

Sphaerographium fraxini produced a few pycnidia in the dark at 30°C.,
whereas none were produced at room temperature in the dark (Leonian,
1924). Pycnidia were produced at room temperature in the light.

SPORULATION 311

Ascochyta nymphaeae, Cytosporella mendax, Endothia parasitica, Keller-
mania yuccagena, Naemosphaera sp., Plenodomus destruens, and Phoma
urens formed more pycnidia at 30°C. in the dark than in the light at room
temperature. The following fungi failed to fruit in the dark at 8°C. but
fruited at the same temperature in the presence of light: Hendersonia
sp., Melanconium hetulinum, Naemosphaera sp., Pestalotia guepinia,
Phoma urens, Phyllosticta opuntiae, Sphaerographium fraxini , and Sphaero-
nema pruinosum. Light favored pycnidial formation by Plenodomus
fuscomacidans (Coons, 191G). The above examples make it clear that
light and temperature may serve as interchangeable stimuli to sporulation
in some, but not all, instances. Since the response (sporulation) is the
same whether light or temperature is the stimulus, this means that these
stimuli in some way brought about the same or equivalent changes in the
internal environment of the fungus.

Drayton (1937) was able to produce the perfect stage of Botryotinia
convoluta by controlling light, temperature, and nutrition. The technique
is somewhat involved, but it should be remembered that in nature the
external environment varies a great deal during the course of a year.
Fluctuations in temperature, moisture, light, and food supply are the
normal result of the procession of the seasons. Drayton found autoclaved
W'hole wheat to be an excellent substratum for this fungus. The most
favorable results w^ere obtained by allowing the culture to develop at
14°C. in the dark for 45 days. At the end of this time the sclerotia w^ere
placed in moist quartz sand at 0°C. for 3 to 4 months, then stored at
5°C. When the apothecial fundaments were 2 to 3 mm. long, the cultures
were moved to a greenhouse and placed under cheesecloth and the tem-
perature held at 7°C. at night and below 15°C. during the day. The
apothecia matured within 4 wrecks.

Yarwood (1936, 1941) observed parasitic fungi under natural condi-
tions and found that the production and liberation of the conidia of
Erysiphe polygoni and the ascospores of Taphrina deformans followed a
definite diurnal pattern in nature.

The combined effects of temperature and light upon sporulation of
Helminthosporium gramineum are clearly shown by Houston and Oswald
(1946). Best sporulation was obtained under outdoor conditions, with
14 to 15 hr. of daylight and the average maximum and minimum tem-
peratures 26.8 and 8.2°C., respectively. No conidia were produced on
potato-glucose agar in the absence of light, either outdoors or inside.
Artificial light apparently was less effective than daylight. However,
continuous light at 13°C. allowed the formation of a few conidia. On
pieces of infected barley leaves, conidia were formed without exposure to
light, over a considerable range in temperature. As an explanation of
these differences, the authors believe that the mycelium in the leaf in

312 PHYSIOLOGY OF THE FUNGI

nature stored up the "necessary potentialities," which then permitted
conidium production in darkness. MyceHum growing from the pieces
of leaf into the agar did not produce spores in darkness. This is an
interesting theory regarding a possible delayed action of light upon sporu-
lation. It also seems possible that the leaf tissue of the host may furnish
some nutrient necessary for sporulation which is not contained in potato-
glucose agar. Perhaps light is essential to the synthesis of this material
by the fungus.

It was demonstrated recently (Barnett and Lilly, 1950) that an isolate
of Choanephora cucurhitarum requires both light and darkness for the forma-
tion of conidia, but these factors have little or no apparent influence upon
the formation of sporangia. This fungus was grown under a number of
conditions, but none was found which overcame the need for either light
or darkness. Cultures incubated in the laboratory under natural alter-
nating light and darkness produced abundant conidial heads during the
second and third nights after inoculation. Exposure to artificial light
for 2 days after inoculation followed by darkness gave similar results, but
an exposure in the reverse order resulted in no conidia. Cultures under
continuous artificial light (65 foot-candles) and those in total continuous
darkness failed to form conidial heads. Continuous light of low intensity
(less than 1 foot-candle) , however, did permit the formation of numerous
conidial heads in the usual period. A summary of the important results
is presented in Fig. 59, together with an outline of a proposed hypothesis
to explain the results. We may assume that light, or its absence, affects
two metabolic reactions, or groups of reactions, which are essential to
conidium formation by C. cucurhitarum. Light, which is essential to
reaction A, apparently inhibits reaction B, which must occur in darkness
or weak light. The reaction in light must be'^followed by the reaction in
darkness, if conidia are to be formed. Continuous bright light favors
only reaction A, while continuous darkness permits only reaction B.
Both reactions occur simultaneously in continuous light of low intensity.

A different isolate of C. cucurhitarum was studied by Christenberry
(1938), who found that alternating periods of light and dark, 12 hr. each,
gave the best sporulation. Red-yellow light was more favorable to
conidium formation than the shorter rays. This isolate formed conidia
in total darkness.

The beneficial effect of alternating light, or a period of light followed
by darkness, was demonstrated (Timnick et al., 1951) for the formation of
ascospores by Diaporthe phaseolorum var. hatatafis. Cultures grown in
continuous darkness formed only a few perithecia, which contained
abundant ascospores. In continuous bright light numerous perithecia
were formed, but relatively fewer ascospores were produced. A long
period of light followed by darkness gave many perithecia with abundant
ascospores.

SPORULATION

313

Marked morphologic differences were found in strains of Fusarium
subjected to different exposures of light and darkness (Snyder and Han-
sen, 1941). Some of the characters affected were color, zonation, type of
colony, presence or absence of sporodochia, occurrence of the perithecial
stage, and size, shape, and septation of macroconidia. Light was usually
found necessary for the formation of macroconidia. Exposures were
made to continuous total darkness but not to continuous light. Evidence
in these experiments indicated that the effect of light is only upon the
actively growing portion of the mycelium.

Bright light

Reaction A

Darkness

Reaction B

= Conidia

Continuous bright light

Reaction A

Continuous darkness

Reaction B

= No conidia

Bright light

Reaction A

= No conidia

Continuous light low intensity

Conidia

Reactions A + B simultaneously
Fig. 59. Conidium formation by Choanephora cucurbitarum under different light
conditions, shownig the possible metabolic reactions controlled by light. Under
variable conditions, the cultures were exposed to the first condition (on the left) for 2
days and to the second condition for 24 hr. (After Barnett and Lilly, Phytopathology
40: 88, 1950.)

The length of exposure necessary to stimulate spore formation may be
very short, as demonstrated by Bisby (1925) for Fusarium discolor sul-
phureum. He observed that brief exposure to light, while Petri dish
cultures were being examined, resulted in the formation of rings of conidia.
Using a photographic shutter, he further demonstrated that an exposure
as brief as }i sec. to outdoor light on a bright day was sufficient to stimu-
late the formation of a ring of conidia.

Coons (1916), in his work with Plenodomus fuscomacidans, reasoned
that the effect of light might be replaced by various oxidizing agents, since
light is known to promote various oxidations. Cultures treated with
hydrogen peroxide and other oxidizing agents produced a few pycnidia.
The age of the culture when these chemicals were added was important.

314

PHYSIOLOGY OF THE FUNGI

For these chemicals to stimulate pycnidium formation, the culture had
to be in such a physiological condition that 1-hr. exposure to light would
induce sporulation.

The sporulation of a number of other species in our laboratory has been
observed to be influenced by the presence or absence of light (Figs. 60,
61). Among these are Dendrophoma obscurans, Trichoderma lignorum,

Fig. 60. The effects of light on the
production of conidia by Trichoderma
lignorum after 3 days at 25°C. A,
exposed to continuous artificial light.
Note the more or less even distribution
of conidia. B, exposed to alternate
ight and darkness, 12 hr. each. Note
the rings of conidia. C, grown in
continuous darkness. Note the ab-
sence of conidia.

Sphaeropsis malorum, Ceratostomella ulmi, Botrytis sp., Endothia para-
sitica, Septoria nodorum. The reaction of some fungi to light is appar-
ently dependent, to a certain extent, upon the composition of the medium.

Still another effect of light should be emphasized, i.e., the inhibitory
effect. The depressing effect of strong light upon growth and length of
sporangiophores of Phy corny ces hlakesleeanus is easily demonstrated.
Elfving (1890) noted that the amount of inhibition of growth by light
varied with the composition of the medium.

Ultraviolet light. The destructive action of sunlight upon micro-
organisms, especially bacteria, was recognized about the time that pure-

SPORULATION

315

culture methods came into wide use. The lethal action of ultraviolet
light is conditioned by the wave length of the irradiation, by the time of
exposure, and by the particular nature of the microorganism. A con-
siderable number of investigators have studied the effect of ultraviolet
radiation upon sporulation. Both favorable and unfavorable results
have been obtained. It should be recognized that length of exposure

Fig. 61. The effect of light on the production of pycnidia by an isolate of Dendro-
phoma obscurans when grown on malt extract-agar plates at 25°C. A, grown under
continuous artificial light, .\lternate light and darkness gave similar results. B,
grown in continuous darkness.

is a very important factor in these experiments. In addition, the medium
used, the age of the culture, and the temperature rise during irradiation
also modify the results.

Stevens (1928) found that ultraviolet radiation induced the formation
of perithecia by various isolates of Glomerella cingulata a few days after
irradiation. While old cultures produced a few perithecia without
irradiation, many more were produced by young cultures within a short
time following irradiation. One effect of such irradiation is the killing
of the aerial mycelium. Short exposures allowed the formation of super-
ficial perithecia, while long exposures prevented their formation. The
majority of the perithecia formed following intermediate dosages were
embedded in the medium. It was noted that the age of the mycelium

316 PHYSIOLOGY OF THE FUNGI

at the time of irradiation had an effect on the number of perithecia formed.
Colonies 4 days old when irradiated produced perithecia, which were most
abundant on mycelium 1 day old at the time of irradiation. Irradiation
of colonies 12 days old led to the formation of but few perithecia. No
evidence was obtained that irradiation of the medium alone had any
effect on perithecium formation. A species of Coniothyrium which
formed pj^cnidia only after the cultures were very old was stimulated to
produce pycnidia within 3 days after irradiation. This work of Stevens
is apparently the first which demonstrated that ultraviolet radiation
stimulated sporulation by fungi.

Spore production by Macrosporium tomato and Fusarium cepae was
greatly increased by the proper exposure to ultraviolet radiation (Ramsey
and Bailey, 1930). A 12- to 15-fold increase in the numbers of spores
produced by these two species was obtained by the optimum exposure.
These investigators also showed that irradiation of the medium before
inoculation had no subsequent effect on sporulation by these two fungi.
The range of wave lengths which stimulated the most abundant sporula-
tion was found to be 2,300 to 2,800 A. Smith (1935) points out that
many workers have neglected the precaution of controlling the tempera-
ture of cultures during irradiation. She found it necessary to control
the temperature of the cultures of Fusarium eumartii in order to separate
the effects of increased temperature and ultraviolet radiation.

Ultraviolet radiation stimulated or depressed sporulation of Diaporthe
phaseolorum var. hatatatis depending on the medium used (Timnick
et al., 1951). Neither stromata nor perithecia were formed on casein
hydrolysate-glucose medium, unless the cultures were irradiated. Cul-
tures grown on potato-glucose agar produced stromata and long-beaked
perithecia without irradiation. Irradiation of cultures on potato-glucose
medium resulted in the formation of fewer and smaller short-beaked
perithecia. Although the mode of action of ultraviolet radiation in
stimulating sporulation is unknown, long exposures are known to be
lethal. We may assume that even short exposures injure or kill some of
the exposed cells. Perhaps some substance is thereby released which
stimulates sporulation. The presence of such a substance in the potato-
glucose medium might explain why irradiation was not necessary for the
production of perithecia by D. phaseolorum var. hatatatis on this medium

Aeration. Although the fungi are aerobic organisms, the amount of
free oxygen that they need to carry out their life processes varies from
fungus to fungus. The amount of oxygen required is less for growth than
for reproduction. The aquatic fungi would be expected to grow and
reproduce in a more limited supply of oxygen than terrestial forms.
While many aquatic Phycomycetes produce their spores under water, a
large number of fungi fail to fruit until some aerial mycelium has been

SPORULATION 317

formed. Examples of the inhibiting effect of insufficient aeration on
sporulation are numerous. Coons (1916) found that lowered oxygen ten-
sion inhibited pycnidium formation by Plenodomusfuscomaculans, though
there was still sufficient oxygen supply to allow some growth. Leonian
(1924) tested the effect of reduced oxygen on pj^cnidium formation by
various Sphaeropsidales. This experiment was carried out by culturing
these fungi in Petri dishes, some of which were placed in desiccators, while
the controls were placed on a table. The following fungi produced fewer
pycnidia in sealed desiccators than in the control cultures: Ascochyta
tiym'phaeae, Phoma urens, Plenodomus destruens, Phyllosticta opuntiae, and
Septosporium acerinum. It is possible that this effect may have been
due to the increased concentration of carbon dioxide in the closed vessels.
Denny (1933) made an accurate study of the effect of oxygen supply on
growth and formation of perithecia by Neurospora sitophila. Only a
trace of oxygen was required for limited growth, for it was necessary to
keep cultures in the presence of alkaline pyrogallol to inhibit growth
entirely. Oxygen concentrations of less than 0.5 per cent inhibited
perithecium formation for 30 days, while perithecia formed in air within
4 days. This paper should be consulted for the details of conducting
experiments of this nature under closely controlled conditions. Some of
Denny's data are given in Table 55.

Table 55. The Effect of Oxygen Concentration on the Formation of Peri-
thecia BY Neurospora sitophila
(Prepared from the data of Denny, 1933. Contribs. Boyce Thompson Inst. 5, 1933.)
Oxygen Cbncentration, % Days Required to

Form Perithecia
20.8 4

9.4 7
3.75 9

1.5 12

0.24 None at 30 days

Conidium production by Choanephora cucurhitarum was poor in tight-
fitting Petri dishes (Barnett and Lilly, 1950). Sealing the dishes pre-
vented conidium formation, while well-aerated dishes allowed abundant
conidial heads to form. Failure to form conidia under these conditions
may be due to (1) insufficient oxygen supply, (2) the accumulation of
toxic, volatile, metabolic by-products, (3) increased carbon dioxide con-
tent, or (4) unfavorable humidity.

Adequate aeration was one of the most important environmental fac-
tors necessary for conidium formation by Piricularia oryzae (Heniy and
Andersen, 1948). The cultures emitted a strong odor of ammonia after
a few days' incubation. It was believed that aeration removed the
ammonia and other volatile metabolic by-products which prevented

318

PHYSIOLOGY OF THE FUNGI

abundant sporulation. Forced aeration of the culture flasks at the rate
of 4 ml. of air per minute per milligram of oats-sorghum medium was
found to be optimum for sporulation. Mader (1943) discussed the
factors inhibiting fruiting of Agnricus campestris and concluded that
volatile substances are important, and that they must be removed by
aeration of mushroom cellars.

Hydrogen-ion concentration. The early workers recognized that the
acidity of the medium influenced sporulation. Lock wood (1937) studied
the formation of perithecia and asci by Penicillium javanicum, Aspergillus
herhariorum, and Chaetomium. globosum in buffered media of various

Fig. 62. The effect of glutamic acid on gametic reproduction of Phycomyces hlakes-
leeanus at 26°C. Left, basal medium; right, basal medium plus 10 mg. d-glutamic
acid, neutraHzed with CaCOs. Note the line of progametes in the plate on the right.
Age, 6 days. (Courtesy of Robbins and Schmitt, A7n. Jour. Botany 32 : 321, 1945.)

hydrogen-ion concentrations and found that the perithecia produced in
the more acid solutions contained few if any asci with ascospores. The
percentage of fertile perithecia increased as the pH was increased to 7 or
8. Similarly, in our laboratory, we have noted that A . rugulosus produces
many perithecia and few conidia at an initial pH value of 6 to 8, while
conidia but no perithecia form at pH 3 to 4.

Robbins and Schmitt (1945) studied the sexual reproduction of Phyco-
myces hlakesleeanus on glucose-asparagine medium and found that mature
zygospores did not form at 26°C. Zygospores formed when various
protein hydrolysates, amino acids (especially glutamic acid), or various
organic acids were added to the medium. These buffers prevented the
pH from falling low enough to inhibit zygospore formation (Fig. 62).
These authors also noted that P. hlakesleeanus on glucose-asparagine
medium produced zygospores at 20°C. This is evidence that the com-
position of the medium has a profound effect on reproduction. In this

SPORULATION 319

instance, it was possible to trace the connection between temperature and
the composition of the medium to a specific factor, i.e., acidity

Perithecia were not formed by Sordaria fimicola until the pH of the
culture medium was 6.5 or greater (Lilly and Barnett, 1947). While
acidity of the medium was not the only controlling factor affecting the
formation of perithecia by S. fimicola, perithecia never formed when the
pH was less than 6.5, however favorable the other external conditions
were.

OTHER PHYSICAL FACTORS

It has frequently been observed that many species of fungi fruit more
readily when grown upon a solid or semisolid substratum than they do in
liquid media. Leonian (1924) reported that only 6 out of 20 species
studied formed pycnidia as readily in liquid medium as on solid medium.
He concluded that the beneficial effect of solid media was due to better
aeration and free transpiration.

The favorable effect of ozone upon the formation of pycnidia and spores
of a limited number of fungi was recently reported by Richards (1949).
The production of viable conidia of three species of Alternaria was greatly
increased on exposure to ozone. Although conidium formation of
Mycosphaerella citrullina was increased by exposure to ozone, the spores
formed did not germinate.

The transformation and elongation of basidia of certain Polyporaceae
in nature and under controlled conditions has been correlated with high
humidity by Bose (1943). It seems likely that the humidity of the
atmosphere may have a greater influence upon conidium formation in the
aerial fungi than is generally supposed. In Rhizopus, for instance, much
more liquid moves upward through the sporangiophore than can be con-
tained within the sporangium. A high percentage of this water must be
transpired in order to condense the protoplasm and food materials stored
in the spores. A change in relative humidity must affect the rate of
transpiration. On the other hand, Ternetz (1900) found that a humidity
of 98 per cent or higher was necessary for fruit-body production by
Ascophanus carneus. Actually, we know little about the influence of
humidity, and much more information is needed on this subject.

Emerson and Cantino (1948) showed that the presence of high concen-
trations of carbon dioxide favored the production of resistant sporangia
by Blastocladia pringsheimii.

Mutilation of the mycelium, which would cause the death and release
of cellular constituents, has been used to stimulate sporulation (see Rands,
1917; Kunkel, 1918; and McCallan and Chan, 1944). Scraping of the
mycelium of Alternaria solani followed by a brief exposure to ultraviolet
rays was used successfully by McCallan and Chan (Fig. 63).

320

PHYSIOLOGY OF THE FUNGI

10,000

<n
o

z
<

cr
o
a.

1,000 r

100 -

640

1280 2560

20 40 80 160 320

EXPOSURE TIME IN SECONDS
Fig. 63. Effect of time of exposure to ultraviolet radiation on the production of
spores from scraped and unscraped cultures of AUernaria solarii. (Courtesy of
McCallan and Chan, Contribs. Boyce Thompson Inst. 13 : 327, 1944.)

NUTRITIONAL FACTORS

The nutritional conditions under which a fungus produces reproductive
bodies and spores are often quite different from those which are optimum
for vegetative growth. Not all media are equally suitable for sporula-
tion. The frequent failure to obtain sporulation of many common fungi
in culture, even though they grow profusely, testifies to the extent of our
ignorance regarding the necessary nutritional factors. However, the
following factors have been shown to be important: concentration of
medium, carbon and nitrogen sources, carbon-nitrogen ratio, micro
essential elements, specific reproductive factors, and vitamins.

Concentration of nutrients. Among the early workers, Klebs (1900)
gave a great deal of attention to the effect of nutrient concentration upon
reproduction. For most of the fungi with which he worked, exhaustion
of the food supply favored sporulation. Klebs (1899) kept a culture of
Saprolegnia mixta in the vegetative condition for 2^^ years by constant
renewal of the nutrient solution. Yet, this fungus produced spores within
a few days when the food supply became exhausted. The same principle
holds true for the Myxomycetes as well as the filamentous fungi. Camp
(1937) grew Physarum polycephalum and studied the effect of the number

SPORULATION 321

of feedings upon the time of fruiting. The protoplasm continued to
grow as long as there was abundant food, but when the food was
exhausted, the sHme mold passed into the fruiting stage.

Leonian (1923, 1924) used a technique in studying sporulation which
consisted in growing a fungus in a medium suitable for vigorous vegetative
growth, and then transferring it to solutions of different concentrations
to stimulate sporulation. When sterile mycelium of Valsa leucostoma
was transferred from a medium containing 1.5 per cent nutrients to a
medium containing 0.37 per cent nutrients the ratio of perithecia to
pycnidia increased. Transferring sterile mycelium to more concentrated
nutrient solutions favored the production of pycnidia and decreased the
number of perithecia formed. Endothia 'parasitica showed a decrease in
the number of pycnidia when the mycelium was transferred from a weak
to a concentrated medium. When the sterile mycelium was grown in a
concentrated medium and transferred to distilled water, the pycnidia
did not mature but an enormous number of pycnidium initials were
formed. If such a culture were then transferred back to a concentrated
medium, maximum sporulation was obtained. A review of the literature
on the effect of concentration on fruiting is given by Leonian (1924).

From the above examples we may conclude that the concentration of
nutrients in a medium may have a profound influence upon fruiting, and
that the different types of fruiting (sexual and asexual) may have different
requirements. Not only the amounts of the different nutrients but the
proper balance between the components of the medium may be essential
for maximum sporulation.

Nitrogen source. The source of nitrogen influenced the formation of
pycnidia and spores by Phyllosticta solitaria (Mix, 1933). The specificity
of the nitrogen source was greater for the production of spores than for the
formation of pycnidia. The different isolates of this fungus responded
differently to the various nitrogen sources. Nitrate nitrogen was the
most favorable. This may have been due to an indirect effect on the pH
of the medium, for this fungus sporulates only between pH 4.2 and 5.8.

In our laboratory we have observed that sporulation of some fungi is
favored by certain sources of nitrogen, which are not necessarily the same
as those which are favorable for growth (Fig. 64) . A few of these species
with the more favorable nitrogen sources for sporulation are Monilinia
fructicola, ammonium tartrate, glycine; Phoma betae, glycine; Neo-
cosmopara vasinfeda, glutamic acid, glycine; Septoria nodorum, glycine;
Diaporthe phaseolorum var. batatatis, asparagine; Choanephora cucur-
bitarum, organic nitrogen.

Carbon source. Not all carbon sources are equally suitable for fruit-
ing of fungi. Some which are favorable for mycelial growth do not favor
sporulation. Hawker (1939) found the number of perithecia produced

322

PHYSIOLOGY OF THE FUNGI

A B C

Fig. 64. The effects of different nitrogen sources on sporulation of three fungi after
19 days on a ghicose-sucrose medium at 25°C. The nitrogen sources are: A, apsaragine;
B, casein hydrolysate; C, potassium nitrate; D, ammonium sulfate; E, ammonium
tartrate; F, glycine. The fungi are: top, Glornerella cingulata; middle, Pleurage

SPORULATION

323

D E F

^^.Jcolla; bottom, Melanospora sp. Note that asparagine and casein hydrolysate
are good sources of nitrogen for spore production of all three fungi. Discharged spores
of P. curvicolla are evident only on these two media.

curvic

324

PHYSIOLOGY OF THE FUNGI

by Melanospora destruens to be influenced by the concentration and kind
of sugar used. Glucose, fructose, or an equimolar mixture of these
sugars, when used at the rate of 5 g. per hter, allowed the production of
perithecia, but no perithecia were formed when 50 g. was used. Many
perithecia were produced when 50 g. per liter of sucrose was used.

The favorable effect of sucrose on perithecial formation was replaced
by various hexose phosphate esters. Glucose-1-phosphate and fructose-
1,6-diphosphate were equally active. In view of their ready enzymatic
interconvertibility in organisms, this would be expected. These results

V ^^'. Warn- ^^MF * ^M

ABC D E

Fig. 65. The effects of different carbon sources on the production of conidia by
Glomerella cingidata after 22 days on asparagine medium at 25°C. The carbon
sources are: A, glucose; B, sucrose; C, maltose; D, sorbose; E, starch. Note that
sporulation is greatest on sucrose and least on starch.

suggest that M. destruens phosphorylates sucrose with greater ease than
either glucose or fructose. This is in line with the experiments of Dou-
doroff (1945) with growth of Pseudomonas saccharophila. Since M.
destruens makes better growth upon glucose than upon sucrose, it may be
suggested that the pathway of carbohydrate utilization is different in
growth and reproduction. These findings emphasize again that the
requirements for growth and reproduction may be different.

Glucose, mannose, fructose, lactose, and sucrose are reported (Mix,
1933) as favorable for pycnidium formation by Phyllosticta solitaria.
Lactose was the most favorable sugar for the production of perithecia by
Diaporthe phaseolorum var. hatatatis (Timnick et al., 1951). Brodie (1948)
induced Cyathus stercoreus to produce normal, fertile fruit bodies on
semisynthetic media containing filter paper.

In our laboratory we have observed that the carbon source affects
reproduction of a number of other fungi, and that the best source for
sporulation is not always the same which yields maximum vegetative
growth (Figs. 65 and 66). Some of these fungi with some more favorable

SPORULATION

325

carbon sources for reproduction are Aspergillus niger, glucose, sorbose,
sucrose; Glomerella cingidata, sucrose; Phoma betae, sucrose; Monilinia
fructicola, sorbose, sucrose; Neocosmopara vasinfecta, maltose, starch,
glucose; Pleurage curvicolla, maltose, starch.

ABC
Fig. 66. The effect of three carbon sources and time on the production of perithecia
by Melanospora sp. on asparagine medium at 25°C. The carbon sources were: A,
glucose; B, sucrose; C, maltose. Above, cultures 11 days old; below, the same cul-
tures 22 days old. Note the poor vegetative growth but presence of perithecia on
sucrose and maltose, and the abundant early vegetative growth but delayed produc-
tion of perithecia on glucose.

For further information on the effects of nutritional factors on sporula-
tion, see Hawker (1950).

Carbon-nitrogen ratio. It seems to be generally held that a proper
balance among the constituents of the medium is quite important in
growth and sporulation. Westergaard and Mitchell (1947) investigated,
among other factors, the influence of the carbon-nitrogen ratio of the
medium on formation of perithecia by Neurospora crassa. Some of their
data are given in Table 56. It is evident that high concentrations of

326

PHYSIOLOGY OF THE FUNGI

glucose and potassium nitrate are unfavorable for the production of
perithecia by N. crassa.

Table 56. The Effect of the Carbon-Nitrogen Ratio of the Medium on the
Production of Perithecia by Neurospora crassa
Production rated on scale of 10. Age, 11 days. (Westergaard and Mitchell,
Am. Jour. Botany 34, 1947.)

Glucose con-

KNO3

concentration, %

centration, %

0.001

0.01

0.05

0.1

0.5

1.0

0.2

0.6

1.0

1.4

1.8

2.2

Micro essential elements. Steinberg (see references in Chap. 5) found
the sporulation of Aspergillus niger to be depressed by the omission of
various of the essential elements. Lockwood and Ward (1936) found
that Rhizopus oryzae sporulated on the thirteenth day of incubation when
zinc was not added to the medium. When zinc was added, sporulation
occurred on the third day of incubation. In general, when any essential
element is low, sporulation tends to be depressed before growth is
inhibited.

Specific reproductive factors. While many factors may influence
reproduction, there is little evidence that the fungi need specific chemical
substances to induce reproduction. Such factors, however, do exist
among certain of the Phycomycetes.

Four specific regulatory substances, called hormones (Raper, 1942, and
Raper and Haagen-Smit, 1942), were shown to initiate and control sexual
reproduction of Achlya hisexualis. A specific substratum is required for
the production of hormone A in large quantities. Hempseed allows a
2- to 10-fold production of hormone A over that produced by similar cul-
tures grown upon corn, rice, lentils, or other substances. Hormone A
has been concentrated 70,000-fold but has not yet been obtained in pure
form. This concentrate of hormone A is active in dilutions of 1 X 10^^^.

Sexual reproduction in Phytophthora cactorum was greatly stimulated by
an extract of garden peas (Leonian, 1936). This substance had no
growth-promoting properties. It was concluded (Leonian and Lilly,
1937) that the sexuality factor was none of the known vitamins, and that
it probably was not carotene or xanthophyll, although it was concentrated
by methods which would concentrate these substances. When vigorous

SPORULATION 327

sterile mycelium of P. cactorum was washed in distilled water and trans-
ferred to the optimum concentration of the sexuality factor in 0.1 per
cent agar, oogonia began to appear within 15 hr., reaching the maximum
development in 3 days. This factor was also effective in inducing sexual
reproduction by P. erythroseptica, P. boehmeriae, and P. megasperma. In
addition, the presence of this sexuality factor induced the formation of
abundant oogonia within a week by 15 of the 20 species of Phythium
tested. However, it failed to induce sporulation of various Zygomycetes,
Ascomycetes, and Basidiomycetes.

7-Carotene is associated with the male cells of the sexual phase and
not with the female cells of Allomyces (Emerson and Fox, 1940). This
specificity of association with the male cells indicates that 7-carotene may
be associated with sexual reproduction in some species of this genus.
No 7-carotene was found in the cells of these fungi in the asexual phase.

Association with other organisms. That one fungus may influence the
sporulation of another has been known for a long time. Sporulation of
Alternaria and Helminthosporium was increased when they were grown
in association with certain other organisms (Porter, 1924). This paper
has a valuable bibliography on associative effects.

The metabolic products of Aspergillus niger are known to promote
conjugation in three species of yeasts belonging to the genus Zygosac-
charomyces (Nickerson and Thimann, 1943). As a result of extensive
investigations, these authors found that part of the activity of Aspergillus
filtrate could be replaced by glutaric acid and riboflavin. While these
substances had some activity w^hen tested separately, the combination of
glutaric acid and riboflavin greatly exceeded the activity of either alone.
Riboflavin was shown definitely to be a component of the Aspergillus
filtrate. An autolysate from Zygosaccharomyces cells had a favorable
influence on sporulation by the same organism. From this experiment
and from the work of Lindegren and Hamilton (1944), who found that
ascus formation in yeast would take place only in portions of the yeast
colony where autolysis had taken place, it may be concluded that auto-
lytic products favor sporulation in some instances. Lindegren and
Lindegren (1944) found that addition of 2 per cent dried brewer's yeast
to a presporulation medium very favorably influenced the sporulation of
Saccharomyces cerevisiae.

The presence of Bacillus weidmaniensis greatly stimulated growth and
production of macroconidia by Microsporum audouini (Benedek, 1943,
and Hazen, 1947). The addition of yeast extract to the medium had a
similar effect. A part of this stimulating effect was attributed to pyri-
doxine. The addition of yeast extract to a basal medium of honey agar
resulted in a marked increase in vegetative growth and macroconidium
production (Hazen, 1947). This stimulation was attributed to the pres-

328

PHYSIOLOGY OF THE FUNGI

ence of growth factors in the yeast extract. The addition of pyridoxine
to the basal medium caused little change in mycelial growth, but a great
increase in abundance of macroconidia resulted. On the other hand, the
addition of thiamine or of a mixture of thiamine and pyridoxine caused
no increase in growth or production of macroconidia.

An interesting observation of the constant natural association between
Nectria coccinea and Gonatorrhodiella highlei is reported by Ayres (1941).
Because of this constant association it was believed that G. highlei was
either parasitic upon A^. coccinea or dependent upon it for some nutri-
tional substance. On potato-glucose, malt extract, and other common

Fig. 67. The stimulating effect of Aspergillus rugulosus (small colony at the bottom)
on the production of perithecia by Sordaria fimicola grown on glucose-asparagine
medium low in biotin. The zone of black perithecia of Sordaria around the colony of
Aspergillus is attributed to the biotin excreted by the latter fungus.

media G. highlei made only slight growth and formed no conidiophores or
conidia. However, the fungus grew well and produced numerous conidio-
phores and conidia on the same media in the presence of A^. coccinea, N.
galligena, or N. cucurhitula. Neither N. cinnabarina nor N . coryli caused
stimulation. G. highlei was cultivated successfully with production of
abundant conidia on oatmeal mush, without the presence of other fungi.
These results strongly suggest a nutritional relation between G. highlei
and N . coccinea, other than that of parasitism.

The beneficial effect of one fungus upon reproduction of another can
easily be demonstrated by placing Aspergillus rugulosus and Sordaria
fimicola on a plate of agar containing little or no biotin (Fig. 67). S.
fimicola, being a more rapid grower, produces a sparse mycelium, which
surrounds the slow-growing colony of Aspergillus. The Sordaria myce-

SPORULATION 329

lium next to the Aspergillus colony soon shows stimulated growth, which
is followed by the formation of abundant perithecia in this area. Since
we know that Sordaria is deficient for biotin and requires an exogenous
supply of this vitamin for reproduction, we may assume that the stimulat-
ing effect is due to the extra biotin produced by A. rugulosus diffusing into
the medium.

Vitamins. Many fungi cannot synthesize suflricient amounts of certain
vitamins and must depend upon an outside source of these vitamins for
optimum growth. Since many nutritional factors may influence both
growth and reproduction, it would be logical to expect that the vitamin
supply would affect the reproduction of vitamin-deficient fungi. Robbins
and Ma (1942) ventured the opinion that, although the sex organs of
certain deficient fungi were formed only in the presence of the growth
factor for which these fungi were deficient, they were doubtful if there
was any direct relation between vitamin supply and the formation of
sex organs. They regarded the failure of Ceratostomella pluriannulata to
produce perithecia in the absence of thiamine to be a disturbance of the
physiology of the fungus and ventured the prediction that the formation
of sex organs in other deficient fungi would be found associated with the
vitamins for which the fungi was deficient.

Melanospora destruens is able to grow in the presence of biotin as the
only growth factor, but it produces perithecia only when thiamine, too, is
added to the medium (Hawker, 1939). The relationship between the
amount of sugar and the amount of thiamine necessary for maximum
fruiting is clearly brought out by the thiamine-deficient fungus, C.
fimbriata (Barnett and Lilly, 1947a). Whether perithecia are formed on a
given medium is determined by the amount of thiamine relative to the
amount of food in the medium (Table 57). In a reduced supply of sugar
the concentration of thiamine necessary to induce fruiting is also reduced.
The abundance of perithecia is conditioned both by the amount of thia-
mine and by the amount of nutrients. Less thiamine is required for
vegetative growth than for the production of perithecia. A similar rela-
tion between amounts of sugar and biotin and sporulation of Memnoniella
echinata was described by Buston and Basu (1948). It should be noted
from Table 57 that, in a medium high in sugar but low in thiamine, no
perithecia were formed even though as much as 30 mg. of mycelium was
present. On the other hand, perithecia were produced on as little as two
mg. of mycelium when the medium contained the same amount of thia-
mine but was very low in sugar. These results are not in accord with the
idea that vigorous or abundant mycelium is essential to the formation of
perithecia; they indicate a more direct relation between vitamin supply
and sexual reproduction.

This direct relationship may be further illustrated by a simple experi-

330

PHYSIOLOGY OF THE FUNGI

ment. Sterile thiamine-starved mycelium may be obtained by growing
C. fimhriata on a synthetic medium containing 25 g. glucose and less than
2 ng of thiamine per liter. When a small portion of this sterile mycelium
is transferred to the surface of distilled water, no perithecia are formed.
How^ever, under the same treatment, but with the addition of thiamine
to the water, fertile perithecia are formed within a few days. We may
assume that, when thiamine is added, the fungus uses this vitamin as a
coenzyme in transforming the protoplasmic reserves into perithecia and

Table 57. The Effect of Various Concentrations of Thiamine upon Growth
AND Estimated Abundance of Perithecia Formed by Ceratostomella fimhriata

in the Presence of Various Concentrations of Nutrients
Growth in milligrams. Abundance of perithecia indicated by: 0 = none; + =

less than 20; ++ = 20 to 200; + + + = 200 to 1,000; + + + + = more than 1,000.

(Barnett and Lilly, Mycologia 39, 1947.)

Thiamine, /xg per

Dilution of i

Tiedium

culture (25 ml.)

Undiluted

2.5

110

+ + + +

_1__L-
1 1

0.156

+ +

+ + + +

+ +

0.04

+ +

+ + + +

0.02

+ + +

ascospores. This experiment has been successfully conducted using other
fungi deficient for thiamine, and similar results also were obtained with
biotin and biotin-deficient fungi. The amount of biotin added to the
medium affected not only the number of perithecia formed by Sordaria
fimicola, but also the time required for this fungus to form mature peri-
thecia (Lilly and Barnett, 1947). This period ranged from 13 to 41 days,
depending on the concentration of biotin used.

There is a pronounced effect of biotin deficiency upon the formation
and development of the ascospores of S. fimicola (Barnett and Lilly,
1947). This fungus is well suited for such a study, for normally nearly
all the ascospores mature at the same time. Figure 68 shows the effects
of biotin starvation upon the formation of ascospores. Severe effects
are evident by the failure of the protoplasm of the asci to be delimited
into ascospores or by the failure of many of the ascospores to mature.
Other conditions being equal, the amount of biotin required for the pro-

SPORULATION

331

duction of mature ascospores is greater than the amount required for
the formation of perithecia.

.4 B

Fig. 68. The effect of biotin starvation on the formation of ascospores by Sordaria
fimicola. A, asci from a perithecium developed in a suboptimum concentration of
biotin. Note the few mature ascospores and the majority of asci in which no spores,
or only aborted ascospores, have formed. B, normal asci with mature ascospores
developed on medium with optimum biotin concentration.

Since the vitamins are not considered as specific reproductive sub-
stances, it may be expected that an adequate vitamin supply may be
necessary for the formation of asexual spores as well as the sexual fruiting
structures. However, it should be kept in mind that asexual reproduc-
tion is more nearly like vegetative growth in its requirements than is
sexual reproduction. A reduction in the supply of a necessary vitamin
to the point where asexual reproduction is inhibited may also allow but
little vegetative growth. Piricularia orijzae is deficient for both thiamine
and biotin (Leaver et al., 1947). Conidia did not form unless both
vitamins were present in the medium. The concentration of biotin
could be reduced to such a level that conidial production was inhibited
but some mycelial growth was still allowed. In an adequate supply of
biotin, growth and sporulation were apparently parallel. It is also

Table 58. The Effects of Concentration of Glucose and Thiamine upon the

Number of Conidial Heads Formed by Choanephora cucurbitarum

(Barnett and Lilly, Phytopathology 40, 1950.)

Glucose,
g. per liter

Thiamine,
Mg per liter

Conidial heads
per plate

210

450

2,000

332 PHYSIOLOGY OF THE FUNGI

evident that a proper balance between the amounts of vitamins and
supply of nutrients is necessary for maximum production of asexual
spores. The effects of the concentrations of thiamine and glucose upon
the production of conidial heads by Choanephora cucurhitarum are illus-
trated in Table 58. On the other hand, we have observed that an excess
of certain vitamins may cause a decrease in sporulation of some fungi.

OTHER FACTORS

Method of inoculation. The method of inoculation and the type of
inoculum used are often important factors affecting sporulation in cul-
ture. Some fungi sporulate more quickly, and often more abundantly,
on agar when the medium is flooded with a spore suspension than when
the inoculum is placed at only one point. This is particularly applicable
to certain pycnidium- or acervulus-producing fungi, which usually pro-
duce abundant mycelium before fruiting.

It is possible that the spores of certain fungi carry over sporulation-
inducing substances to the next generation. The production of conidia
of CoUctotrichum lindeviuthianum was greater on agar media inoculated
at one point with spores than when bits of mycelium were used as inocu-
lum (Mathur et at., 1950).

Method of sterilizing media. The most common method of sterilizing
culture media is the use of steam pressure in an autoclave. The standard
time is usually 15 to 20 min. at 15 lb. pressure. This temperature is
known to cause a breakdown of certain sugars, with an accompanying
change in acidity of the medium. When natural media are used, other
chemical changes occur, which may or may not be beneficial to reproduc-
tion of the fungi. Shanor (1936) reported that fruiting structures of
Cordyceps militaris were not formed when autoclaved insects were inocu-
lated, whereas the fungus produced stromata and perithecia when living
pupae were inoculated. A new approach to the problem of sterilization is
suggested by Hansen and Snyder (1947), who propose the use of propylene
oxide. This method has great possibilities but has not been used exten-
sively enough to determine all its virtues and limitations.

Influence of the host. Numerous parasitic fungi which have been
grown on artificial media have not been induced to form the sexual repro-
ductive stages under any conditions in culture. Some of these will
produce the reproductive stages when grown on their respective hosts.
For instance, most smuts will produce mycelium in culture, but few have
produced teliospores (''chlamydospores") under these conditions. Claviceps
has never been induced to form sclerotia (which necessarily precede the
perfect stage) in artificial culture. Other fungi seem to lose their ability
to sporulate profusely in culture but may regain that ability when grown
on the appropriate host. It may be pointed out that many of the fungi

SPORULATION 333

pathogenic on man do not produce the same reproductive stages in culture
that they do in their host (Conant et al., 1944). Much more intensive
study needs to be made of these pathogens.

SUMMARY

Reproduction in the fungi, particularly sexual reproduction, with all the
necessary preliminary metabolic activities, is a complex phenomenon.
Some fungi appear to be relatively indifferent to and independent of their
environment with respect to sporulation, while others appear to require
a special combination of environmental conditions. If we assume that
all fungi require similar internal conditions for sexual reproduction —
and we do not believe this to be an unreasonable assumption — we must
recognize the existence of numerous fundamental differences in the
metabolic activities of the many different fungi. Some are capable of
creating the necessary internal conditions in spite of external conditions
which may be limiting factors for other fungi. Other species may not
have the ability to create the necessary internal conditions without
specific action of certain external factors, such as light, proper nutrients,
vitamins, and others. As has been previously emphasized, these proc-
esses are often dependent upon the enzyme systems of the fungus, and
these systems and their activity vary widely among the different species.

Many fungi reproduce sexually only as the vegetative growth is near or
past the maximum. At this time many of the cells of the mycelium are
dead or dying. Autolysis follows, and the cells that remain alive absorb
certain of these products of autolysis and are thus enabled to increase
their concentration of certain essential substances to such a degree that
reproduction is possible. If these speculations are valid, it might be
expected that nearly any type of injury which causes death of some of the
cells would have a favorable effect on reproduction of some fungi. Ultra-
violet radiation, which inhibits growth and often favors sporulation, may
act by killing some of the cells. Heat may also act by speeding up the
life processes of a fungus so that maturity is reached more quickly, which,
followed by death of cells and autolysis, would furnish the necessary
stimulus for reproduction. Treatment with hydrogen peroxide and
certain other chemicals may also result in death and autolysis of some
cells. No claim is advanced that these speculations are the true explana-
tion in all instances, but they do provide a hypothesis for interpreting
certain puzzling problems connected with sexual reproduction in the
fungi.

Factors which may initiate or stimulate sporulation of fungi which
grow well but fruit only sparingly or not at all under the usual conditions
of artificial culture (assuming that genetic factors are favorable), are as
follows: (1) A change in the concentration of one or more of the nutrients.

334 PHYSIOLOGY OF THE FUNGI

A reduction in sugar alone may be effective. (2) A change in the source
of carbon. Replacing glucose by the same amount of sucrose, lactose,
starch, or other carbon source may favor reproduction. (3) A change
in the source of nitrogen. (4) A change in the carbon-nitrogen ratio.
(5) The addition of an adequate supply of vitamins for vitamin-deficient
fungi. (6) The addition of certain micro elements to the medium, if it
is made up of highly purified chemicals. (7) The addition to the medium
of certain natural products, such as pieces of stems or leaves. (8) The
addition of culture filtrate containing metabolic products from the same
or other fungus. (9) The addition of special compounds, such as glutaric
acid, which has been shown to be effective in a few cases. (10) The
addition of certain specific sexual factors, or hormones, known to be
effective for certain Phycomycetes. (11) The addition of certain chem-
icals, such as hydrogen peroxide, to the mycelium. (12) Exposure of
cultures to ozone. (13) The use of spores instead of mycelium as inocu-
lum. Flooding of agar plates with spore suspension has given excellent
results with some fungi. (14) The sterilization of media by means other
than heat. (15) Transfer of certain parasitic fungi to their living hosts.
(16) Growing the fungus in the presence of certain bacteria or other
fvmgi. (17) A change in pH of the medium, (18) A change in the
temperature of incubation. (19) Adequate aeration. (20) Exposure to
light or alternate light and darkness. (21) Short exposures to ultraviolet
radiation. (22) Variation in the intensity and wave length of light. (23)
Mechanical injury to the mycelium. (24) Gradual desiccation of the
cultures. (25) Allowing the cultures to age. (26) The proper combina-
tion of any two or more of the above factors. The secret of the sexual
reproduction of many fungi no doubt lies in the proper combination of
factors which singly are known to favor reproduction in other fungi.

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for use as culture media, Phytopathology 37: 369-371, 1947.
*Hawker, L. E.: The influence of various sources of carbon on the formation of

perithecia by Melanospora destruens Shear in the presence of accessory growth

factors, Ann. Botany (N.S.) 3: 455-468, 1939.
Hawker, L. E.: Stimulation of the formation of perithecia of Melanospora destruens

Shear by small quantities of certain phosphoric esters of glucose and fructose,

Ann. Botany (N.S.) 12: 77-79, 1948.
Hawker, L. E.: Physiology of Fungi, University of London Press, Ltd., Bickley,

Kent, England, 1950.
Hazen, E. L. : Microsporum audouini: The effect of yeast extract, thiamine, pyrido-

xine, and Bacillus weidmaniensis on the colony characteristics and macroconidial

formation, Mycologia 39 : 200-209, 1947.
*Herny, W. B., and A. L. Andersen: Sporulation by Piricularia oryzae, Phytopa-
thology 38 : 265-278, 1948.
Houston, B. R., and J. W. Oswald: The effect of light and temperature on conidium

production by Hebtiinthosporium gramineum in culture, Phytopathology 36:

1049-1055, 1946.
Jones, R. C: Factors affecting the production of resistant sporangia of Allomyces

arbuscula, Mycologia 38: 91-102, 1946.
■*Kauffman, C. H.: Klebs' theory of the control of developmental processes in

336 PHYSIOLOGY OF THE FUNGI

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Lilly, V. G., and H. L. Barnett: The influence of pH and certain growth factors on

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*Lindegren, C. C., and E. Hamilton: Autolysis and sporulation in the yeast colony,

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SPORULATION 337

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Jour. Botany 28 : 355-357, 1941.

CHAPTER 15
SPORE DISCHARGE AND DISSEMINATION

While the production of mature viable spores may be considered the
climax in the life of a fungus, the wide dissemination of these spores is
often a requisite to the perpetuation of the species. Frequently, this is
assured by the production of enormous numbers of spores, which increases
their chances of falling into favorable environment. The chief agent of
dissemination among the fungi is air currents. Water, insects, and other
animals play lesser roles in the natural dispersal of spores. Light, dry
spores are usually disseminated by air currents, which may be strong
enough to loosen them from the fruiting structures on which they are
produced. On the other hand, spores borne in a gelatinous matrix are
better adapted to transmission by rain, by insects, or by other animals
which come in contact with them. Of particular interest are those fungi
which possess certain special mechanisms for discharging their spores
away from the fruiting structures. A study of the functions of these
special adaptations must of necessity be based upon a knowledge of the
structure of the fruit bodies which produce the spores.

METHODS OF SPORE DISCHARGE

The discharge or liberation of spores from the reproductive structures
which produce them may take place by (1) violent expulsion of the spores
or sporangia, due to internal pressure, (2) motility, as in the zoospores of
the aquatic Phycomycetes, and (3) external forces of the environment.

The violent discharge of spores, sporangia, or other reproductive bodies
depends upon the development of considerable pressure within the fungus.
The structure of the fungus cell is very similar to that of algae or the
parenchyma of the higher plants. The vacuole is filled with water and
its dissolved compounds, such as sugars, salts, and amino acids. Foods
in the cell m^ay be in the form of sugar, glycogen, or oil. If the amount of
soluble materials, such as sugar, is increased, the cell has a tendency to
absorb water. As a result, the cell becomes more distended and may
continue to swell until the elasticity of the cell wall is exceeded. The
increase in sugar concentration may be the result of the hydrolysis of
glycogen.

Discharge of sporangia. The genus Piloholus illustrates a remarkable
combination of adaptations for the production, discharge, and subsequent

338

SPORE DISCHARGE AND DISSEMINATION 339

dissemination of its spores. These include (1) the dependence upon Hght
for the production of sporangia, (2) the positively phototropic response
of the sporangiophores (see frontispiece), (3) the violent discharge of the
sporangium into the air toward the source of light, (4) the sticky nature
and the heavy black wall of the sporangium, and (5) the dissemination
of the spores by the passage through the digestive tract of animals which
ingest them.

We owe much of our knowledge regarding the structure of Piloholus, its
physiology, and its life history to the careful study and comprehensive
descriptions of Buller (1934). Much of his work was done with P. kleinii
and P. longipes. Brefeld (1881) showed that, in the absence of light, no
sporangia were formed, but that the sporangiophores continued to grow
for 10 to 14 days and reached the length of 8 to 10 in. A 2-hr. exposure to
light was sufficient for partially formed sporangiophores to complete their
development in the dark. Sporangiophores and sporangia developed
normally in blue light but did not develop in red-yellow light.

Under natural conditions, Piloholus produces successive daily crops of
sporangiophores and sporangia. Each crop requires approximately 24 hr.
for its development. The sporangiophores begin to form near midday
or early afternoon. By evening they have received enough light to allow
the further development and production of the sporangia during the
night. By the following morning, the sporangia are completely formed.
During the morning the sporangiophores react phototropically, directing
the sporangia toward the source of light. From midmorning to early
afternoon the sporangia are discharged violently into the air for a con-
siderable distance. The horizontal distance, according to Buller, may
be as great as 8 ft. 7 in.

To understand the mechanism of sporangium discharge in Piloholus, it
is first necessary to know the structure of the sporangium and the spo-
rangiophore (Fig. 69). The entire sporangiophore consists of a single
large cell, with a rather slender lower portion, a subsporangial swelling,
and a conical columella, which projects upward into the sporangium. A
rather thin layer of cytoplasm lies next to the cell wall and surrounds a
large central vacuole. At the base of the subsporangial swelling there is
a thick perforated ring of protoplasm, which is reddish in color, containing
carotene.

When the sporangiophore is pointed directly toward the source of light,
the parallel rays of light which strike the black hemispherical sporangium
are screened out. The subsporangial swelling acts as a lens, and the rays
falling upon it are bent so that they converge on, or uniformly near, the
red mass of protoplasm at the base of the swelling; this results in an
equilibrium, i.e., no bending occurs. When the sporangium is directed
at an acute angle away from the source of light, the side of the sub-

340

PHYSIOLOGY OF THE FUNGI

Fig. 69. A, a median longitudinal section of Pilobolus kleinii just before discharge oi
sporangium. The gun is pointed at an acute angle away from the light source. The
light rays that strike the sporangium are screened out. The rays that strike the side of
the subsporangial swelling are bent and concentrated on the oposite side. This presum-
ably gives a photochemical stimulus which is conducted to the motor region below the

SPORE DISCHARGE AND DISSEMINATION

341

A.H.RB,

subsporangial swelling. Bending toward the source of light then occurs until an
equilibrium is reached and the light rays are concentrated at the base of the sub-
sporangial swelling. B, at this time the gun is pointed directly toward the light
source. (After Buller, Researches on Fungi, Vol. VI, pp. 91, 92, 1934. Reproduced
by permission of Longmans, Roberts and Green,)

342 PHYSIOLOGY OF THE FUNGI

sporangial swelling, acting as a lens, causes the light rays to converge on
the side of the swelling away from the source of light (Fig. 69A). Pre-
sumably, this causes a photochemical reaction in the protoplasm, and the
stimulus is transmitted downward to the motor region, the portion of the
sporangiophore just below the subsporangial swelling. The growth of
this region is more rapid on the side away from the light source, which
results in a bending of the sporangiophore until an equilibrium of light is
again reached; i.e., when the sporangium is pointing directly toward the
source of light (Fig. 695).

There is a thicker layer of protoplasm near the upper portion of the
subsporangial swelling. This layer also contains some carotene. Buller
believes that it is photochemical ly reactive and may serve to bring about
chemical changes which result in the increase in the osmotic pressure of
the cell. When a culture with nearly mature sporangia is placed in the
dark, a much greater time is required for the discharge of the sporangia
than when it is left in the light.

The weakest place in the wall of the Pilobolus structure is located just
below the sporangium, and it is here that the wall of the subsporangial
swelling breaks circularly as the sporangium is discharged. The increased
osmotic pressure becomes too great for the resisting elastic wall, and the
system is ruptured. A drop of cell sap is squirted out of the tip of the
subsporangial swelling as discharge occurs. The conical columella is also
carried away with the sporangium.

In nature the sporangia adhere to the surface of vegetation, where they
may be ingested by herbivorous animals. The spores are released in the
digestive tract and pass out in the feces unharmed. It seems probable
that exposure to gastric juices helps to break dormancy of the spores and
favors immediate germination. The sporangium adheres to the vegeta-
tion by the lower gelatinous part, with the black, hemispherical, non-
wettable portion outward. Thus, the injurious ultraviolet rays are
screened out, and the spores remain viable, although they may not be
eaten for weeks or even months.

Basidioholus ranarurti shows a great many characters similar to those of
Pilobolus, namely, the general structure of the sporangiophore, the
mechanism of discharge of the sporangia, the coprophilous habit, and the
general method of dissemination. The sporangiophore consists of a
slender lower portion and cylindrical enlarged upper portion, which
supports a spherical sporangium. The osmotic pressure in the spo-
rangiophore increases to the point that it exceeds the tensile strength of
the resisting wall, which is suddenly ruptured circularly near the base
of the enlarged portion (Ingold, 1934). At this instant, the upper portion
contracts and causes the cell sap to be squirted backward, giving a rocket-
like effect. The dissemination of spores is accomplished after the spo-

SPORE DISCHARGE AND DISSEMINATION 343

rangia are eaten by beetles, which in turn are eaten by frogs or hzards.
After the beetles are digested and the sporangia are released, the spores
are formed. These spores are then capable of germination and produc-
tion of mycelium on frog or lizard excreta.

A somewhat different method of discharge is described for the genus
Entomophthora by Fitzpatrick (1930) and more specifically for E. sphaero-
sperma by Sawyer (1931). Instead of being due to a squirting action of
the cell contents, as in the case of Piloholus and Bastdiobolus, the discharge
of the sporangium in Entomophthora involves the opposing forces of
osmotic pressure and the adhesive power between the two walls separating
the sporangium and sporangiophore. As the sporangium matures, the
pressure on both sides of the separating walls becomes so great that the
outer wall is suddenly broken, and the sporangium is thrown into the
air. The sporangia, being sticky, readily adhere to the objects which
they strike.

The theory of violent discharge of sporangia in certain downy mildews
was advanced by De Bary (1887), using Peronospora parasitica and
Phytophthora infestans as examples. Later, Pinckard (1942) found the
forcible adiection of sporangia in Peronospora tabacina to be the same as
that described by De Bary. As the mature sporangiophore dries out,
the entire crown, with its branches and sporangia, begins a counterclock-
wise rotation. Each portion of the sporangiophore, including the
sterigma, rotates independently. The sporangiophore is hygroscopic,
and as the air becomes more moist, the movement is reversed. The effect
is a sudden release of the mature sporangia. During the course of rota-
tion many of the branches become entangled with others, and the spo-
rangia are dislodged by the spring-like action as the branches are dis-
engaged. The discharge of sporangia was verified by observations on
single isolated sporangiophores, showing that it is not dependent upon
the intermingling of the sporangiophores. No sporangia were released
in a saturated atmosphere, since no hygroscopic movement took place.
Other species which were observed to react similarly were Peronospora
parasitica, P. geranii, P. halstedii, and P. effusa. Similar rotation of
conidiophores upon desiccation is apparently not uncommon among fungi
of other groups, particularly those with long conidiophores.

Discharge of ascospores. In the majority of fungi (except those whose
asci deliquesce), ascospore discharge is accomplished by the building up
of osmotic pressure of the ascus to a point where it exceeds the resistance
of the elastic ascus wall. In one type of expulsion, the ascus wall is
suddenly ruptured, usually throwing the ascospores outward into the air
simultaneously. In other species, the ascospores are discharged succes-
sively through an apical pore in the ascus. In the latter case, the ellipsoid
or fusoid shape of the spore is apparently important. The spore pushes

344 PHYSIOLOGY OF THE FUXGI

pai't way through the pore to its broadest point and is then suddenly
squeezed out by the contraction of the ascus tip (Ingold, 1933).

The increase in the osmotic pressure within the maturing ascus must
be preceded by an increase in the soluble materials in the cell sap. This
is believed to be accomplished by the digestion of glycogen, which is
known to occur in the young ascus. In the majority of t he Pyrenomycetes
the asci are produced within a spherical or flask-shaped perithecium.

There are three general ways by which the ascospores are released
through the ostiole of the perithecium, two of which depend upon the
explosi^•e rupture of the ascus wall. In the first type, which is the most
common and believed to be the most primitive, the ascus wall remains
attached at its base, while the spores are discharged. This is accom-
plished by the elongation of the elastic ascus. until the tip reaches or
protrudes through the ostiole. The ascus then explodes, throwing the
ascospores into the air. The wall of the empty ascus contracts to the
base of the perithecium. and another ascus elongates. The process is
repeated successively as the asci mature. This type is illustrated by
Sordaria, Pleurage, and many other common fungi. An interesting
parallelism exists between the method of dissemination of Pilobolus and
that of Pleuragc, Sordaria, and other coprophilous Pyrenomycetes. The
short beaks of the perithecia are positi\ely phototropic and, as they
develop, are directed toward the source of light. In nature the ascospores
fall upon vegetation and are subsequently eaten and disseminated bj'
herbivorous animals. The vertical distances to which ascospores may
be shot have been reported as 6 cm. for S. fimicola and 45 cm. for P.
curvicolla (Weimer. 1920).

A second general type of ascospore discharge occurs more commonly in
species with long perithecial beaks. The asci become detached from the
base of the perithecium and are pushed up through the beak to the ostiole,
where the spores are released simultaneously or successively. This is a
rapid method of spore discharge. Examples of this type are Etidothia
parasitica. Gnomonia rubi, Guignardia bidivcllii, and CcraiosfomclJa anipid-
lax:ea. !Most of these species are adapted to AA"ind dissemination of
ascospores. Some idea of the tremendous numbers of ascospores dis-
charged is given by Heald and Walton (1914), who reported that some
specimens of E. parasitica expelled ascospores every day for 168 days.
The rate of spore discharge from one perithecium was found to be as high
as one ascus explosion about every 2 sec. At this rate approximately
14,000 ascospores may be discharged per perithecium per hour. On the
basis of these figures, it is little wonder that the fungus spread so rapidly
among the American chestnuts.

A third group includes the nonexplosive type of ascus, in which the
ascospores are released by the deliquescence of the ascus wall. Thej' are

SPORE DISCHARGE AND DISSEMINATION 345

embedded in mucilage, and as they accumulate in the body of the peri-
theciu.m, some spores ooze out through the ostiole, much like tooth paste
from the tube. Examples of this type are Chaetomium spp., Cerato-
stomeUa fimhriata, and C. ulmi. These spores are not adapted to wind
dissemination but may be carried in moist weather by insects (C. ulmi),
by other contacts (such as C. fimhriata on stored sweet potatoes), or by
rain.

The Discomycetes, in general, show a marked response to the stimulus
of light in orienting the asci so that the ascospores may be discharged
into the air away from the apothecium. The apothecium of Ascobolus
is small, and only a few asci mature at one time. As an ascus matures,
it enlarges greatl}^ and extends well beyond the surface of the hymenium.
It then reacts phototropically so that the tip is pointed directly toward
the source of light. When the ascus bursts, the operculum at the tip is
forced open, and the spores are expelled simultaneously. Most species
of Ascobohis are coprophilous and are disseminated in much the same
manner as Piloholus and Sordaria.

The phenomenon of ''puffing" in manj^ of the larger Discomycetes is
described in most textbooks of mj^cology and plant pathology. It is due
to the simultaneous violent spore discharge from many asci, so that a
cloud of spores may be seen to rise a few inches from the apothecium.
This may be so violent that a faint hissing or fizzing sound can be heard.
If the asci were to explode singly as they mature, the ascospores would
be shot up into the air only bj^ the initial force of the explosion. For most
species, this distance would probably not exceed 1 or 2 in. However,
when a great many asci explode simultaneously, an air blast is created
which carries the ascospores vertically to a much greater height, as great
as 5 to 7 in. (Buller, 1934). This additional distance above the fruit
body, which is commonly located on or near the ground, increases the
chances of dissemination by air currents.

In nature, the puffing of ascospores may be initiated by a sudden
change from shade to open sun, by the passing of a cloud, or by swaying
of a branch. Strong sunlight is not the only stimulus, for the phenomenon
has been observed in the laboratory under uniform light conditions. A
sudden jar of the fruit body, when it is tapped or picked up, may cause
spore discharge in some species. Likewise, an instant's exposure to
alcohol fumes may serve as the stimulus.

Buller (1934) has shown that in the cupulate or V-shaped apothecia,
such as those of Sarcoscypha protracta, many of the asci are pointing
directly toward the opposite side of the cup; yet the ascospores are dis-
charged upward, free from the fruit body. The operculum of this species,
instead of being centrally located at the tip of the ascus, is obliqueh^
placed toward the upper side of the ascus. As the discharged ascospores

346

PHYSIOLOGY OF THE FUNGI

leave the ascus, they are directed vertically. Biiller })elieves that the
oblique position of the operculum is a physiological character formed as a
response of the ascus end to the stimulus of light. Seaver (1928), how-
ever, believes that the position of the operculum is not determined by

light.

Fig. 70. Sections through the hymeniuni of Ascobolus magnificus, showing the photo-
tropic response of the ascus tips to Hght. Discharge of the ascospores is then directly
toward the source of hght. (After Buller, Researches on Fungi, Vol. VI, p. 272, 1U34.
Reproduced by permission of Longmans, Roberts and Green.)

In the development of the apothecium the paraphyses are formed
before the asci mature, and the developing asci push their way upward
among the paraphyses. In some species (Ascobolus spp., Lachnea scuiel-
lata) the paraphyses are straight, and only the portion of the ascus extend-
ing beyond the paraphyses tips responds phototropically (Fig. 70). The
paraphyses of others {Peziza hadia, Aleuria vesiculosa) bend toward the
light, and the developing asci are likewise bent as they elongate. Aleuria
repanda sometimes shows a coarse adjustment toward light, by the turn-
ing of the entire apothecium, and a fine adjustment, by the bending of the
ascus tips in the same direction.

SPORE DISCHARGE AND DISSEMINATION

347

Discharge of peridioles. The discharge of the peridiole (gleba-con-
taining basidiospores) of Sphaerobolus depends largely upon the unique
structure of the fruit body (Fig. 71). The spherical fruit body measures
but 2 to 3 mm. in diameter. The peridium is made up of six distinct
layers. At maturity, the peridium breaks open at the top, in a stellate
manner, through all but the sixth, or innermost, layer, which surrounds

Fig. 71. Structure of fruit body and mechanism of discharge of peridiole of Sphaero-
bolus stellatus. A, section of mature sporocarp, with six layers (1-6) that invest the
central peridiole (7). B, dehiscence of sporocarp at apex. The inner membrane is
liquefied. C, eversion of the remaining two layers by which the peridiole, D, is sud-
denly discharged. (Reproduced by permission from Wolf and Wolf, The Fungi, Vol.
II, p. 203, John Wiley & Sons, Inc., New York, 1947.)

the peridiole. This layer deliquesces, and the peridiole then rests in the
watery substance produced. A split then occurs between layers 3 and 4,
beginning at the base but not progressing to the very top. Laj^er 4, the
fibrous layer, is composed of small, rather closely packed cells, while layer
5, the palisade layer, is made up of comparatively large, somewhat
elongated cells. The cells of the palisade, which is on the concave sur-
face, increase in turgor and in size and are held under great tension by
the relatively inelastic fibrous layer. These inner layers are suddenly
everted, acting as a catapult, throwing the peridiole violently upward.
Walker (1927) has reported that the peridiole of S. stellatus may be
thrown to a vertical distance of 14 ft., while Buller (1934) reports a maxi-
mum horizontal distance of 18 ft. 7 in.

348 PHYSIOLOGY OF THE FUNGI

The force which causes the discharge is apparently located in the
palisade layer. It has been demonstrated by microchemical tests that
the palisade cells of the unopened fruit body are densely filled with
glycogen, which disappears before the discharge of the peridiole (Walker
and Andersen, 1925). The glycogen is converted to reducing sugars, one
of which is maltose, and this leads to the increase in osmotic pressure.
Light hastens the opening of the fruit body and the discharge of the
peridiole and is believed to speed up the conversion of glycogen into
sugars. S. stellatus is a coprophilous or lignicolous species, and the
peridioles may be eaten and disseminated by herbivorous animals.

Dodge (1941) reports his own observations as well as those of others
upon the presence of peridioles of the bird's-nest fungi attached to leaves
and branches as high as 10 to 15 ft. above the ground. Dodge describes
the attachment of the peridioles of Cyathus striatus to the peridium but
offers no theory to explain the mechanism of the peridiole discharge or
the force which is responsible. The slender mucilaginous threads which
attach the peridioles in the fruit bodies also serve to attach the discharged
peridioles to certain objects.

Discharge of basidiospores. The mechanism and the force involved
in the discharge of basidiospores in the Hymenomycetes and of the
sporidia of the smuts and rusts have not been satisfactorily explained.
There is no evidence that the explanation used for any of the types
described above can be applied to the discharge of basidiospores. How-
ever, certain structural features are present which may be adaptations
for this special method of spore discharge.

In all Basidiomycetes in which the spores are shot off forcibly, the
sterigma is attached slightly to one side of the tip of the spore (Fig. 72).
Just before a spore is discharged, a small drop of liquid appears at the
tip of the sterigma. Its invariable presence is believed to be an important
feature in the process of spore discharge. After discharge, there appears
to be no pore present, either in the spore or in the tip of the sterigma.
Buller (1922, 1924) suggests that the spore may be shot from the sterigma
by hydrostatic pressure, but that the amount of liquid ejected as the
explosion occurs at the sterigma may be so minute that it may not be
detected by the microscope. He further suggests that the surface ten-
sion of the drop of water may in some way bring about the abjection of the
spore. While neither theory is completely satisfactory, they are the best
yet offered.

Discharge of aeciospores. Experimental work showing that the
aeciospores of the rust fungi are violently discharged was reported by
Buller (1924) and Dodge (1924, 1924a). They have reported this phe-
nomenon in seven species, including Puccinia coronota, P. graminis, P.
podophijlli, Gytnnoconia peckiana, and Uromyces pisi. The maximum

SPORE DISCHARGE AND DISSEMINATION

349

distance above the aecia to which the spores were shot varied from 6 to
15 mm., being about 8 mm. for P. graminis. The exact mechanism of
aeciospore discharge is not known, but it probably depends largely upon
turgor pressure of the mature aeciospores, which are formed in chains.
The double wall between two aeciospores is at first flat, but near maturity
of the spores the osmotic pressure increases and tends to make these w^alls
convex. It is assumed that the adhesive force between the two walls is
suddenly overcome by the increased osmotic pressure, and the terminal
spore or group of spores is thrown outward into the air.

Fig. 72. Discharge of the third basidiospore from basidium of Agaricus campestris,
following the formation of a drop of water at the tip of the sterigma. (After Duller,
Researches on Fungi, Vol. II, p. 12, 1922. Reproduced by permission of Longmans,
Roberts and Green.)

Liberation of zoospores. In most of the aquatic Phycomycetes and in
some terrestrial forms which show definite aquatic affinity, zoospores are
the primary means of reproduction. The characteristic motility of the
zoospores may be more correctly considered as a means of local dissemina-
tion, but motility is also involved in the liberation from such large spo-
rangia as those of Saprolegnia and related fungi.

INFLUENCE OF EXTERNAL CONDITIONS

The effect of light upon the discharge of spores by Piloholus, Ascoholus,
Sordaria, and Sphaeroholus has been discussed briefly. Light is necessary
for the production of spores in a number of fungi in which it plays no
direct part in spore discharge.

Favorable temperature is a prerequisite for all biological activity.
Its effect upon spore formation and discharge is often not clearly defined.
The maximum temperature permitting spore discharge is appreciably

350

PHYSIOLOGY OF THE FUNGI

Fig. 73. Stills from ultra-high-speed film showing impact of drop of water with a fruit
body of Lycoperdon perlatum and the subsequent puff of spores. The drop, 5.0 mm. in
diameter, fell with a velocity of -440 cm. per sec. The time elapsed between the con-
tact of drop with peridiimi until the last photograph was 0.046 sec. (Courtesy of
Gregory, Trans. Brit. Mycol. Soc. 32 : 14, 1949. Published by permission of Cam-
bridge University Press.)

SPORE DISCHARGE AND DISSEMINATION 351

lower than that for viabiht}^ of the fungus. Low temperatures usually
merely slow down spore production and discharge. Duller (1909) found
that Daedalea unicolor, Lenzites hetulinus, Polyporus versicolor, and P.
hirsutus discharged spores when the air temperature was 0°C. Schizo-
phyllum commune shed spores vigorously at 5°C., but not at 0°C.

Andersen et al. (1947) showed that few conidia of Piricularia orijzae
were liberated when the host plants were dry. Continued wetting greatly
increased the secondary spread of the fungus in experimental trials.
Sporidia of rusts are formed and discharged only during periods of high
humidity. The need of the downy mildew fungi for changing conditions
of humidity has been pointed out. The hygroscopic character of the
capillitium of certain slime molds aids in pushing the spores to the surface,
where they may be disseminated by various agents. Many other species
of fungi appear to be independent of the air moisture, as long as there is
sufficient moisture in the fruit body. Gravity is believed to have little
effect upon spore discharge, except in the proper orientation of the fruit
bodies.

The spores of some puffballs are enclosed within the nearly spherical
peridium, which opens by an apical pore. When sudden pressure is
applied to the peridium, the spores are puffed out of the pore in clouds of
"smoke." Gregory (1949) has shown, by use of ultraspeed photography,
that raindrops falling on the thin peridium of Lycoperdon perlatum cause
a puffing of spores (Fig. 73). The velocity of the puff as the spores
emerged from the ostiole was approximately 100 cm. per sec. Under the
conditions of the experiment, it was estimated that a drop of water falling
130 cm. caused the ejection of approximately 15 million spores. The
endoperidium and the spores inside remain dry, and the puffing is not
hindered, even during a rain. The impact of raindrops is believed to
be an important means of spore discharge from the ostiolate puffballs.

SPORE DISSEMINATION

Many other fungi do not have any special method of spore discharge
and must depend upon physical or biotic agents in nature for getting their
spores away from the fruit body where they are produced. Some of these
possess some special adaptations for dissemination by certain agents.
The most important agent of dissemination is air currents. The uredio-
spores of the rusts are not violently discharged. They accumulate in
the sorus and must be dislodged by the wind or movement of the host
plant. Many of the Monihales which produce dry conidia also depend
upon air movement to shake them loose from the conidiophores. Con-
vection currents are responsible for local spread of dry spores, while
splashing rain is important in the dissemination of spores with matrix.

352 PHYSIOLOGY OF THE FUNGI

It is well known that spores of some of these fungi may be blown for
hundreds of miles and remain viable.

Dissemination of the zoospores of the aquatic fungi may be accom-
plished locally by means of the flagella or for greater distances by the
movement of water, which may carry the spores both in the motile and
in the encysted stages. The condition of diplanetism, which involves
two motile stages separated by an encysted stage, may be advantageous
for the greater dissemination in water. The translocation of mud and
moist soil by means other than water may also be important. Although
the motility of zoospores of certain parasitic fungi, such as Phytophthora
infestans, can scarcely be considered an important means of dissemina-
tion, it does enable the spore to move short distances in a drop of water
on the host and facilitate penetration through stomata.

Insects are likewise important agents of dissemination of fungus spores.
The insect may be attracted to spore masses by odor or color and feed
upon the spores, or the insect may be merely an incidental carrier of
spores adhering to the external parts of the body. Leach (1940) states
that "in the majority of cases where the question has been investigated,
spores have been found to pass through the intestinal tract of insects
uninjured." The conidia of Claviceps purpurea, being produced in sweet
droplets of liquid, also emit an odor which attracts insects. Similarly,
the stinkhorn fungi produce their spores in a malodorous matrix and are
frequently visited by flies. These adaptations ensure insect dissemina-
tion. The blue-stain fungi {Ceratostomella spp.) produce spores in sticky
droplets in the tunnels of bark beetles, which act as the principal agents
of dissemination.

The symbiotic relationship between species of Septohasidium and scale
insects represents a highly evolved adaptation for the dissemination of
the fungus spores. Although these fungi produce basidiospores, they are
unlike most Basidiomycetes in that the spores are not forcibly discharged
from the basidium. Couch (1938) has made a comprehensive study of
this genus and has described the life history in detail. The fungus forms
a layer over the bodies of scale insects, some of which are parasitized,
while others are not. The uninfected female insects under the fungus
may produce young, many of which crawl over the sporulating surface
at the time of sporulation. These may become infected, crawl about, and
settle down some distance away. Such infected young insects are solely
responsible for the dissemination of the fungus.

Spore dissemination is also unique in the Tuberales (truffles), whose
fruit bodies are formed entirely underground. There are no direct means
of getting the spores up to the air for dissemination. These fruit bodies,
which give off an odor, are dug up and eaten by rodents. In this process,
pieces of the fruit bodies are dropped, and the spores are thus disseminated.

SPORE DISCHARGE AND DISSEMINATION 353

SUMMARY

Many fungi have no means of forcibly discharging their spores but must
depend upon the physical and biotic factors in nature for liberation and
dissemination of spores. Others possess special mechanisms for dis-
charging their spores away from the fruiting structures which bear them.
In most cases this violent discharge depends upon high osmotic pressure
within certain cells of the fungus. Increased osmotic pressure usually
is a result of the digestion of glycogen to soluble sugars.

Many coprophilous fungi, such as Pilobolus, Ascobolus, Sordaria, and
Pleurage, forcibly discharge their spores for some distance toward the
source of light. In nature this adaptation is of great advantage to the
fungus in its dissemination by animals, which ingest the spores with the
vegetation. Some Discomycetes exhibit a puffing of the spores when
many asci discharge their spores simultaneously.

In Pilobolus, Basidiobolus, and Entomophfhora the sporangia are forcibly
abjected from the sporangiophores. Ascospores may be ejected either
simultaneously or successively from the ascus. The ascus may elongate
to reach the surface of the ascocarp and discharge its spores, or the asci
may become detached in some Pyrenomycetes and, after being forced
through the ostiole, may explode to release the spores. In other species
the ascus walls are deliquescent, and the ascospores ooze out of the
ostiole.

The basidiospores of most Basidiomycetes are forcibly discharged by a
mechanism which is not well understood. A drop of liquid is extruded at
the tip of the sterigma just prior to discharge and is believed to affect the
process in some way. The peridioles of Cijathus and Sphaerobolus may
be thrown several feet away from the fruit bodies. The latter fungus
exhibits a unique catapult action by a portion of the fruit body. Other
mechanisms act in the forcible discharge of the aeciospores of many rusts
and the sporangia of Peronospora.

Air currents are the most common agent of dissemination of dry spores.
Spores borne in a sticky, malodorous, or sweet matrix are well adapted to
insect dissemination. Some fungi, such as Septobasidium, have estab-
lished a symbiotic relationship with certain insects, which are the sole
agents of dissemination.

REFERENCES

Andersen, A. L., W. B. Henry, and E. C. Tullis: Factors affecting infectivity,

spread and persistence of Piricularia oryzae, Phytopathology 37: 9-4-110, 1947.
Brefeld, O.: Botanische Untersuchungen iiber Schimmelpilze, Heft 4, Verlag von

Arthur Felix, Leipzig, 1881.
*Buller, A. H. R.: Researches on Fungi, Longmans, Roberts and Green, London.

Vol. I, 1909; Vol. II, 1922; Vol. Ill, 1924; Vol. VI, 1934.
Couch, J. N. : The Genus Septobasidium, The University of North Carolina Press,

Chapel Hill, 1938.

354 PHYSIOLOGY OF THE FUNGI

De Bary, a.: Comparative Morphology and Biology of the Fungi, Mycetozoa and

Bacteria (trans. H. E. F. Garrney, rev. I. B. Balfour), Oxford University Press,

New York, 1887.
Dodge, B. O.: Aecidiospore discharge as related to the character of the spore wall,

Jour. Agr. Research 27: 749-75G, 1921.
Dodge, B. O.: Expulsion of aeciospores by the may apple rust, Puccinia podophylii

Schw., Jour. Agr. Research 28: 923-92(i, 1924a.
Dodge, B. O.: Discharge of the sporangioles of bird's nest fungi, Mycologia 33:

650-654, 1941.
FiTZPATRiCK, H. M.: The Lower Fungi, McGraw-Hill Book Company, Inc., New

York, 1930.
Gregory, P. H. : The operation of the puff-ball mechanism of Lijcoperdon perlatum

by raindrops shown by ultra-high-speed Schlieren cinematography, Trans.

Brit. Mycol. Soc. 32: 11-15, 1949.
Heald, F. D., and R. C. Walton: The expulsion of ascospores from the perithecia

of the chestnut blight fungus, Endothia parasitica, Am. Jour. Botany 1 : 499-522,

1914.
*Ingold, C. T.: Spore discharge in the ascomycetes, New Phytologist 32: 178-196,

1933.
Ingold, C. T.: The spore-discharge mechanism in Basidiobolus ranarum, New

Phytologist 33: 274-277, 1934.
*Leach, J. G.: Insect Transmission of Plant Diseases, McGraw-Hill Book Company,

Inc., New York, 1940.
*PiNCKARD, J. A.: The mechanism of spore dispersal in Peronospora tabacina and

certain other downy mildew fungi. Phytopathology 32: 505-511, 1942.
Sawyer, W. H. : Studies in the morphology and development of an insect-destroying

fungus, Entoniophthora sphaerosperma, Mycologia 23: 411-432, 1931.
Seaver, F. J.: The North American Cup Fungi. Operculates, published by the

author, New York, 1928.
* Walker, L. B.: The development and mechanism of the discharge in Sphaerobolus

iowensis and S. stellatus, Jour. Elisha Mitchell Set. Soc. 42 : 151-178, 1927.
Walker, L. B., and E. N. Andersen: Relation of glycogen to spore ejection,

Mycologia 17: 154-159, 1925.
Weimer, J. L.: Some observations on the spore discharge of Pleurage curvicolla,

Am. Jour. Botany!: 75-77, 1920.
Wolf, F. A., and F. T. Wolf: The Fungi, Vol. II, John Wiley & Sons, Inc., New

York, 1947.

CHAPTER 16
SPORE GERMINATION

Spore germination, in general, implies a change from an inactive to an
actively growing condition. This is accomplished in most fungi b}^ the
formation of a germ tube, which continues to elongate and ultimately
leads to the formation of the vegetative body of the fungus. In the
Phycomycetes the germination of oospores and of some sporangia may
take place by the internal formation of zoospores. Certain higher fungi
also produce secondary spores externally without the formation of
mycelium.

Among the universal requirements for the germination of spores are
(1) suitable temperature, (2) adequate moisture supply, (3) adequate
oxygen supply, (4) suitable hydrogen-ion concentration, and (5) viable
spores.

Some of these factors may be measured quantitatively, and for each
there is an optimum for germination for a given fungus. The maximum
percentage of spore germination in the shortest time will occur when all
the influencing factors are at or near the optimum. This is a situation
which might seldom occur in nature. Germination will occur or not
depending upon the number and relative importance of the favorable
factors.

Clayton (1942) suggests that the differences in nutritional require-
ments for germination shown by the various species may be due to dif-
ferences in the spore wall and in the composition and quantity of reserve
foods. Some spores contain stored food in the form of oil, while others
contain glycogen. It is believed that water enters the spore by imbibition
or osmosis and activates the enzyme, glycogenase, which hydrolyzes
glycogen to sugars.

Spores which do not germinate after being exposed to the usually
favorable conditions for a reasonable length of time are said to have a
period of dormancy, which may be broken by the presence of a special
set of conditions. Among these are (1) exposure to high or low tempera-
ture, (2) the presence of certain nutrients or stimulants in natural prod-
ucts, (3) exposure to chemical stimulants, (4) alternate wetting and dry-
ing, and (5) aging. These same factors may also influence the percentage
of spore germination of many species which do not have a definite dormant
period. Dormancy may be due, at least in some cases, to the failure of

355

356 PHYSIOLOGY OF THE FUNGI

the usual favorable conditions to activate a certain enzyme. Some
special stimulus may then be required to perform this function.

The present discussion deals primarily with the factors which influence
some physiological phase of spore germination. The main emphasis
will be placed upon a discussion of these factors and how they act, or
what part they play in germination. Doran (1922) gives a good discus-
sion of the more important factors affecting germination and lists many
references to the earlier work. A more recent discussion of this subject
is given in the excellent review by Gottlieb (1950).

PHYSICAL FACTORS

Temperature. Temperature is one of the most important external
factors which influence germination. It not only affects the percentage
of germination but also the length of time required for germination, and,
in certain fungi, it often determines the method of germination. The
literature contains a great many references to spore germination at dif-
ferent temperatures, but none of these have attempted to explain the
intricate effects or responses within the spore. We may assume that a
favorable temperature permits certain enzymatic activities essential to
germination. Different species of fungi have different temperature
requirements for germination. The cardinal temperatures (minimum,
optimum, and maximum) for spore germination may be found for many
fungi in scattered reports. These are based mainly on casual observa-
tions made during the study of other problems. Few comprehen-
sive studies have been made of the effects of temperature upon spore
germination.

The cardinal temperatures of a few selected species are presented as
examples: AUernaria solani, 1 to 3°C., 26 to 28°C., 37 to 45°C. ; Cronartium
rihicola aeciospores, 5°C., 12°C., 19°C.; Phyllosticta antirrhini, 18°C.,
25°C., 'i7°C. ■,Phytophthora infestans, 2 to 3°C., 12 to 13°C., 24 to 25°C.;
Puccinia graminis teliospores, 5 to 9°C., 20 to 22°C., 23 to 25°C. ; Venturia
inaequalis conidia, 3°C., 14 to 15°C., 31°C. For other examples, see
Doran (1922).

The general optimum temperature for spore germination of certain
species of Agaricaceae and Nidulariaceae is near 30°C. (Kauffman, 1934).
Walker and Wellman (1926) found that, when the soil temperature was
above 25°C., there was low percentage of "chlamydospore" germination
of Urocystis cepulae, while spore germination ceased entirely at 29 or
30°C. They attribute the low percentage of infection above 25°C. to the
direct inhibitory effect of the higher temperature upon the parasite.

The presence of nutrients and the supply of oxygen caused variation
in the minimum temperature for spore germination in Colletotrichum
lagenarium (Gardner, 1918). The minimum was 14°C. in water with the

SPORE GERMINATION 357

hanging-drop technique, 7°C. in exposed drops of water, and 4°C. in
prune decoction. This may serve to exphiin, in part, the differences in
cardinal temperatures reported by different authors.

The method of spore germination may be determined by temperature.
Below 20°C. the sporangia of Phytophthora infestans germinate more
frequently by the formation of zoospores, while above this temperature
production of a germ tube is more common.

The optimum temperatures for germination for the various species of
Myxomycetes were from 22 to 30°C., with an over-all range of 2 to 36°C.
Certain species had narrower ranges. Below 10 and above 30°C. the
rate of germination was greatly reduced. When the spores of Enteridium
rozeanum were held for a time at a temperature above maximum and then
returned to optimum, the spores germinated explosively through a thin
area in the wall (Smart, 1937). Under uniform optimum temperature
an irregular pore was formed and the protoplast slowly squeezed out.

It must be emphasized that temperature affects the time required for
germination, as well as the percentage of germination and the growth
rate of the germ tubes. All three have been used as measurements of
spore germination. It seems likely that temperature might have a
greater effect upon the time of germination than upon the percentage
of germination.

Heat treatment and breaking of dormancy. The effectiveness of pre-
heating ascospores of Neurospora tetrasperma in breaking their dormancy
has been discussed by several authors. Heating the spores to 50°C. for
a few minutes induced germination 2 or 3 hr. after they were returned to
a favorable temperature. Goddard (1935) found that spores thus
"activated" could be "inactivated" (returned to the dormant condition)
by placing them under anaerobic conditions for a short time. They
remained dormant when brought back into air. The respiration rate of
the activated spores was greatly increased and germination occurred
only after 2 to 3 hr. of continuous high respiration. Further work by
Goddard and Smith (1938) led to the conclusion that the heat activated
carboxylase, which is latent in the dormant spores, and that two different
respiratory systems are in operation : one, the dormant system, functions
in the absence of carboxylase; and the second, the active system, functions
after the spores are heated. Similar stimulation of germination of
Ascoholus ascospores by heat was found by Dodge (1912). Only a few
spores germinated in water without being preheated. Heating the spores
to 65 to 75°C. for approximately 15 min. and then returning them to
favorable temperature allowed good germination of most species.

Moisture. The spores of many species of fungi will not germinate
unless they are in contact with liquid water. Others are capable of
germination on dry surfaces in an atmosphere of high humidity, usually

358 PHYSIOLOGY OF THE FUNGI

95 per cent or above. A third group is represented by some of the pow-
dery mildews, whose spores are able to pat out short germ tubes under
conditions of extremely low relative humidity. Comparatively little
careful work has been done to determine the moisture requirements for
spore germination. Doran (1922) reviewed some of the earlier reports
and gives the results of his own experiments. Among the species whose
spores have been reported as requiring contact with liquid water for
germination are the following: Sderotinia Jnicticola, Peronospora pygmaea,
Phyllosticta antirrhim, teliospores of Gymno sporangium juniperi-vir-
ginianae, Cylindrocladium scoparium, and Plasmopara viticola.

Numerous fungi whose spores may germinate in the absence of liquid
water have been reported. Some of these have been germinated on a dry
glass slide in a moist chamber, where the humidity is assumed to be at
100 per cent, the saturation point. However, Clayton (1942) showed
that a humidity of 100 per cent sometimes gave visible condensation of
water vapor, w^hereas a relative humidity below 99.85 per cent gave no
condensation at constant temperature. The spores of this group of
fungi usually show a much higher percentage of germination in liquid
w'ater if a plentiful supply of oxygen is present. Some representative
fungi reported in this group with the approximate minimum humidity
are Puccinia glumarum urediospores, 99 per cent; Venturia inaequalis
ascospores and conidia, 98.7 per cent; Ustilago nuda, 95 per cent; and
Penicillium glaucum, 84 per cent. The minimum relative humidity for
Aspergillus niger is near 70 per cent (Bonner, 1948). Figure 74 shows
germination curves.

The germination of the conidia of certain species of the Erysiphaceae
in relative humidity at or near zero has been reported by several investi-
gators (Brodie, 1945; Brodie and Neufeld, 1942; Yarwood, 1936; and
Clayton, 1942). These species are Erysiphe polygoni, E. graminis, and
Microsphaera alni. The mechanism for spore germination under these
very dry conditions must be quite different from that of other spores,
which require liquid water or high humidity for germination. The
"apparent osmotic pressure" of the cell sap of the conidia is reported by
Brodie (1945) as about 63 atm. for E. polygoni and 68 atm. for E. graminis
hordei. It is likely that these high osmotic pressures may be an aid in
absorbing moisture from a relatively dry atmosphere. Brodie believes
that the conidia contain little free water but that imbibition may be
partly brought about by hydrophilic colloids.

Brodie and Neufeld (1942) offer a tentative theory to explain "germi-
nation" under conditions of 0 per cent humidity. They believe that, as
germination begins, free water is released by respiration and by changes
in colloidal materials containing bound water. No changes in the length
or width of the conidia could be detected during germination. The

SPORE GERMINATION

359

formation of the germ tube was calculated to add approximately 2 or 3
per cent to the volume of the ungerminated conidium. It is believed
possible that this slight increase in volume might be accounted for by
one or both of the above factors. Yarwood (1936) offers no explanation
of the process of germination at such a low humidity but reports a decrease

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Fig. 74. Germination curves for Aspergillus niger under variable temperature and
humidity. Note that the optimum temperature for germination varied with the
relative humidity, being near 30°C. at relative humidity of 100 per cent and near 40°C.
at 93 per cent. As the temperature or humidity digressed from the optimum, ^the
time required for germination increased. (Courtesy of Bonner, Mycologia 40 : 733,
1948.)

of about 24 per cent in volume of the Erijsiphe conidia during germina-
tion. Spores of all other fungi (except other powdery mildews) which he
tested showed increases in volume during germination.

Dormancy of some spores may be broken by alternate wetting and
drying. This treatment apparently makes the thick resistant wall more
permeable to water.

Oxygen supply. Since respiration is greatly accelerated during spore
germination, it follows thg^t an adequate supply of oxygen is a prerequisite

360 PHYSIOLOGY OF THE FUNGI

for germination. Brief reports of a number of observers on oxygen
requirements are given by Doran (1922). It is generally agreed that
reduced oxygen supply decreases spore germination. Spores germinate
better on or near the surface of a li(iuid than when submerged deep in the
liquid. In some cases the spores may germinate under water, but only
abnormal germ tubes are formed. Aerated water gives better germina-
tion than nonaerated water. The spore load in a drop of water, whether
all of the same species or of mixed spores, influences greatly the percent-
age of germination. This is believed to be due primarily to the competi-
tion for the limited supply of oxygen, rather than to toxic substances
produced by other germinating spores.

According to Jones (1923), spore germination of Ustilago avenae is
greatest in soil with 30 per cent of water-holding capacity and is greatly
reduced at 80 per cent. This was probably due to the amount of avail-
able oxygen. The spores failed to germinate in water when exposed to
an oxygen-free atmosphere. The " chlamydospores" of Ustilago zeae
do not germinate in the absence of oxygen, and at least 5 per cent oxygen
must be present to allow germination as high as in the open air (Platz
et at., 1927).

The supply of oxygen may influence the method of spore germination
(Uppal, 1926). Germination by zoospores was possible in the absence
of oxygen for the sporangia of Phytophthora mfcstans, P. colocasiae, P.
palmivora, and P. parasitica. Germination by germ tubes does not take
place in these species in the absence of oxygen. However, the presence
of oxygen is essential for zoospore formation by sporangia of Alhiigo
Candida, Plasmopara viticola, and Sclerospora graminicola. The two
methods of germination are different processes, the direct method more
nearly resembling vegetative growth.

Hydrogen-ion concentration. Under natural conditions acidity is not
usually a limiting factor for spore germination. In general, spores will
germinate within a wide pH range. It seems significant that, in most
species of fungi, germination is favored by an acid medium, often at a
pH considerably lower than the optimum for vegetative growth or sporu-
lation. The effects of acidity of the medium upon a number of species,
including Botrytis cinerea, Aspergillus niger, Penicillium cyclopium, P.
italicum, Puccinia graminis urediospores, Lenzites saepiaria, Colleto-
trichum gossypii, and Fusarium sp., are reported by Webb (1921). The
spores of the Fusarium germinated equally well in alkaline and acid
media, while CoUetotrichuni gossypii was the only species of the group
studied in which germination was better in an alkaline medium. At
pH 2.5 spore germination was prevented in all species, and the optimum
for most species was 3.0 to 4.0. In sucrose-nitrate (Czapek's) solution,
two maxima usually occurred, the primary one at pH 3.0 to 4.0 and a

SPORE GERMINATION 361

secondary one between G.O and 7.0. Of all the media tested, beet decoc-
tion gave the maximum germination imder the widest range of conditions.
Webb also clearly demonstrated that the range of pH favoring germina-
tion is influenced by temperature and by the constituents of the medium.

All the species of Myxomycetes studied by Smart (1937) germinated
within a pH range of 4.0 to 8.0. Spores of Fuligo septica germinated
from pH 2.0 to 10.0. Optimum for all species ranged from 4.5 to 7.0,
with some germinating better near 4.5 and others near 7.0. The spores
of Urocystis occulta germinated between pH 5.0 and 8.9, with the optimum
at 6.8 (Ling, 1940). This optimum is higher than those for most fungi.
Kauffman (1934) found the range for spore germination of several species
of Basidiomycetes (Agaricaceae and Nidulariaceae) to be pH 5.0 to 8.5
with the optimum near 7.5.

It is interesting that Doran (1922) in his review of spore germination
makes no mention of acidity as a factor. It would appear that acidity
is of more or less importance as a modifying factor, even though it is
seldom a limiting factor for spore germination. This may explain, at
least in part, the fact that we often find abundant ungerminated spores
in fruiting liquid cultures. Some fungi sporulate only in neutral or
alkaline media, which, in general, are not favorable to spore germination.

NUTRIENTS AND STIMULANTS

The constituents of the substrate are known to influence spore germina-
tion of some species of fungi. Some species germinate well in distilled
or tap water, while others require certain special nutrients such as sugar,
salts, or even a particular nitrogen source. No one medium has been
found which will allow good germination of all fungi, although certain
natural media, such as beet or bean decoction and soil infusion, seem to
favor germination in a large number of fungi. When such media con-
taining natural products are used, it is difficult to determine whether the
higher percentage of spore germination is due to the nutrients or to some
stimulant which is not used in the metabolism of the fungus.

Duggar w^as one of the foremost American workers interested in spore
germination as a primary subject of experimentation. Prior to his work,
most of the study on spore germination was only incidental to other
problems. Duggar (1901) demonstrated that species differ in their
nutrient requirements for germination by placing spores in water, bean
decoction, nutrient-salt solution, and cane-sugar solution. A portion of
his data showing the percentage of germination after 15 hr. is given in
Table 59.

Among some of the compounds Duggar found to influence sporulation
of Aspergillus flavus and A. niger were varying amounts of peptone,
ammonium nitrate, and magnesium sulfate. Ammonium nitrate at a

362

PHWSIOLOGY OF THE FUNGI

particular concentration gave abundant germination of A . flavus but had
no effect upon ^4. niger.

Brefeld (1905) was perhaps the first to observe the germination of the
spores of various smuts in culture. He noted that the spores germinated
poorly or not at all in water, while excellent germination occurred in
nutrient solutions (probably dung infusion). Brefeld expressed surprise
at the vigorous saprophytic development which followed, especially since
the species had previously been known only as obligate parasites.

More recently it was noted that pretreatment with dung infusion
markedly stimulated germination of spores of Ustilago striiformis (Cheo

Table 59. Percentage of Spore Germination after 15 Hours
(Duggar, Botan. Gaz. 31, 1901.)

Spores of

Aspergillus niger

Penicillium glaucum

Monilia fructigena

Mucor spinosus

Phycomyces nitens

Coprinus jimetarius

C. comatus

C. micaceus

Uromyces caryophyllinus

Water

0
0
75
0
0
0
0
0
100

Bean
decoction

100

5-10

100

Xutrient-salt
solution

100

Sucrose
solution

75
1

100
1

2-10
0
0
0

100

and Leach, 1950). Untreated spores in distilled water germinated only
after 5 to 8 days, and the total germination was less than 1 per cent.
Spores soaked in a concentrated horse-dung infusion for 15 days or more,
then placed in distilled water, germinated within 5 hr., with a total
germination of 50 per cent or higher. The exposure to the dung infusion
is believed to increase the permeability of the spore wall, allowing the
more rapid absorption of water. It might also be pointed out that the
dung infusion evidently contains substances which prevent spore germina-
tion until highly diluted or removed entirely.

Although the spores of the Myxomycetes germinate in distilled water,
the percentage may be greater in weak decoctions of the natural sub-
strate, such as rooting wood, bark, leaves, or humus (Smart, 1937).
Similarly, the conidia of PhyUosticia solitaria germinate more profusely
in apple-bark decoction and potato-dextrose broth than in distilled water
(Burgert, 1934). While it is possible that increased spore germination
is due primarily to some stimulating substance, it seems likely that certain
nutrients are also involved.

The conidia of Glomerella cingulata apparently have special nutritional
requirements for germination. There was little or no germination in

SPORE GERMINATION

363

distilled water and in dextrose solution lacking minerals (Lin, 1945).
From his experiments involving various inorganic compounds, Lin
concluded that carbon, magnesium, nitrogen, and phosphorus, are
required (Table 60). The need for sulfur was not so evident as that for
the other elements, and sulfur was not essential. The minimum require-
ments of nitrogen and phosphorus were calculated to be of the order of
10"' Mg per spore. No evidence was found that an external supply
of any organic substance, other than sugar, is necessary for spore
germination.

Table 60. The Essentiality of Various Ions for the Germination of the

CoNiDiA OF GlomereUa cingulata
(Lin, Am. Jour. Botany 32, 1945.)

Chemical substance applied*

Element
lacking

Germination,

None (redistilled water)

dnrosR

Carbon and minerals

Minerals

None

Nitrogen

Potassium

Phosphorus

Sulfur

Magnesium

Carbon

0.0
0.0

Glucose, KNO3, KH2PO4, MgS04

Glucose, NH4CI, KH2PO4, MgS04

Glucose, KCl, KH.,P04, MgS04

Glucose, NaNOs, NaH,P04, .MgS04

Glucose, KNO3, KCl, MgS04

Glucose, KNO3, KH2PO4, MgCh

Glucose, KNO3, KH.POj, ^aSO,

KXO,, KH2PO4, MgS04

80.4
92.8

3.9
84.1

1.5
79.3

0.9

0.7

* In all cases, the concentration of glucose is 0.01 per cent, that of each of the mineral salts 1.0 milli-
mole.

The constituents of the medium may modify the effects of pH on spore
germination. This is illustrated in Fig. 75 by the germination of Lenzites
saepiaria on 2 per cent bacto-peptone, in sucrose-nitrate (Czapek's)
solution, and in beet decoction (Webb, 1921).

Emerson (1948) showed that D-xylose as a carbon source gave a high
percentage of germination of ascospores of Neurospora crassa without
heat treatment. Xylose was more effective when autoclaved than when
filtered. This was believed to be due to the slight conversion to furfural,
which was also shown to be active in increasing spore germination.

From this brief discussion it is evident that little is known about the
effects of nutrition upon spore germination. This is no doubt due, in
part, to the lack of planned experimental work along this line. Many
of the favorable effects of natural products may in fact be due to the
presence of stimulants rather than to the nutrients. At the present time
we have no conclusive evidence that spores require an external source of
vitamins for germination. In the light of the recent discovery of Ryan

364

PHYSIOLOGY OF THE FUNGI

■^V

■s \

V^ ■

'; /
• /

\
^

(1948) that the amino acids leucine, lysine, and proline favored spore
germination in mutants of Neurospora deficient for those amino acids, it
also seems likely that spore germination in certain vitamin-deficient fungi
may be aided by the addition of the vitamins in question. A careful
study of the effects of vitamins is needed.

The spores of some fungi, such as Botrytis cinerca, germinate much
better Avhen in contact with plant tissue than in distilled water (Brown,
1922). It was concluded that certain substances diffuse out of the host
plant into the infection drop containing the spores and stimulate germina-
tion and infection. Leach (1923) believes that a similar situation may

100

c
,o

■| 60

a>
o>

§ 40
I
20

» 23456789

pH of medium

Fig. 75. The effect of the pH and kind of medium on the percentage of germination
of spores of Lenzitcs sacpiaria at 20 to 23°C. A, in sugar-beet decoction; B, in 2 per
cent bacto-peptone sohition; C, in Czapek's full nutrient solution. (Redrawn from
Webb, Ann. Missouri Botan. Garden 8: 325-327, 1921.)

exist with Colletotrichum Undemuthianum. The spores of this fungus
germinated poorly in distilled water alone, but distilled water plus a
piece of fresh bean tissue gave a high percentage of germination. Fresh
bean juice was equally effective, but boiled bean decoction did not stimu-
late germination. However, green-bean agar made from a similar decoc-
tion gave excellent germination, as did potato-dextrose agar. These
results led Leach to conclude that two distinct stimulating factors may
be involved. A portion of Leach's data is summarized in Table 61.

Some know^n stimulants may eliminate the need for certain factors
ordinarily supplied by natural media for the germination of spores of
Phycomijces (Robbins et al., 1942). Germination of spores was about
12 per cent or less on mineral-dextrose agar with thiamine. The addition
of an extract of potatoes, or of other natural products, of hypoxanthine,
acetate, or some other organic acids increased germination to nearly 100
per cent. Treatment of spores with aqueous pyridine had the same
favorable effect. These authors believe that certain factors (called Z
factors) are essential in spore germination. One of these (factor Zi)

SPORE GERMINATION 365

has been identified as hypoxanthine, while the identity of factor Z2 i»
still unknown. An explanation of the effects of these stimuli is given by
these authors:

The dormant spores are considered to lack sufficient available Z factors for
germination. The extracts of natural products or the Z factors furnished in the
medium supply this deficiency, which may also be met by treatment with heat,
cold, acetate or pyridine. These treatments are thought to change the Z factors
in the spores from an unavailable to an available form.

The effects of certain gases and volatile compounds upon germination
have also been demonstrated. It has been observed that spores of num-
erous fungi germinate better in a container in which some living plant
part is also present. This was demonstrated for Basisporium gallarum
by Durrell (1925), who also found that the introduction of carbon dioxide

T.^ELE 61. The Effect of Various Media and Plant Tissues on Spore Germina-
tion OF Colletotrichuni lindemuthianum
(Leach, Minn. Agr. Expt. Sta. Bull. 14, 1923.)

Medium Germination, %

Distilled water 3-6

Sucrose-nitrate (Czapek's) solution 5-11

Sucrose-nitrate (Czapek's) solution plus bean decoction 10

Bean decoction 8

Distilled water plus fresh bean tissue 83-95

Distilled water plus sunflower tissue 5

Distilled water plus wheat tissue 12

Distilled water plus corn tissue 10

Distilled water plus tomato tissue 2

Sucrose-nitrate (Czapek's) solution plus bean tissue 95

Green-bean agar 97

Potato-glucose agar 98

into the container enclosing the spores gave the same increase in germina-
tion. The same effect was demonstrated for Ustilago zeae (Platz et at.,
1927). An atmosphere containing 15 per cent carbon dioxide was found
to be optimum for spore germination. Such a condition gave a pH of
the medivmi from 4.9 to 5.6. These authors conclude that the stimulating
effect is apparently due to "a definite action of carbonic acid." Is it
possible that this is an example of heterotrophic utilization of carbon
dioxide?

While the release of carbon dioxide into the atmosphere by various
living plant parts may explain the stimulation of spore germination in
many cases, the presence of carbon dioxide alone will not explain certain
results obtained by some workers. For instance, spore germination of
Botrijtis cinerea was stimulated by the presence of living tissues of apples
or leaves of Ruta or Eucalyptus in the same container, while tissues of
potato tuber and onion scales inhibited germination (Brown, 1922).

366 PHYSIOLOGY OF THE FUNGI

Distillates of these leaves increased germination four to ten times. Ethyl
acetate likewise gave similar results. The possibility of specific activity
was suggested by the fact that apple tissues distinctly stimulated germina-
tion of B. cinerea spores, while they inhibited germination of spores of
Colletotrichum lindemuthianum. The stimulation was greater with old
spores.

Presoaking and the subsequent addition of a stimulating volatile agent
gave optimum germination of Urocystis tritici spores (Noble, 1923).
The expressed sap of wheat placed in the same container with germinating
spores, but in separate dishes, proved to be a good stimulating agent.
Uninjured seedlings of certain nonsusceptible hosts likewise stimulated
spore germination. Benzaldehyde, salicylaldehyde, butyric acid, and
acetone in certain concentrations stimulated germination of presoaked
spores. Noble believed that presoaking increased permeability of the
spore and allowed the more rapid intake of the stimulatory volatile sub-
stance, which increased the permeability of the protoplasmic membrane
by changing its physical condition.

Likewise, a solution of benzaldehyde (3/2,000,000) stimulated germina-
tion of Urocystis occulta spores, which germinated very poorly in water
(Ling, 1940). Ethyl alcohol stimulated spore germination in Aspergillus
flavus; methyl alcohol was slower and less effective (Duggar, 1901). A.
niger was stimulated by oxalic acid, whereas .4. fiavus was not. It is
understood that the stimulatory power of these chemicals depends upon
the concentration.

An interesting situation exists in the germination response of some
spores to the presence of other fungi, or even to the medium in which
other fungi have grown. The few experiments conducted along this line
suggest that the constant association with other organisms may be highly
beneficial to spore germination as well as subsequent growth of some fungi
in nature.

The germination of a number of species of Myxomycetes was increased
by the addition of the filtrate of a medium in which spores had previously
been germinated (Smart, 1937). Smart calls the stimulatory factor an
"autocatalytic agent." A portion of Smart's data is presented in Table
62.

Fries (1941, 1943) obtained almost phenomenal results with spores of a
number of Hymenomycetes, which previously had germinated poorly
or not at all, by sowing the spores on malt agar with living cultures of
Torulopsis sanguinea. Spores of ten species of Tricholoma, which ger-
minated only with difficulty without the yeast, were found to germinate
readily in its presence. One species of Tricholoma gave only negative
results. In Amanita mappa, A. porphyria, and A. rubescens germination
occurred only when Torulopsis was present. Germination of two other

SPORE GERMINATION

367

species of Amanita was considerably improved by the presence of the
yeast. None of the seven species of Boletus germinated on malt agar
without the yeast. On the same medium and in the presence of Torulopsis
sanguinca, germination was obtained with spores of B. bovinus, B. elegans,
B. flavidus, B. granulatus, B. luteus, B. variegatus, and B. viscidus. Some
germination of Boletus spores was also obtained in the presence of living
colonies of certain other fungi, but none was so effective as Torulopsis.
Spores of certain other fungi {Hijdnum repandum, H. imhricatum, Craterel-
lus lutescens, Lycoperdon umhrinum, L. echinatum., L. nigrescens, L. pra-
tense, L. pyriforme, and Scleroderma aurantium) germinated in Fries's

Table 62. Germination of Single Myxomycete Spores
(Smart, Aryi. Jour. Botany 24, 1937.)

Number of spores germinating

Species

Lot 1 (10 spores)
(fresh medium)

Lot 2 (10 spores)

(previous germination

medium)

Vulino sevtica,

9 after 3 hr.
6 after 3 days

3 after 2 days

4 after 1 day
0 in 2 weeks
0 in 2 weeks
3 after 2 days
6 after 6 days
2 after 18 days
9 after 7 days

10 in 45 min.

Physarum polycephahini

Stemonitis fusca

S ciTifero,

8 in 15 hr.

9 in 1 day
8 in 8 hr.

Enteridium rozeanum

Reticularia lycoperdon

Lycogala epidendrum

Arcyria denudata

Dictydium cancellaium

Physarum cinereum

10 in 30 min.
8 in 15 min.

8 in 3 hr.

G in 6 days
2 after 18 days

9 in 6 days

experiments only in the presence of T. sanguinea. He also tested the
effects of mycelial extracts on spore germination and found that extracts
of certain species of Boletus stimulated germination of spores of the same
species. Many of the fungi studied by Fries are believed to be mycorhizal
and may require the presence of a special set of conditions, perhaps the
roots of certain plants (or conditions which simulate their presence),
before germination will occur.

The time required for a spore to germinate after being subjected to
favorable conditions is a reflection of the interaction and relative impor-
tance of all the various influencing factors. The nearer all these factors
are to the optimum, the shorter will be the time required for germination.
Time is an important factor for the subsequent infection of the host. In
nature the near-optimum environmental conditions, principally tempera-
ture and moisture, may persist for bvit a short time, and a change in but
one of these factors may inhibit spore germination.

3G8 PHYSIOLOGY OF THE FUNGI

LONGEVITY OF SPORES

The length of Hfe of spores is usually measured by their ability to
germinate after various periods of time. It is affected by environmental
conditions, principally temperature and moisture. The greatest period
of longevity reported for fungus spores appears to be among the Myx-
omycetes. Smith (1929) succeeded in germinating spores from herbarium
specimens of Myxomycetes 5 to 32 years after they were collected. A
few of the common species whose spores germinated after approximately
30 years are Physarum cinereum, Fuligo septica, Hemiirichia clavata, and
Stemonitis ferruginea. Smut spores also have a long period of viability
(Lowther, 1950). Spores of Aspergillus orijzae germinated after 22 years
in a sealed tube at room temperature (McCrea, 1923).

In contrast to long periods of longevity, some fungus spores die very
soon after they are liberated. The sporidia of Cronartium rihicola lived
less than 10 min. at room temperature with a humidity of 90 per cent
(Spaulding, cited by Doran, 1922). Sporidia of Gymno sporangium
juniperi-virginianae lived no longer than 6 days in dry air. Eight weeks
is reported as the maximum longevity of aeciospores of C. rihicola, with
only 5 per cent germination after 7 weeks. In general, aeciospores of the
rust fungi remain viable about 50 per cent longer than the urediospores,
whose average longevity ranged from 30 to 60 days (Doran, 1922).

Other factors have been reported to influence longevity of spores.
Ascospores of Endothia parasitica remained viable for a year when dried
in the bark, but when removed from the bark, they lost the ability to
germinate within 5 months (Anderson and Rankin, 1914). Similarly,
conidia in dry spore horns retained viability for at least a year, but when
placed in water, separated, and then dried, the time was less than 1
month. It seems likely that one of the functions of the gelatinous matrix
of the conidia of certain fungi, such as Gloeosporium, Colletotrichum, and
Cytospora, is to increase the longevity of the spores through its water-
holding capacity.

Light is apparently only of minor importance as a factor influencing
longevity. No doubt ultraviolet light in nature plays an important part
in reducing the period of viability and even in killing many of the hyaline
spores. Spores having dark walls are protected somewhat against the
penetration of the ultraviolet rays.

SUMMARY

Spore germination represents a change from an inactive to an active
phase in the life cycle of a fungus. Since it involves the first stages of
growth, it is reasonable to expect that many of the factors which influence
vegetative growth also affect spore germination. On the other hand,
the spore, being a resting cell, may contain stored materials not usually

SPORE GERMINATION 369

present in appreciable quantities in vegetative cells. Since the metabolic
activity of a resting spore is at a minimum in contrast with that of actively-
growing vegetative cells, the internal responses to the environmental
factors may be quite different.

The variability of the needs of spores of different fungi for germination
is adequately illustrated in the literature. Certain general conditions
are essential for all spores, while some require a special set of conditions.
Water is essential to activate certain enzyme systems, to initiate other
internal chemical changes, and to increase the volume of the germinating
spore. WTien the temperature is near the optimum, the enzymatic
activity and the rate of spore germination are increased. The supply of
oxygen must be adequate to meet the demands of the greatly increased
rate of respiration. The acidity of the substrate must be favorable.
Variability in the period of viability of spores is striking, but longevity
is greatly influenced by the environment. Much information is yet to be
gained regarding the longevity of spores, particularly of the plant
pathogens.

Certain special conditions are required for germination of some spores.
These may act as a stimulant in breaking dormancy or may supply needed
nutrients. The effects of other living organisms, or even of the substrate
upon which they have grown, are of particular interest, for such associa-
tion is the usual condition under which germination occurs in nature
One might suppose that the secretions of certain plants would exert a
selective action on spore germination and affect the pathogenicity of
certain fungi, but evidence on this point is lacking.

The production of short germ tubes by spores of some species of Ery-
siphe in an absolutely dry atmosphere is unusual. If this is to be con-
sidered as true germination, it must represent a unique method among
fungi. The Erysiphales, however, are excellent examples of fungi whose
spores germinate in atmospheres of lower relative humidity than most
fungi can endure.

Under the changing conditions of nature, the period of time during
which a factor is active is of utmost importance. Germination is the
result of the action of all the influencing factors operating at the same
time. Most of these factors vary in intensity or concentration, so that
the combined optima of all factors are seldom, if ever, reached at any
given time in nature. As a result, an extremely low percentage of the
spores formed by a fungus ever germinate, while still fewer give rise to
extensive mycelium.

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370 PHYSIOLOGY OF THE FUNGI

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SPORE GERMINATION 371

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York, 1947.
Yarwood, C. E.: The tolerance of Erysiphe polygoni and certain other powdery

mildews to low humidity, Phytopathology 26 : 845-859, 1936.

CHAPTER 17
THE PHYSIOLOGY OF PARASITISM AND RESISTANCE

A discussion of parasitism occupies an important position in any
treatise on the physiology of fungi, particularly for those students who are
interested in plant diseases or the fungi which cause them. This phase
of study offers many challenging unsolved problems. Parasitism involves
primarily two living organisms, the parasite, whose actions are offensive,
and the host, whose reactions are defensive. If the defenses of the host
plant, either before or after penetration by the parasite, are successful,
the plant is resistant; if not, it is susceptible. To be successful, a parasite
must find the nutritional and environmental conditions favorable for its
development. If even a single important factor is unfavorable to the
parasite, the fungus may fail to establish a parasitic relationship with its
proposed host. Such factors may exert their influence either before or
after penetration by the fungus. Environmental factors acting before
penetration may in reality bring about an escape from a disease rather
than true resistance to it.

The present discussion is divided into three main parts: (1) penetra-
tion; (2) parasitism, the action of the parasite in becoming established and
obtaining its food ; (3) resistance of the host to penetration or against the
parasite after penetration. The comprehensive reviews of the physiology
of the host-parasite relationship given by Brown (1936, 19-48) should be
read by all students. Similar reference is made to Arthur et al. (1929),
who give an excellent discussion of the parasitic relations of the rusts,
and to the treatise of Gaumann (1946, 1950) on the principles of plant

infection,

PENETRATION

A parasite may gain entrance into the host (1) through the natural
openings, such as stomata or lenticels, (2) by direct penetration through
the uninjured epidermis, or (3) through wounds.

Through stomata. Viable spores may fall upon a host plant and pro-
duce germ tubes, which by chance grow over or near stomata. The
outer walls of the epidermal cells of aerial plant parts are covered with
cutin, which is somewhat resistant to penetration by some fungi. The
germ tube which enters through a stoma may then be favored by the moist
atmosphere in the substomatal cavity. In some cases, the unspecialized
hyphae may penetrate the host cells; in other fungi, haustoria, which

372

PARASITISM AND RESISTANCE 373

arise from the intercellular mycelium, penetrate the host cells and absorb
food. Water vapor has been suggested as the stimulus which causes the
germ tube to turn inward and enter a stoma. This, however, cannot be
the case with zoospores which are immersed in water and which have been
noted to cluster around stomata. The fungi which normally enter the
host plant through stomata include the cereal rusts (aeciospore and
urediospore stages), Cercospora heticola, Phytophthora infestans (zoospore
stage), the Peronosporales, Albugo Candida, and others.

The cereal rusts have received a great deal of attention in resistance
studies. It has been reported (Hart, 1929) that Puccinia graminis
apparentl}^ requires the open stomata of wheat plant for penetration.
On the other hand, Caldwell and Stone (1936) have shown that the germ
tubes of Puccinia triticina are able to force their way between the guard
cells of closed stomata of wheat leaves. A germ tube from a urediospore
may start to enter an open stoma, but as it forms an appressorium, the
stoma closes. Further penetration is accomplished between the guard
cells by a slender hypha. Allen (192G) believes that the appressorium
probably secretes some toxin which harms or even kills the guard cells,
causing the stoma to close. Caldwell and Stone, however, do not believe
that this injury to the guard cells is necessary for entry of germ tubes.
The appressorium seems to function as a special organ to apply the pres-
sure needed for the forced entry between the closed guard cells.

Penetration through lenticels more often occurs in the underground
parts of the host which are in a more or less moist situation. Potato
tubers may become infected by Actinomyces scabies and by germ tubes
from sporangia of Phytophthora infestans, chiefly through the lenticels.

Direct penetration. A large number of fungi are capable of penetrating
the unbroken epidermis of a plant, directly through the cutinized outer
walls. The spore may germinate on the surface of the plant in a drop of
water. The germ tube grows over the epidermis and by some stimulus
is caused to turn inward and penetrate the cell. Brown (1922) demon-
strated that there is a certain amount of exosmosis of materials from host
tissue into a drop of liquid on the surface. In some cases this may lead
to a chemotropic response by the fungus. However, in most cases the
stimulus of contact is believed to initiate appressorial formation and
penetration. The formation of appressoria is common among many fungi
when the germ tubes come in contact with the epidermal cells. The
fact that the appressoria are often formed on a glass slide is further
evidence that their formation is in response to contact with a solid sur-
face. Appressoria are usually bulb-like or disk-like in shape and are
believed to serve as an adhesive disk against which the slender infection
hypha may push in penetrating the cell wall. Brown (1915, 1922)
presents evidence that the host cells are not killed by Botrytis cinerea,

374 PHYSIOLOGY OF THE FUNGI

Sclcrotinia sclerotiorum, and Collctoirichum lindemuthianum until after the
fungus penetrates the cuticle of the host. In other words, there is little
or no diffusion of the toxic materials through the cuticle. Direct penetra-
tion through cutinized walls is believed to be entirely by mechanical
pressure, since no cutin-dissolving enzyme has been demonstrated in the
fungi.

The rhizomorphs of Armillaria mellea usually gain entrance directly
through the sound cork layer of comparatively old roots (Thomas, 1934).
Penetration is believed to be accomplished partly by mechanical pressure
and partly by chemical means. There is evidence that a suberin-dis-
solving enzyme aids in the destruction of some of the cork cells. Some
fungi may enter the same host by more than one method. Fusarium
lint may enter through young epidermal cells of the root, root hairs,
stomata of seedlings, and perhaps through wounds.

The penetration of noncutinized cell walls may be either by mechanical
pressure or by the dissolving action of enzymes secreted by the fungus.
Hawkins and Harvey (1919) concluded that the hyphae of Pythium
debaryanum penetrated the cell walls of susceptible potato tubers by
mechanical pressure, and that the resistant varieties in general showed
greater resistance to mechanical puncture. They found no evidence of
cellulases which might aid in penetration by dissolving the cellulose cell
wall. Using cane sugar as the plasmolyzing solution, they found that
the hyphae of P. debaryanum were capable of exerting as high as 54 atm.
osmotic pressure. These hyphae would have a strong tendency to absorb
water, and as a result greater internal pressure would be exerted against
the hyphal wall. Apparently the hyphal wall is capable of withstanding
this pressure at all points except its tip, where growth occurs. The
pressure exerted by the growing tip is believed to be sufficient to cause
penetration of the host cell wall. By direct microscopic examination
Hawkins and Harvey observed that, just after the hyphal tip came in
contact with the host cell wall, it formed a swelling, back of which a bend
developed. This was followed by penetration of the wall by a small tube.

Penetration through noncutinized cell walls by chemical means has
been described for Spongospora suhterranea by Kunkel (1915). It seems
likely that other nonfilamentous fungi penetrate cell walls in the same
way. Likewise, wood rot fungi penetrate the cellulose and lignified cell
walls by enzymatic action, as evidenced by the boreholes in decaying
wood. It may be significant that the hyphal walls of Pythium, as well
as of other Oomycetes, contain cellulose, while the hyphal walls of other
fungi are composed principally of chitin, which would not be acted upon
by cellulases.

It must be emphasized that penetration of the host in itself does not
necessarily lead to the establishment of the fungus in the host and the

PARASITISM AND RESISTANCE 375

production of a disease. In some cases it is known that a fungus may
enter resistant or immune plants, as well as susceptible ones, but find the
conditions unfavorable for its establishment and further development.
Through wounds. A number of fungi apparently are unable to pene-
trate a healthy plant except through wounds. These may be insect
wounds, broken branches of trees, broken roots, etc. In addition, some
fungi which are capable of entering the host by other means may also
penetrate through wounds. Phymatotrichum omnivorum, the cause of
numerous root rots, commonly enters roots through wounds, although
these are not necessary. Fusarium, causing dry rot of potato, apparently
enters the tubers only after they have been wounded. Likewise most
of the wood-rotting Basidiomycetes enter the host only through wounds,
principally at broken or dead branches and at pruning or lightning and
fire scars. Here, the air-borne basidiospores must find suitable moisture
for germination and for penetration of the wood. Endothia parasitica
is said to enter the chestnut tree only through wounds that extend through
the corky layer. Ceratostomella ulmi is transmitted by the European
bark beetle, which introduces the spores into its feeding wounds. Bruises
and wounds of fruits and vegetables are common ports of entry for numer-
ous rot-producing fungi, such as Rhizopus nigricans on sweet potato.
Monilinia fructicola on stone fruits, Penicillium expansum on apple, and
P. italic^im. and P. digitatum on citrus fruits.

PARASITISM

A discussion of the action of the parasite after it enters the host is so
closely correlated with the defense of the host that it is difficult to discuss
each topic separately. For the sake of convenience, however, it seems
desirable to discuss some of the outstanding effects of fungi upon their
hosts and the methods by which the parasites obtain their food under a
separate heading of parasitism.

Parasitism in plants. Parasitism may begin as soon as a fungus hypha
enters the host. The primary consideration is the securing of suitable
nutrients and water by the fungus. This may be accomplished by two
general methods, (1) by killing the cells of the host and obtaining food
from the dead cells, or (2) by establishing a close nutritional relationship
with the living host cells and absorbing the soluble nutrients without
causing necrosis. The fungi falling in the first group are the destructive
parasites, while those belonging to the second group have been called the
balanced parasites (Bessey, 1935). The latter group includes those fungi
known at present as obligate parasites (such as the Uredinales, Erysiphales,
and Peronosporaceae), and some other fungi (such as the Ustilaginalesand
Taphrina) which in their hosts obtain food only from living cells.

The destructive parasites, as a whole, are strong producers of enzymet;

376 PHYSIOLOGY OF THE FUNGI

and toxins but may be weak in mechanical action. Some of these cause
rapid rots of fruits or vegetables but are unable to penetrate the unbroken
epidermis and must depend on wounds for their entrance. Others, which
are seldom, if ever, found as pathogens in nature, may cause rot when
artificially inoculated into succulent plant tissues.

Rotting of the tissue is due to two distinct effects of the fungus on the
host: (1) death of the cells, and (2) dissolution of the middle lamellae.
The separation of the cells is due to the action of the enzymes proto-
pectinase, pectinase, and pectase on the middle lamella. These three
enzymes are often collectively referred to as pectinase. There is some
evidence that pectinase may also cause a change in permeability of the
cell membranes and the death of the cells, but it is possible that some
other toxic substance may be closely associated with pectinase. How-
ever, no such substance has been isolated. Extracts of rotted tissues
have been shown to cause the same effects as the fungi themselves. These
effects are described by De Bary (1886) for Sclerotinia sclerotiorum and
by Brown (1915) for Botrytis cinerea. Higgins (1927) believes that
oxalic acid produced by Sclerotium rolfsii is the principal agent of destruc-
tion. The death of the host cells well in advance of the invading hyphae
indicates rapid diffusion of the toxic substance in the case of fungi produc-
ing soft rot. Brown (1948) believes that the enzyme pectinase acts as a
cytolytic toxin. For a discussion of the identity of enzymes and toxins
of species of Clostridium, see Smith (1949).

Thatcher (1942) has shown that B. cinerea and S. sclerotiorum cause
a fourfold increase in the permeability to water of the host cells just
beyond the discolored necrotic zone. Some substance other than pecti-
nase may bring about this change in permeability and be a contributing
factor to the "action in advance" of many fungi. PhytophtJwra infestans
caused a change in permeability in host cells beyond the extent of the
hyphae which penetrated the living tissue. The identity of the substance
causing a change in permeability is unknown, but it is likely a weak toxin
or an enzyme which alters the structure or activity of the plasma mem-
brane. The increase in permeability may concern water alone or both
nutrients and water.

An osmotic pressure higher in the fungus cells than in the surrounding
host cells is apparently characteristic of the host-parasite relationship
(Table 63) . This is necessary before the parasite can absorb water from
the host cells.

The production of pectinase and its activity under different conditions
were studied by Vasudeva (1930) and Chona (1932), who showed that
the amount produced by Botrytis allii varied with the medium in which
the fungus was grown. B. allii did not secrete a demonstrable amount of
pectinase when grown on apple extract, but when asparagine, potassium

PARASITISM AND RESISTANCE

377

nitrate, or ammonium sulfate was added to the apple extract, there was a
decided increase in the amount produced. This is not surprising, for
there are numerous reports that the available nutrients and the pH of the
culture medium affect both the kind and amount of metabolic products of
a fungus.

The activity of the enzymes produced by a pathogen varies with the
conditions of the host cells. It seems probable that the inhibition of the
fungus enzymes by the host cells is an important factor in resistance.
Klotz (1927) proposed this hypothesis to explain the greater resistance
of sour orange and the greater susceptibility of lemon to Pythiacystis
citrophthora and Phomopsis calif or nica, the causes of certain bark diseases.

Table 63. The Osmotic Pressures of Host and Parasite
(Thatcher, Can. Jour. Research 20, 1942.)

Fungus

Ave.
osmotic
pressure,

atm.

Host

Ave.
osmotic
pressure,

atm.

Uromyces fabae, germ tubes . . .
haustoria

44.25

21.9

18.6

29.8

23.5

18.9
18.0

17.4
15.5
18.1
41.3

Pisum sativum, leaf

petiole

Dianthus, leaf base

9.15
10.1

U. caryophyUinus, haustoria. . .

Botrytis cinerea, hyphae

Sclerotinia sderoliorum, hyphae.
Puccinia graminis, haustoria
(race 21)

11.2

Apium graveolens, petiole

A. graveolens, petiole

Mindum wheat, leaf

Brassica, leaf

Solanum tuberosum, tuber

netiole

8.3

13.4

9.4

Erysiphe polygoni, hyphae

Phytophihora infestans, hj^phae
(aerial)

10.6
10.6

hyphae (intercellular)

sporangia

8.9

Brassica, root

Phoma lingam, hyphae

11.3

The greater pathogenic action of a destructive fungus occurs in the host
whose cells are favorable for the activity of the enzymes of the fungus.
Further evidence of enzyme inhibition of certain plant tissues was
presented by Chona (1932), who studied the rotting action of B. cinerea,
the cause of a soft rot of various plant tissues, and Pythium sp., a rot
producer of potato tubers. Vigorous germination of spores of Botrytis
and even some sporulation took place in artificial wounds in potato
tubers, but no decomposition followed. The pectinase produced by B.
cinerea was active against apple tissue, but the presence of potato tissue
inhibited its activity. It was then found that the mineral salts, particu-
larly KH2PO4 and MgS04, in the potato were the inhibiting factors. On
the other hand, Pythium spores germinated well on apple tissue but failed

378 PHYSIOLOGY OF THE FUNGI

to rot it. The inhibition in this case was traced to the mahc acid in
the apple. The pectinase produced by Pythium was most active in an
alkahne medium, near pH 8.0, while that of B. cinerea was more active
in an acid medium, at pH 5.0 to 5.5.

In contrast with the destructive fungi which rot the host tissue are
those which cause wilting and certain types of necrosis without disintegra-
tion of the host cells. These fungi produce little or no pectinase. Some
common fungi which cause wilting of mature plants are species of
Fusarium, Verticillium, Cephalosporium, and Ceratostomella. It is now
generally believed that in most cases wilting caused by fungi is due to
toxins or to the plugging of the vessels by polysaccharides or other similar
metabolic products of the fungus, rather than to plugging by the excessive
mycelial growth in the vessels. Extracts of the mycelium or the culture
filtrate of a number of these fungi cause effects that are the same as or
similar to those caused by the fungi themselves in their respective hosts.

A definite correlation between the pathogenecity of two strains of
Fusarium lycopersici and the toxicity of their metabolic products was
demonstrated by Haymaker (1928). There was similarity of symptoms
and of the effect of temperature on wilting. The culture filtrate was more
toxic when the fungus was grown at 28°C. than that obtained at any other
temperature. The toxic substance was not identified. Other workers
(Plattner and Clausson-Kaas, 1945; Woolley, 1946) have reported that
the wilt-inducing compound produced by F. lycopersici is lycomarasmin,
a peptide of aspartic acid. Gaumann and Jaag (1947) reported that
clavacin exerted a wilting effect on detached tomato shoots similar to
that of lycomarasmin. But, whereas lycomarasmin acted chiefly on the
cells of the leaf blade, clavacin is toxic mainly to the phloem and paren-
chyma of wood and cortex of the stem and petiole. The action of both
compounds is believed to be similar, destroying the semipermeability of
the plasma membranes, thereby decreasing the water-holding capacity of
the cells and inducing wilting.

Various polysaccharides have been shown to produce wilting in tomato
cuttings (Hodgson et al., 1949). Since there was a direct relationship
between molecular weight and wilt-inducing action of these compounds,
it was concluded that their action was mainly by mechanically interfering
with the transportation of water. Dimond (1947) also reported wilting
of elm leaves due in part to a polysaccharide produced by Ceratostomella
ulmi in culture. Its action is believed to be similar in naturally infected
elm trees.

More recently, Feldman et al. (1950) have presented evidence to show
that the primary wilt-inducing agent produced by C. ulmi is not the
polysaccharide, but a toxin. The production of toxin in liquid culture
filtrate was greatly influenced by the pH of the medium, being greater in

PARASITISM AND RESISTANCE

379

buffered media at pH 4.25 than at 5.25, although growth was more rapid
in the less acid medium (Fig. 76). The toxin was shown to be irreversibly-
inactivated at pH G or above. The introduction of calcium hydroxide
into trees and the application of basic chemicals to the soil have been
somewhat successful in retarding the disease. Presumably, these chem-
icals act by raising the pH of the sap of the tree.

Days
Fig. 76. Growth of Ceratostomella ulmi and production of toxin, as measured by wilt
of tomato seedlings induced by culture filtrate, in buffered media at different pH
levels. Note that toxin production is favored by the more acid medium, while
growth is greater in the less acid medium. (Courtesy of Feldman, Caroselli, and
Howard, Phytopathology. 40: 348, 1950.)

The varieties of oats susceptible to toxic culture filtrates of Helmin-
thosporium victoriae were the same that were susceptible to the fungus in
nature (Meehan and Murphy, 1947). Plants of Boone variety were
killed, but Clinton plants were unaffected when grown in the same con-
centrations of the filtrate. The toxic substance, which was not identified,
was produced when the media contained either organic or inorganic
nitrogen. This species differs from H. sacchari, which was reported by
Lee (1929) to reduce nitrates to nitrites, which were toxic to sugar cane.

380 PHYSIOLOGY OF THE FUNGI

The toxicity of the metabohc products of Fusarium vasinfectum was
found to be dependent upon the medium on which the fungus was cultured
(Rosen, 192G). Filtrates of cultures grown in a medium containing
potassium nitrate and sucrose were highly toxic to cotton plants, while
filtrates from cultures grown in a medium containing ammonium lactate,
sodium asparaginate, and glycerin were not toxic. The filtrate of the
nitrate-sucrose medium contained nitrites. Solutions of chemically pure
sodium nitrite were also decidedly toxic to cotton plants. We may
assume that the action of this fungus in converting nitrates to nitrites
is the same within the host plant as it is in the culture vessel.

Thus, there seems to be abundant evidence that the metabolic products,
including enzymes and toxins, of a given fungus vary both in kind and in
amount with the pH and composition of the culture medium. On the
other hand, the evidence that the same situation exists in nature is
extremely scarce. One may speculate, however, that the types of nutri-
ents furnished by the host cells and the pH of the cell sap may also influ-
ence the metabolic products of the fungus in the host plant. If this is
true, a given fungus may find the nutrients and environment supplied by
one host particularly favorable for the production of a disease-inducing
toxin or enzyme. If the host is unable to inhibit the action of these
substances, disease may result. The natures of both the pathogen and
the host determine the severity of the disease. This hypothesis may
help to explain, in part, the variation in intensity of parasitism of a
fungus on its different hosts. While there is little evidence to support
this idea at present, it is hoped that experimental work will be conducted
to test its merits.

The possibility that the presence of vitamins may affect pathogenicity
has been suggested (Pehrson, 1948; Prasad, 1949). There is no evidence
that deficiencies for vitamins are correlated with either parasitism or
pathogenicity, and vitamin deficiency may be excluded as a factor leading
to the parasitic habit. Likewise, there seems to be little or no correlation
between the nitrogen requirements of fungi and the parasitic habit.
Nonliving organic materials in nature are sources of vitamins and organic
nitrogen just as are the living plants. For example, Ustilago striiformis,
a highly parasitic fungus, is self-sufficient with respect to vitamins, and
some isolates are capable of utilizing nitrate nitrogen, while Phycomyces
hlakesleeanus, an obligate saprophyte, is deficient for thiamine and is
unable to utilize nitrate nitrogen.

Opposed to the destructive parasites discussed above are the balanced
parasites, which, in general, have a strong power to penetrate mechan-
ically but whose chemical actions on the host are relatively weak. Most
of the filamentous balanced parasites produce intercellular mycelium,
sending haustoria into the host cells. These serve as food-absorbing

PARASITISM AND RESISTANCE 381

structures, but the exact mechanism of the transfer of food is not so well
understood. The haustorium of the filamentous parasite is very similar
in its behavior to the intracellular nonfilamentous parasite, being sur-
rounded by the protoplasm of the host cell. Haustoria may be of several
forms, simple and nearly spherical, coiled, and branched in various ways.
Most cytologists agree that there is a cellulose wall, or sheath, around the
older haustoria. It is presumably formed by the host cell and suggests
a weak mechanism of defense against the invading parasite, yet it does
not prevent the diffusion of soluble food into the haustorium.

The haustorium commonly comes into contact with the nucleus of the
host cell. In 23 of the 35 cases (host-parasite combinations) reported
(Rice, 1927, 1935), habitual contact was observed between haustorium
and nucleus. Two theories as to the meaning of this contact have been
suggested. One is that the haustorium seeks out the region of the cell
nucleus in order to facilitate the absorption of food from the cell. The
second theory is that the action of the cell nucleus is defensive and that
in some cases it may cause the death and degeneration of the haustorium.

In the case of Synchytrium (Chrysophylyctis) endohioticum the swarm
cells migrate into close proximity w^th the nucleus of the host cell (Orton
and Kern, 1919). In the majority of cases the nucleus is engulfed at the
time or soon after the swarm cells unite to form the vegetative body of the
parasite. The host nucleus disappears as the sporangia develop. The
exact significance of this close relationship between parasite and host
nucleus is not clear, but it apparently represents a more or less unique
method of parasitism among the fungi.

It is generally believed that the balanced parasite causes harm to a
susceptible host primarily through its demand upon food and water.
There is little or no evidence that the protoplast is attacked chemically,
although host cells may be killed by growth pressure. There are numer-
ous reports of the disappearance of food in the region of haustoria. Butler
(1918) reported that starch is absent in cells containing haustoria of
Sclerospora graminicola, and at the time of sporulation the host cells
collapse and die. The only abnormal effect observed by Mains (1917)
on the cells of corn parasitized by Puccinia sorghi was the absence of
starch in the bundle sheaths near the rust pustules. He interpreted this
to mean that the parasite uses the food materials before they reach the
bundle sheath where they are normally stored. Similar disappearance
of starch in the host cells near infection by Synchytrium endohioticum has
been reported by Orton and Kern (1919).

On the other hand, starch may accumulate in the infected tissues dur-
ing early stages of development of rusts but usually disappears in later
stages of development. This may be due to some disruption of the host's
physiology. The physiological reactions of the host are known to involve

382 PHYSIOLOGY OF THE FUNGI

translocation of food, transpiration, respiration, and photosynthesis.
Increased respiration has been reported for some hosts, while a decrease
has been found for others. The rate of transpiration is usually increased.
An early infection of orange rust on Ruhus may even cause the formation
of stomata on the upper epidermis, where they are normally lacking
(Dodge, 1923).

The reactions of the chloroplasts of the host cells are believed to indicate
the degree of adjustment between the host and parasite (Rice, 1935).
Local chlorosis and streaking are common symptoms of a number of
diseases caused by haustoria-forming parasites.

Thatcher (1939, 1942) has shown that certain obligate parasites cause
an increase in permeability of the cell membranes of susceptible hosts.
There was a decided reduction in osmotic pressure of the tissues of Pisum
surrounding the rust hyphae. If the fungus is unable to bring about an
increase in permeability so that it can obtain its necessary nutrients, the
host is resistant. Thatcher found evidence that the plasma membranes
of some resistant varieties of wheat may actually become less permeable
as a reaction to the rust hyphae, and starvation of the fungus may result.

The change in permeability incited by the balanced parasites seems
to be similar to the action of the destructive parasites, except for the
matter of degree. Thatcher (1939) believes that parasitism in the rusts
has become highly specialized, and the intensity of the effect on permea-
bility of the cell membranes has been reduced. The substance involved
is apparently a metabolic product of the fungus.

If the conditions afforded by a certain variety of host are favorable
for the production of a comparatively large amount of toxin (assuming
that this substance is a toxin) , the host cells may be killed and the further
development of the obligate parasite would be prevented. The sudden
death of the host cells is the condition described by Stakman (1914) as
hyper sensitiveness. Hypersensitive hosts are highly resistant or immune
to the pathogenic action of the obligate parasites. Stakman reported
that, in varieties of wheat resistant to Pucciiiia graminis tritici, when
the hyphae of the fungus come in contact with the host cells, the latter
often show plasmolysis, disintegration, and finally death. After the
death of a few surrounding cells the tips of the hyphae die. However, it
was discovered that in some cases the hyphae may die before the host cells
are killed. Stakman concluded that the problem of resistance to rvists
is one of toxins of the parasite or the host, or both, and can best
be explained by what he terms the "toxin or enzyme theory." Brooks
(1948) also concluded that the death of the parasite is due to the lethal
action of the host rather than to starvation.

Opponents of the toxin (or enzyme) theory of parasitism in the rusts

PARASITISM AND RESISTANCE 383

point out that no toxin has ever been demonstrated experimentally.
Leach (1919) believes that each physiologic race of Puccinia graminis
has its own characteristic food requirements which are met by only a few
varieties of the host. According to this hypothesis, if a race of rust
enters a host which does not meet its specific nutritional requirements, it
dies, and enzymes which are injurious to the host cells are released. This
hypothesis is supported by Wellensiek (1927) who worked with Puccinia
sorghi.

While it is evident that the food supply varies with the varieties of the
host, it seems equally possible that the difference in nutrients may have a
more indirect effect in determining whether the fungus survives. Is it
merely that the fungus starves if the host does not provide the appropriate
food, or are the conditions in the host unfavorable for the production of
certain metabolic products which are essential to the pathogenic actions
of the fungus?

The type of host-parasite relationship found in Phyllachora graminis
seems to be unique (Orton, 1924) . This fungus apparently has the power
of digesting and absorbing the tissues within the leaf, producing cavities
in which the ascocarps later form. The hyphae bore their way through
the cell walls of any of these tissues and, in doing so, absorb a portion of
the wall. The parenchyma cells become disorganized, and their contents
disintegrate. The vascular cells may be invaded and partially absorbed
and become filled with hyphae. The most striking physiological charac-
teristic of this fungus is its ability to absorb, replace, and engulf the
tissues of the host leaf without any external evidence of necrosis of the
host. This would seem to indicate the presence of highly active cellulo-
iytic enzymes (and perhaps others) confined to the area near the fungus,
without the presence of toxic substances, which would cause necrosis of
the leaf tissue.

Actually, comparatively little is known about the activities which
lead to parasitism, particularly of the balanced parasites. It is hoped
that more planned experiments will be conducted in an attempt to gain
more knowledge regarding the mode of parasitism of plant pathogens.
Only by understanding the action of the parasite can we understand the
basic facts underlying resistance and susceptibility.

Parasitism and symbiosis with insects. There are numerous reports
of the parasitic and symbiotic relations of fungi with insects. For a
more complete discussion than this text offers, see Leach (1940) and
Steinhaus (1946). In many cases the relationship is solely to the advan-
tage of the fungus (true parasitism), but a number of cases of mutualistic
symbiosis do exist. The fungi may be disseminated by the insects which
serve as their hosts. One can only speculate regarding the basic nutri-

384 PHYSIOLOGY OF THE FUNGI

tional requirements of these fungi, since very little is known. We may
assume that rather specific nutritional needs, either for growth or for
reproduction, are satisfied by the relation with insects.

Among the fungi parasitic on insects the genus Entomophthora is the
most common. Various common species attack houseflies, grasshoppers,
and other insects. A direct correlation between the amount of precipita-
tion and the number of infections on houseflies was reported by Yeager
(1939). Massospora cicadina infects the seventeen-year cicada and
produces spores inside the abdomen. The posterior portion of the abdo-
men sloughs away, exposing the spores w^hile the insect is still able to crawl
about. This is apparently the chief method of dissemination of the
spores. The mode and time of infection are unknown. Species of
Cordyceps are common on pupae and larvae of certain insects. The fact
that C. militaris produces abundant mycelial growth on a variety of
synthetic media in the laboratory suggests the possibility that in nature
this fungus may grow on other substrata, requiring the insect association
only to fruit.

Fawcett (1910) described the use of a fungus, which he named Aegerita
webberi, in controlling whitefly in the orange groves of Florida. Ascher-
sonia aleyrodis has also been used for the same purpose. A chytrid,
Myrophagus ucrainicus, is reported (Karling, 1948) as a parasite on scale
insects in Bermuda, Louisiana, and Ontario. In severe outbreaks as
many as 45 per cent of the female insects may be killed. It has also been
transmitted to mealy bugs. Another group of fungi parasitic on insects
is the Laboulbeniales. These are minute fungi developing almost entirely
on the surface, sending short haustoria into the insect to obtain food.

The symbiotic relationship between Septobasidium and scale insects
is interesting because of the high degree of specialization on the part of
the fungus (Couch, 1938) . The dependence of the fungus for its distribu-
tion upon the migrating young scale insects was previously mentioned in
Chap. 15 under Spore Dissemination. The fungus forms a crust over
scale insects, some of which are parasitized while others are not. The
uninfected females give rise to young insects, which may remain under
the fungus crust, crawl out through tunnels under the fungus, or crawl
out over the sporulation surface of the fungus. The young insects are
infected only by the bud cells from the basidiospores, never by the older
fungus hyphae. The bud cells germinate on the surface of the insect and
apparently enter principally through the natural openings. The fungus
then produces coiled haustoria, which absorb food directly from the
circulatory system of the insect, which in turn sucks its food from the
host tree. Some infected insects may settle down on the bark, while
others crawl under a nearby protective fungus colony. Only the former
are responsible for distributing the fungus, while the latter are responsible

PARASITISM AND RESISTANCE 385

for the survival of the ah-eady formed fungus colonies. Connections are
then made by anastomoses of the hyphae from the insect and the hyphae
of the fungus crust under which the insect has come to rest. Thus, the
fungus colony does not originate from one individual but from the aggre-
gation of several individuals by anastomosis, or grafting. The parasitized
insects are dwarfed and do not reproduce but may live as long as the
uninfected insects. The fungus covers the insect's body but is in contact
with it only by the numerous coiled haustoria. The insect in turn
receives protection from severe weather conditions, from parasitic wasps,
other insects, and birds. Certain species, particularly S. hurtii, are easily
cultivated on liquid or agar media. Couch believes that failure of the
fungus to fruit in culture may be due to lack of proper nutrition, which
is furnished by insects in nature, or to a complicated heterothallic condi-
tion of the fungus.

Fungi parasitic on other fungi. The parasitic habit of many of the
chytrids upon other aquatic or semiaquatic fungi and algae is apparently
quite common. A number of these genera are described and illustrated
by Fitzpatrick (1930) and Karling (1942). Practically nothing is known
regarding their nutritional requirements. There appear to be fewer
filamentous fungi parasitic upon fungi. The mention of only a few of
these will serve as examples. Species of Piptocephalis, Chaetocladium,
and Syncephalis are parasitic on other Mucorales. A number of fungi are
reported to be parasitic on members of the Agaricaceae and other higher
fungi. Among these are species of Spinellus, Mycogone, Hypomyces,
Nyctalis, and some Myxomycetes. A species of Penicillium is parasitic
upon an Aspergillus (Thorn and Raper, 1945). Of particular interest
are the hyperparasites, fungi parasitic upon other parasitic fungi. Cicin-
noholus cesatii is parasitic on the Erysiphales, and Darlucafilum is parasitic
on Uredinales. So far as is known, no study of the basic nutritional
requirements of these fungi has been attempted.

Fungi parasitic on man and animals. Many of the fungi which cause
disease of man and animals show distinct differences in morphology when
grown under different conditions. The spore forms produced on artificial
media may be quite different from those developed in the host. This
may be a response to certain nutritional factors, to temperature differ-
ences, or to the presence of certain chemical substances which inhibit
or limit the production of certain spore forms.

In general, the pathogens of man and animals have no unique nutri-
tional requirements. Some are able to utilize inorganic nitrogen, while
others are not; some are deficient for certain vitamins. Nickerson (1947)
points out that there is no direct correlation between nutritional require-
ments and pathogenicity. In fact, there is little concrete evidence
regarding the mode of action of these fungi in causing disease.

38G PHYSIOLOGY OF THE FUNGI

In the case of the dermatophytes, Nickerson has suggested that growth
and sporulation in vivo may be affected by a chemical supplied to the
hair and scales of the skin by diffusion from the adjacent resistant tissues.
There is some evidence that resistance of skin to fungus infection may be
influenced by the nutrition of the individual.

For more complete discussions of the fungus diseases of man and
animals, the student is referred to Nickerson (1947), Wolf and Wolf
(1947), Conant et al. (1944), Emmons (1940), and Dodge (1935). The
pioneering work of Sabouraud (1910) should also be consulted.

Cultural characteristics and pathogenicity. Studies of numerous
isolates of a given species or genus have indicated a possible correlation
between pathogenicity and some particular cultural characteristic. The
recognition of such relations and definite knowledge regarding them would
be of great value to plant pathologists. One such study was made by
Houston (1945) on 52 isolates of Corticium solani from various hosts.
These isolates fell into three culture types based upon the characteristics
of the mycelium and sclerotia. There was a certain degree of correlation
between culture type and pathogenicity and symptoms on certain hosts.
He concluded that the culture type of C. solani is more important in
predicting the pathogenicity of an isolate than the host from which it was
isolated.

During a study of the physiological characteristics and pathogenicity
of 143 isolates of Actinomyces, mostly from scabbed potato tubers, it was
found (Taylor and Decker, 1947) that certain isolates produced a dark
ring of growth at the surface of separated milk. This characteristic was
correlated perfectly with the ability to produce typical scab lesions on
potato tubers. No attempt was made to explain the basic relation of
these two apparently unrelated physiological characteristics.

RESISTANCE

Resistance is the ability of a host to prevent or oppose the entrance or
subsequent growth and development of a parasite. It may be effective
either before or after penetration of the host. A host which cannot
successfully prevent such actions of the parasite is susceptible. Studies
in the nature of resistance have been only partially enlightening, and in
many cases the nature of disease resistance is still obscure. Some of the
present theories are based on what might be termed "circumstantial
evidence," such as a general correlation between resistance and some
characteristic of the host. There is sufficient evidence, however, that it
is dangerous to generalize about the nature of resistance. It seems likely
that in many cases the cause of resistance may be specific, being common
perhaps to but one or only a few host-parasite combinations.

The types of resistance may be placed for convenience into three

PARASITISM AND RESISTANCE 387

groups: (1) mechanical, the prevention of penetration or of unlimited
spread by the structure of the host; (2) functional, the prevention of
penetration by stomatal action of the host; (3) physiological, chemical
action against the parasite or incompatible food relations. The relative
importance of these factors is difficult to determine, but Butler (1918)
states that physiological characters are much more important as a factor
for resistance than the anatomical characters of the host.

Mechanical resistance might be considered as the first line of defense
by the host. According to Melander and Craigie (1927) resistance of
species of Berheris to infection by sporidia of Puccinia graminis is due
to the thickness of the cuticle. B. thunbergii, which is immune, has a
heavy layer of cutin, while in general the susceptible species have a
thin layer. These conclusions were reached after anatomical studies and
after using a mechanical device to measure the resistance of the epidermis
to puncture. The thickness of the cuticle increases with age, as does the
resistance to mechanical puncture and to infection. The same is true
with the apple scab fungus and powdery mildew of barley; young leaves
are susceptible but become more resistant with age.

Resistance in some cases is apparently due to layers of cork cells formed
by the host in advance of the invading parasite. Varieties of flax resistant
to wilt {Fusarium lini) and of cotton to black root rot {Thielaviopsis
hasicola) seem to be successful in walling off the parasite by forming such
a layer of cork which it cannot penetrate. Varieties of potatoes resistant
to scab {Actinomyces scabies) form cork more quickly when wounded than
do susceptible varieties and are believed to owe their resistance to this
characteristic. Thomas (1934) found that the newly formed layer of
cork cells was penetrated by invading hyphae of Armillaria mellea and
that the cork layer did not successfully stop the advance of this fungus.
Brown (1936) states that there is some doubt as to whether the cork layer
really functions at all or whether it is formed after the fungus has been
stopped by some chemical means.

Lignified tissues offer more mechanical resistance than nonlignified
cells. Certain varieties of wheat resistant to stem rust have a compara-
tively greater amount of sclerenchyma and a correspondingly lesser
amount of collenchyma and parenchyma in the stem, as compared with
susceptible varieties. The maturity of host tissue may be a factor in
resistance, even though the tissue does not become lignified or suberized.
Some of the systemic smut fungi in cereals are able to grow and penetrate
the cell walls in meristematic tissue but are apparently unable to pene-
trate the cellulose walls of mature parenchyma cells. After infection
in the embryo or seedling stage, the fungus must continue to grow in the
growing tip of the shoot if it is to reach the flower parts. Conditions
which favor slow growth and delay the maturity of the host favor the

388 PHYSIOLOGY OF THE FUNGI

fungus, while conditions which favor rapid maturity of the host cells may
cause the fungus to be left behind in the mature tissues which it cannot

penetrate.

Hart (1929) studied the nature of resistance of wheat varieties to stem
rust and described a type of resistance that she terms funciio7ial resistance,
which is dependent upon the stomatal movements of the host, and con-
cluded that the parasite enters the wheat only through open stomata.
There has been frequent discussion regarding the importance of the
acidity of the cell sap of the host and its effect upon resistance. The
effects of cell-sap acidity may be threefold: (1) an increase in the hydrogen
ions; (2) the toxicity of the organic acids; (3) the influence upon the
chemical changes and the possible formation of toxic products by the host
cells. In some cases these effects have not been satisfactorily distin-
guished. Numerous examples may be found in the literature in which
resistance has been attributed to the acidity of the host or host part.
Butler (1918) refers to investigations showing that the leaves of varieties
of grape resistant to powdery mildew contain three to five times as much
acid as the nonresistant varieties. He also showed that the red rot
fungus of sugar cane, Colletotrichum falcatum, was present in infected
canes from sowing time but usually did not develop severely until matu-
rity of the canes. He attributed this to either the increase in sugar or the
decrease in acid. The more acid lemons are less attacked by the fruit-
rotting fungi. The amount of acid in the fruit, as indicated by chemical
analysis, may be greater than the amount necessary to check the growth
of the fungus in culture (Cook and Taubenhaus, 1911). A number of
workers have considered cell-sap acidity as a possible cause for resistance
of wheat to stem rust, but this factor now is believed to be of little impor-
tance. No correlation was found between resistance and acidity of the
expressed sap (Hurd, 1924). Similarly, there was no correlation between
resistance and hydrogen-ion values or the titratable-acid values of the
juice of wheat plants resistant to Ustilago tritici (Tapke, 1929).

Some of the most complete experimental evidence showing the correla-
tion between acidity and resistance is presented by Reddy (1933) for
different inbred lines of corn in relation to Basisporium gallarum. Briefly,
he found that when the pH of the cob was below 5.0, resistance to cob
infection was high. Resistance was notably lower at high pH values.
Table 64 gives a summary of some of Reddy's experiments. Reddy also
believes that the influence of pH may explain why the seedlings, which
are acid, are resistant to infection by B. gallarum, while the dry kernels,
which are neutral or alkahne, are susceptible. On the basis of evidence
previously discussed, it is likely that the pathogenic activities of certain
enzymes produced by B. gallarum are inhibited in media having pH of
5.0 or less.

PARASITISM AND RESISTANCE

389

On the other hand, greater acidity of the cell sap may favor the develop-
ment of some diseases. The susceptibility of certain varieties of grape to
Guignardia bidwellii has been correlated with a greater amount of tartaric
acid (Butler, 1918). This author points out that leaves are susceptible
only while they are young and rich in tartaric acid.

Table 64. Hydrogen-ion Readings of Apparently Healthy Cobs of 75 Inbred

Lines of Corn and Incidence of Basisporium Ear Infection Following

Both Natural and Artificial Inoculation

(Redd3^ loim Agr. Expt. Sta. Research Bull. 167, 1933.)

CobpH

No. of inbreds in

No. of in-

No. of ears

Ears

class interval

breds infected

observed

infected, %

4.4-4.7

116

4.8

121

2.5

4.9-5.0

312

7.4

5.1-5.2

313

22.7

5.3-5.4

258

38.0

5.5-5.6

175

41.7

5.7-5.8

185

33.5

5.9-6.3

173

48.6

According to Smith et al. (1946), there is evidence that slight variation
in pH may have a greater influence upon disease resistance of a plant than
is generally believed. Such resistance is not due directly to the number
of hydrogen ions. These authors state:

The observed behavior of hydroquinone and catechol, representatives of the
widely occurring ortho- and para-dihydroxyphenolic compounds, suggested that
hydrogen ion differences also may influence toxicity by affecting the rate or
extent of conversion to the more toxic quinones on invasion by pathogens or by
other injur5^

The possibility that the presence of the pathogen may alter the pH
of the host cells, making it more favorable to extensive invasion, should
not be overlooked. Apparently this situation exists in the relation of
Erwinia carnegieana to its host, the giant cactus of Arizona. Boyle
(1949) reported that the freshly expressed sap from uninfected plants
gave pH readings of 5.0 to 5.5, while the healthy-appearing tissue from
infected plants had pH values of 7.0 to 7.4, and the pH of discolored tissue
not yet broken down was 8.7 to 9.0. These differences could not be
attributed to genetic variation and were believed to be a result of the
pathogen. The possibility that similar relations exist between fungus
pathogens and their hosts seems to merit greater consideration than it
has received.

390 PHYSIOLOGY OF THE FUNGI

That resistance is due to the presence of some toxic substance, perhaps
an organic acid or some related compound, in the living host cell is one
of the most popular theories. However, detailed proof of the effective-
ness of such a compound, even though present in the plant, is often diffi-
cult to obtain. Cook and Taubenhaus (1911) list some organic acids in
order of their toxicity as follows: tannic, gallic, malic, tartaric, and citric.
They state that vegetable juices contain an enzyme which acts upon
gallic acid to produce tannin or a tannin-like compound which is toxic
to fungi. The amount of the enzyme decreases with maturity and
ripening of the fruits (apples, pears, persimmons, etc.), which accordingly
become more susceptible to rot fungi.

An outstanding example of chemical resistance is that described by
Link and Walker (1933) for onion smudge caused by CoUetotrichum
circinans. The cell sap of the colored varieties (resistant) is much more
toxic to the fungus than the cell sap of the white-skinned varieties (sus-
ceptible) . Furthermore, the sap of the colored varieties contains catechol
and protocatechuic acid in amounts that would account for the resistance
of these varieties to the fungus. The action of volatile and nonvolatile
antibiotics in the fleshy scales of the onion is believed to be a definite
factor in relative resistance of onion varieties to C. circinans, Aspergillus
niger, and Botrytis allii (Hatfield et al., 1948). Reynolds (1931) explains
resistance of flax varieties to Fusarium lini as being due to the higher
amounts of glucosides, which upon hydrolysis yield hydrocyanic acid.
Similarly, the resistance of species of Solamim to Cladosporiuni fulvum
is believed to be due to the presence of higher amounts of solanine
(Schmidt, 1933; cited by Brown, 1936). Rochlin (1933) believes that
there is a direct connection betw^een resistance of crucifers to clubroot
and the amount of glucosides, which on fermentation give rise to pungent
mustard oils. The isolation of 2-methoxy-l,4-naphthoquinone from
Impatiens balsamina was reported by Little et al. (1948). This substance
had a high antibiotic activity against several fungi and was nontoxic to
tomato and bean plants. This may be an example of a naturally occur-
ring antibiotic as a factor in resistance. Fontaine et al. (1947) suggest
that tomatin may be a factor in the resistance of certain tomato varieties
to Fusarium lycopersici.

An interesting theory of resistance to obligate parasites is presented
by Dufrenoy (1936). He divides the hosts into three groups: (1) highly
resistant, (2) moderately susceptible, (3) extremely susceptible. He
believes that, when a fungus enters the highly resistant host, it kills the
cells it penetrates and that the death of these cells alters the metabolism
of the surrounding cells, so that their cell sap becomes rich in phenolic
compounds, which prevents the further invasion by the pathogen. In
the moderately susceptible host the host cells and their living contents

PARASITISM AND RESISTANCE 391

are so modified that they revert to the embryonic condition and may even
divide. When the obhgate parasite enters the extremely susceptible
host, it causes so little disturbance that, at least in the first stages of
infection, the metabolism of the host is afTected but little or not at all.
Walker and Link (1935) caution against jumping at conclusions regard-
ing the importance of phenolic compounds as factors in resistance. They
point out that

. . . the mere piesence of phenolic substances in a host plant does not warrant
the conclusion that they play a role in the resistance of that host to a given
parasite or parasites. Toxic phenolic substances might be present in concen-
trations so low that their inhibitory effects are negligible, or they might also be
present in concentrations that have a stimulative effect. When a phenolic sub-
stance with a specific toxicity toward a given organism is present in the host in an
appropriate concentration, it may be regarded as a part of the disease resisting
mechanism of that host.

The four fungi studied by Walker and Link {Colletotrichum circinans,
Gibber ella saubinetii, Botrytis allii, and Aspergillus niger) reacted quite
differently to the various phenolic compounds. Protocatechuic acid
inhibited C. circinans at 1/800 and retarded growth at 1/12,800, while
it did not affect A. niger at 1/200. Colored onions containing this acid
are resistant to C. circinans but quite susceptible to A. niger.

The immunity of monocotyledonous plants to Phymatotrichum omniv-
orum is due to certain unidentified toxic materials present in monocots
but apparently absent in most or all dicots (Ezekiel and Fudge, 1938).
Growth of the pathogen was prevented by the expressed juices from a
number of monocots but not by juices of susceptible dicots. Ether
fractions of monocot roots, or other underground parts, were highly
potent against the pathogen, while similar extracts from susceptible dicot
plants were uniformly nonpotent.

In some other highly parasitic fungi the action of the fungus causes
the death of the surrounding cells, which then prevents the further spread
of the parasite. Leach (1923) found that in a highly resistant variety
of bean the hyphae of Colletotrichum lindemuthianum seldom attack more
than one or two cells of the host. Both the host cells and the fungus
hyphae then die, and the entire cell contents are stained a reddish brown.
In less resistant varieties the parasite attacks more host cells, but sooner
or later the mycelium disintegrates. Leach interprets this as "a nutri-
tional phenomenon," which results in death of the fungus by starvation,
and the products of autolysis then kill and stain the host cells.

It has been pointed out previously that certain fungi are able to pene-
trate some plants but are then unable to establish themselves (Stakman,
1914; Jones, 1919; Salmon, 1905). These plants may be either closely

392 PHYSIOLOGY OF THE FUNGI

related or unrelated to those which serve as the natural host of the fungus.
In such cases the failure to cause disease may be due to unfavoraVjle
nutritional relations. The theory of a toxin-antitoxin, or toxin-counter-
toxin, between parasite and host has been suggested by a number of
investigators (Ward, 1905; Marryat, 1907; Stakman, 1914; Allen, 1923;
Walker, 1924) as a possible explanation for resistance to the rusts.

Cytological studies of Puccinia graminis tritici infections of both
susceptible and resistant varieties of wheat were made by Allen (1923),
who concluded that secretions from the fungus stimulate the metabolic
activities of the susceptible host to produce more food, while in the
resistant host the same secretions cause disintegration and death of the
host cells near the infection. More distant cells may be stimulated.
The haustoria usually die soon after the host cells are killed. Leach
(1919) believes resistance to P. graminis tritici and P. graminis tritici-
compacti can best be explained on the basis of specific food requirements
of the parasite and specific food production by the host. It was sug-
gested that the injury to the host cells might be due to an excess in amount
of enzymes stimulated by a limited supply of food in resistant hosts.
Similarly, Wellensiek (1927) believes that this theory best explains the
resistance of corn to strains of P. sorghi. He suggests that the difference
between susceptibility and resistance is of a quantitative nature and that
the amount of the specific nutrient determines resistance or susceptibility.

Walker (1924) points out that resistance may be due to the action of a
number of factors and that a clear understanding of resistance must be
based upon a thorough understanding of parasitism. Walker's excellent
discussion of the nature of disease resistance gives many references to the
literature on this subject.

Host nutrition and its effect on the development and severity of disease
is a relatively new phase of study, and much more investigation is neces-
sary before general conclusions can be drawn. The fungi vary widely
in their reactions to differences in host nutrition, the type of parasitism
apparently being a determining factor. The action on the pathogen is
believed to be principally indirectly through the effects of nutrition on
the host, although it is possible that some of the vascular parasites may
be directly affected by the nutrients which pass through the xylem. An
increase in the salt concentration of the nutrient solution increased the
development of clubroot, w^hile it decreased the severity of cabbage
yellows (Walker, 1946). The development of Fusarium wilt of tomato
was affected in a way similar to cabbage yellows. More recently, Gallegly
(1949) reported that the development of Verticillium wilt of tomato was
reduced with a reduction in salt concentration of the balanced solution
used to grow the tomato plants. Stakman (1914) and Ward (1902) came
to the conclusion that deficiencies in nitrogen and phosphorus salts avail-

PARASITISM AND RESISTANCE 393

able to the host had no appreciable direct effect upon the resistance to
rusts. A summary of the work on the effect of soil nutrients and environ-
ment upon resistance to disease has been presented by Wingard (1941).

The carbon metabolism of a plant likewise influences resistance to
certain rusts. Waters (1926) found that urediospores of Uroniyces fabae
developed on detached leaves floating on 5 per cent sucrose solution in the
dark, while none formed when leaves were floated on water. These
observations were confirmed by Yarwood (1934) for rust and powdery
mildew of clover. It follows that active carbon assimilation increases
susceptibility of the host to the obligate parasites.

Although the environmental factors are of great importance in deter-
mining the resistance or susceptibility to a disease, their effects are usually
upon the host and only indirectly upon the parasite. Abundant refer-
ences on this subject can be found in the literature. The effect of tem-
perature upon the metabolism and resistance of certain hosts may be
illustrated by Gihherella zeae on wheat and corn (Dickson, 1923). Seed-
ling infection of wheat was found to occur at high temperatures and of
corn at low temperatures; i.e., the temperatures unfavorable to host
development. In the germination of w^heat at low soil temperatures the
starch of the endosperm is hydrolyzed more rapidly than the proteins,
which results in abundant sugar but little available nitrogen for seedling
growth. Thus, the cell walls are thickened and more resistant. At
higher temperatures both starch and proteins are rapidly hydrolyzed;
there is a greater supply of available nitrogen, and growth is more rapid.
The cell walls remain longer in the pectic condition and are more suscepti-
ble to attack. In corn the situation is reversed. At high temperatures,
which favor the corn, the cell w'alls develop more rapidly and are more
resistant.

Sharvelle (1936) concludes that the resistance of flax to flax rust cannot
be attributed to any single factor but probably results from a number of
factors operating together. Doubtless, the same statement could be
applied to many other diseases to which the nature of resistance is not well
understood.

SUMMARY

Some of the different types of parasitism may be summarized as fol-
lows: (1) The parasite produces extracellular enzymes, particularly
pectinase, w'hich dissolves the middle lamellae of the host cells, allowing
the cells to separate (rotting). This may or may not be accompanied
by toxic substances but results in the death of the cells. The soluble
food materials are then free to be absorbed by the fungus. The insoluble
foods stored in the host cells may be digested by other extracellular
enzymes. This type is illustrated by the rots of fruits and vegetables.

394 PHYSIOLOGY OF THE FUNGI

(2) The parasite may produce toxic materials or other substances whi( ii
may be active at some distance from the fungus, but it usually does not
cause the rotting of the tissue. This is illustrated by a number of wilt
diseases and by some others. (3) The third type depends upon a con-
genial nutritional relationship between the parasite and the host cells.
In susceptible hosts of this type there is little or no apparent effect upon
the host cells. The resistant hosts may show a high degree of sensitivity
to the parasite, which may result in the death of the invaded cells and
starvation of the parasite. This type of parasitism is characteristic of
the balanced parasites. The balanced parasite enters the susceptible
host cell and establishes a compatible food relationship, absorbing the
soluble nutrients elaborated by the host, without disturbing the metabolic
activity of the host in the early stages. In this respect, the relationship
of parasite and susceptible host represents the most highly specialized
type of parasitism.

The destructive parasites, as a rule, are strong producers of toxins
and exoenzymes, while the balanced parasites must be quite weak in this
respect. In many host-parasite relations studied, there is a change in
the permeability of the host cells surrounding the invading hyphae. This
is believed to be a direct response to substances secreted by the parasite.
Increased permeability would allow greater diffusion of water and nutri-
ents from the host cells to the parasite. The metabolic products of the
fungi involved in parasitism are for the most part undetermined, but they
are known to include toxins, enzymes, and polysaccharides. Since the
kind and amounts of such products are known to vary with the composi-
tion of the medium in the laboratory, it is believed that like variation
may occur in different hosts in nature.

The basis of resistance to disease may be mechanical, functional, or
physiological. Some of the known or proposed causes of physiological
resistance are (1) cell-sap acidity; (2) toxic substances of the host; (3)
inhibition of the activity of certain enzymes of the parasite by the host ;
(4) hypersensitiveness ; (5) incompatible nutritional relationship; (6)
decreased permeability of the cell membranes of the host, resulting in
partial or complete starvation of the parasite; (7) a combination of
various factors acting together.

The obligate parasites, principally the rusts, offer some challenging
unsolved problems for the future students of parasitism. Probably the
principal one involves the culturing of such fungi under controlled condi-
tions on media of known composition. All of the many attempts to solve
this problem have met with failure, yet few investigators doubt that it
can be solved. The phenomenon of heteroecism among the rusts is of
great interest from the standpoint of food relationships. For instance,
sve must either assume that the wheat and the barberry furnish the same

PARASITISM AND RESISTANCE 395

nutrients for Puccinia graminis tritici, and the white pine and Ribes for
Cronartium ribicola, or that the nutrient requirements of the haploid
mycelium are different from those of the diploid mycelium.

Much more investigation is needed to increase our knowledge of possi-
ble correlations between pathogenicity and metabolic products. This
should lead to a better understanding of parasitism. The possible role
of antibiotics occurring naturally in host plants as a factor in disease
resistance has received some attention recently, but much more knowl-
edge of this type is desired. Many of the problems of today may come
nearer to solution with a clearer understanding of the enzyme systems of
the parasitic fungi and the basic principles of specific enzymatic action.

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Houston, B. R.: Culture types and pathogenicity of isolates of Corticium solani,

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398 PHYSIOLOGY OF THE FUNGI

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1941.

PARASITISM AND RESISTANCE 399

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of northern Idaho, Mycologia 31: 154-156, 1939.

CHAPTER 18

PHYSIOLOGICAL VARIATIONS AND INHERITANCE OF
PHYSIOLOGICAL CHARACTERS

Variation in the results of experimental work with fungi is of frequent
occurrence; it is perhaps even more frequent than uniformity. Different
investigators conducting the same experiments with the same species of
fungus have often failed to obtain the same results. Such variation may
be attributed to (1) genetic differences in the strains or isolates used, (2)
slight nutritional differences in the experiments, or (3) differences in the
physical environment. Examples of the second and third groups of
factors have been pointed out frequently in the earlier chapters. A brief
discussion of the genetic differences involving physiological expression
and the general mode of inheritance of these factors (in so far as they are
known) will be given.

PHYSIOLOGICAL VARIATION

Variation in physiological behavior of different species of fungi has
been noted in the preceding chapters. The present discussion emphasizes
the physiological variation within a species, i.e., between different isolates,
strains, or races, which show little or no morphological difference.

Nutritional requirements. Variations in the nutritional requirements
of different isolates of the same species are numerous. Differences in
vitamin requirements or in carbon and nitrogen utilization may serve as
examples.

Differences in deficiencies for one or more vitamins have been reported
for different isolates of Fusarium avenaceuni (Robbins and Ma, 1941),
Sclerotinia minor (Barnett and Lilly, 19-47), Saccharomyces cerevisiae
(Leonian and Lilly, 1942; Burkholder and Moyer, 1942), Sordaria fimicola
(Hawker, 1939; Barnett and Lilly, 1947a), and numerous others. For
example, certain isolates of Sordaria fimicola from nature are totally
deficient for biotin alone, while others are deficient for both biotin and
thiamine. A somewhat different type of variation is reported by Thren
(1941) for Ustilago nuda. The haploid mycelium showed no deficiency
for vitamins, while the diploid mycelium required an external supply of
thiamine or pyrimidine. The plus and minus strains were also found to
differ in their nutritional requirements.

400

VARIATION AND INHERITANCE 401

Different isolates of Ustilago striiformis have shown strikingly different
responses to sources of carbon and nitrogen (Cheo, 1949). The isolates
from bluegrass segregated into two groups based on mycelial type, ''frag-
menting" and "mycelial." The "fragmenting" type grew well only
on media containing sucrose and organic nitrogen, while the "mycelial"
type could utilize a number of sugars and nitrate nitrogen. Single-spore
(haploid) isolates from the same fruit body of Lenzites trdbea collected in
nature varied nearly fourfold in their ability to synthesize thiamine
(Lilly and Barnett, 1948).

Induced deficiencies for a number of vitamins and amino acids have
been demonstrated by Beadle (1946) in mutants of Neurospora and by
Bonner (1946) in mutants of Penicilliiim. The mutations were induced
by exposure of spores of these fungi to ultraviolet and X-ray radiation.
Mutants that showed deficiencies for thiamine and differences in nitrogen
requirements were also reported for Aspergillus terreus (Thorn and Raper,
1945). One mutant differed from most species of Aspergillus in its
inability to utilize nitrate nitrogen. Fries (1948) describes spontaneous
mutations of Ophiostoma which yield the same strains and in the same
proportion as those induced by X rays. These results lead us to conclude
that similar mutations are the principal cause of variation in the isolates
obtained from nature.

Response to environment. Isolates of the same species frequently
vary in their physiological responses to some environmental factors,
among which are temperature and light. For example, isolates of
Phytophthora infestans were found to vary in their resistance to high
temperature (Martin, 1949). Of the eight isolates studied, four from
Louisiana withstood exposure to 36°C. for 6 days, while three isolates
from Minnesota were killed after 4 days and one isolate from New York
was killed in less than 6 hr. at the same temperature. The presence of
the high-temperature strain is believed to be responsible for the prevalence
of late blight in Louisiana during the past few years. Houston (1945)
found that, for one group of isolates of Corticiiim solani, the optimum
temperature for growth was 24 to 25°C. and the maximum was 33°C.
For two other groups the optimum and maximum temperatures were 28
to 29°C. and 40°C., respectively. The three groups also varied in growth
rates.

Variation in response to light is illustrated by Choanephora cucur-
hitarum. This was indicated first by Wolf (1917) for two isolates. The
isolate used by Christenberry (1938) produced conidia in continuous
total darkness, while two isolates used in our laboratory failed to produce
conidia in continuous darkness (Barnett and Lilly, 1950).

Metabolic products. Both qualitative and quantitative variations in
the metabolic products of different isolates of the same species are com-

402 PHYSIOLOGY OF THE FUNGI

mon. Industries involved in the commercial production of alcohols,
certain organic acids, and antibiotics are in constant search for higher
yielding "strains" of the species in present use, as well as of other species
of fungi. Such a search led to the discovery of PenicilHum chrijsogenum
Q176 and its variants, which are high producers of penicillin. Brewer's
yeast is said to grow in media with an alcohol content as high as 14 to
17 per cent, while the baker's yeast is checked in about 4 per cent alcohol
(Wolf and Wolf, 1947). Both yeasts belong to the species Saccharomyces
cerevisiae.

A different type of variation, apparently linked with sexuality, is
reported in Mucor racemosus (Harris, 1948). Here, the production of an
undetermined antibiotic seems to be confined to the minus strain. Varia-
tion in bioluminescence is reported for Panus stypticus (Macrae, 1942).
The fruit bodies and mycelium of the strain found in North America
are luminescent, while those found in Europe are not (Fig. 77). Variants
of the same species commonly differ in pigment production (Christensen
and Graham, 1934; Leonian, 1929). Mutants, or saltants, are commonly
lighter in color than the parent type.

Sporulating ability. Many investigators have noted the spontaneous
development of nonsporulating cultures or sectors from a sporulating
mycelium. Variation in abundance of spores produced by different
isolates from nature is also common. For example, some of the species
which illustrate this variability are Fusarium spp., Phytophthora spp.,
Phoma terrestris, Gibber ella zeae, Glomerella cingulata, Lenzites trabea,
Monilinia fructicola, and Ustilago striiformis. Variations in fruit bodies
of Cyathus stercoreus produced in culture are described by Brodie (1948).
Variation in production of sclerotia has been observed in isolates of
Sclerotinia trifoliorum by Kreitlow (1949) and of S. sclerotiorum in our
laboratory.

Pathogenicity. Variability in the metabolic products such as enzymes
and toxins and in the ability to establish compatible food relations with
the host may be of great importance in determining pathogenicity. Das
Gupta (1936) discusses the pathogenicity as well as other characteristics
of "saltants." Such soil-inhabiting fungi as Fusarium spp. are notorious
for their variability in pathogenicity within a species. Species of Helmin-
thosporium (Christensen, 1922; Dickinson, 1932) and Cortidum solani
(Houston, 1945) are likewise highly variable. In the highly parasitic
fungi, such as the smuts, rusts, and powdery mildews, there is a high
degree of physiologic specialization of races.

The determination of physiologic races is based on infection types of
several varieties or species of the host. Dickson (1947) reports the
existance of 189 known physiologic races of Puccinia graminis tritici and
128 physiologic races of P. rubigo-vera tritici. Genetic studies indicate

VARIATION AND INHERITANCE

403

that the physiologic races may vary in Init a single gene and that they may
arise by hybridization or by mutation.

There is abundant evidence that the haploid and diploid stages of some
fungi may differ in pathogenicity. The haploid phase of a number of

Fig. 77. Panus stypticus grown on malt agar. A, diploid mycelium, 4 weeks old, from
a pairing between a haplont of the luminous American form and a haplont of the
nonluminous European form, photographed by reflected light; B, the same culture as A
photographed by its own light; C, a 2-weeks-old pairing between a nonluminous
haplont, on the left, and a luminous haplont, on the right, photographed by reflected
light; D, the same pairing as C photographed by its own light. (Courtesy of Macrae,
Can. Jour. Research, Sec. C, 20: 424, 1942.)

smuts is apparently unable to cause infection, while the diploid mycelium
is pathogenic. Since the haploid and diploid mycelia of the heteroecious
rusts parasitize different hosts, we must conclude that they also differ in
pathogenicity.

404 PHYSIOLOGY OF THE FUNGI

INHERITANCE OF PHYSIOLOGICAL CHARACTERS

The genetics of the fungi has been, in general, a neglected study.
Numerous papers have appeared on the sexuality of the fungi, particu-
larly with regard to the various sexual or compatibility groups in the
Basidiomycetes. The sexuality of the Mucorales has been studied to a
lesser extent. Genetic studies of morphological characters have been
decidedly fewer. Perhaps this is due to the failure to recognize definite
morphological differences betw^een individuals of opposite sex but of the
same species. An equally great handicap to such studies lies in the
difficulty in obtaining the perfect stage of many of the fungi which other-
wise might be suitable. Studies dealing with inheritance of physiological
characters (if sexuality is excluded) are comparatively few and recent.

The basis of inheritance. The physical basis of inheritance is the
gene, located at a specific position on a certain chromosome. In mitosis
the chromosomes and their genes divide, and half of each goes to each
daughter nucleus. With the exception of parthenogenesis, all perfect
stages of the fungi arise as a result of the union of two nuclei. These
two nuclei may arise from the same haploid individual (homothallism)
or from separate haploid thalli (heterothallism). The union of the two
haploid nuclei, each with a single set of chromosomes, initiates the diploid
nucleus, or the syncaryotic stage, in which the chromosomes are paired.
The syncaryotic stage in fungi is usually short in duration, being followed
closely by meiosis, which involves the separation of the two chromosomes
(and genes) of each pair. Certain pairs of chromosomes may separate in
the first division, while others separate in the second. Therefore, two
successive nuclear divisions are necessary to complete the reduction of
all pairs of chromosomes (and likewise all the pairs of genes). In the
Ascomycetes and the Eubasidiomycetes karyogamy and meiosis occur
in the same cell, the ascus and the basidium, respectively. In the smuts
and rusts, meiosis typically takes place in a promycelium, while kary-
ogamy occurs in the teliospore. When a single pair of genes is considered,
half the haploid ascospores or basidiospores carry one gene and half carry
the other gene.

Inheritance in the Ascomycetes. Some of the outstanding genetic
work has been done by Dodge (1927, 1928) and others with Neurospora,
by Ames (1934) and Doweling (1931) with Pleurage anserina, by Edgerton
et at. (1945), Chilton and Wheeler (1949), and their associates with
Glomerella, and by Lindegren (1945, 1948) and his colleagues with yeasts.
Most of these investigations have been concerned primarily with sexual
or morphological characters. The life cycle of Neurospora is shown
diagrammatically in Fig. 78.

Beadle and his associates have contributed much to our knowledge

VARIATION AND INHERITANCE

405

of the inheritance of physiological characters in the Ascomycetes. Beadle
(1946) believed that, if the ability to synthesize a certain amino acid or
growth factor were due to the action of a single gene, it should be possible
to modify the gene in such a way that the fungus could no longer syn-
thesize that compound. Previous work of other geneticists with corn,
Drosophila, and other organisms had shown that exposure to X rays or
ultraviolet radiation caused mutations by either destroying the gene or
modifying it so that it could no longer function normally. Beadle found
that exposure of conidia of Neurospora crassa and A^. sitophila to X rays
or ultraviolet rays had the similar effect of causing mutations that were

Germinating

a SCO spore- .-^

Germinating
ascospore

Conidia

Protoperittiecium A —

~- Protoperithecium a

Hypnal fusion

Fig. 78. Diagram of life cycle of Neurospora. (Courtesy of Beadle, Am. Scientist
34 : 36, 1946, and Science in Progress, 1947. Published by permission of the Society of
the Sigma Xi.)

expressed in the inability of the fungus to synthesize vitamins, amino
acids, and other essential metabolites.

The 'Svild type" of Neurospora is deficient for biotin but is self-suffi-
cient for all other vitamins and for its necessary amino acids. The
conidia were exposed to the ultraviolet rays of a Sterilamp for such a
time that most of the spores were killed. The spores were then
sown over the surface of agar plates in such concentration as to give
individual "colonies," which were isolated and allowed to grow. When
these were transferred to a minimal medium, containing sucrose, nitrate,
mineral salts, and biotin, the failure of an isolate to grow showed an
induced variation from the wild type in its capacity to synthesize essen-
tial metabolites.

The variant cultures were then selected and crossed with the wild

406

PHYSIOLOGY OF THE FUNGI

strain of the opposite sex to determine if the changes were inherited. The
ascospores from these crosses were planted on both the minimal medium
and a complete medium. The appearance of the deficiency in half of
the cultures was considered as evidence that the change was of genetic
origin; i.e., a mutation. Transfers of the mutant to four different media
(minimal, with amino acids, with vitamins, and complete) then deter-
mined whether the deficiency was for an amino acid or a vitamin. All
media contained biotin. For a diagrammatic scheme of the procedure
see Fig. 79.

X-ravs or
ultraviolet

-^ © © © -

Coniolict
(asexuoil spores)

Wild fv/pe

J-

\.y

Crossed
with wild
t^pe of
opposite
sex

Frui+inq body

-<r-

Sexut^l spore

Complete

meolium

(with vitoimins,

oimino cicids,

etc.)

Minim(7il Complete

MinirriCTl Vitoimins Amino

medium acids

Fig. 79. Experimental procedure by which biochemical mutants are produced and

detected in Neurospora. (Courtesy of Beadle, Am. Scientist 34 : 37, 1946, and Science

in Prug'-ess, 1947. Published by permission of the Society of the Sigma Xi.)

7 he identification of the specific deficiency involved the growth of the
iiiyt2,nt upon the minimal medium plus each of the amino acids and
vitamins added singly. This procedure is shown in Fig. 80. Figure 81
shows the proof of inheritance of the deficiency for pantothenic acid.

Mutations involving tli'^ following vitamins have been described:
thiamine, riboflavin, pyridoxin'^, niacin, pantothenic acid, p-amino-
ben.^oic acid, inositol, and choline. All mutants, as well as the wild
type, are deficient for biotin. In addition, mutations have appeared
which cannot synthesize tlis following amino acids: arginine, isoleucine,

VARIATION AND INHERITANCE

40;

Q Q Q Q Q

Thiamin

Complete
medium

Ribo-
flavin

Pyri-
doxin

Panto- Niacin
thenif
acid

p-Amino-

benzoic

acid

Inositol Choline

Folic
acid

I ©

Nucleic
acid

Minimal
control

Fig. 80. Tests of mutant on individual vitamins or growth factors. Growth only on
pantothenic acid indicates a single deficiency for this vitamin. (Courtesy of Beadle,
Am. Scientist 34: 39, 1946, and Science in Progress, 1947. Published by permission
of the Society of the Sigma Xi.)

I
f

Fruiting

QQOOQOQO

Uhu) Mb* iHi iki iSS

|r jF I I p

KJ kJ \J \J kJ

With pantothenic oicid

i } 1 I 1 1 1 1

QOOOQOQQ

Panfothenicless

Wild
t:ype

Without pantothenic acid
Fig. 81. Scheme by which the inheritance of a mutant type is determined. The 1 to
1 ratio with regard to need for pantothenic acid indicates simple Mendelian inheritance.
(Courtesy of Beadle, Am. Scientist 34: 40, 1946, and Science in Progress, 1947. Pub-
lished by permission of the Society of the Sigma Xi.)

408 PHYSIOLOGY OF THE FUNGI

leucine, lysine, methionine, phenylalanine, proline, threonine, trypto-
phane, and valine.
Beadle (1946) states:

The list of compounds that Neurospora can be made to require from an external
source is remarkably similar to a list of chemicals that we cannot make and
require in our food suppl3^ It is clear, therefore, that the substances the bread
mold needs in its metabolism are very much the same as those we need. The
difference is only an apparent one and results from the fact that bread mold
makes them whereas we let some other organism make them for us. By inacti-
vating the right genes the bread mold can be made very similar to man in its
nutritional requirements.

Using a technique similar to that described above for Beadle's work,
Bonner (1946) exposed conidia oiPeniciUium. notatum and P. chrysogcmnn
to X rays and ultraviolet rays. Of a total of 85,595 "strains" tested,
398 were found to be deficient in synthetic ability. Since these species
of PeniciUium are imperfect, the genetic basis for the biochemical changes
cannot be proved, but it seems likely that this is the case, just as in
Neurospora.

Inheritance in the Basidiomycetes. In the life cycle of the Basidio-
mycetes there exists a distinct diploid (dicaryotic) vegetative phase of
extended duration, in which the cells usually contain two haploid con-
jugate nuclei. Buller (1941) cites the results of numerous experiments
by himself and others to furnish ample proof that one nucleus of the
conjugate haploid pair in the diploid mycelium may affect the expression
of the other nucleus and thus exhibit dominance. This is true for physio-
logical as well as morphological characters. Experimental evidence
indicates that the genetic behavior of a cell containing two conjugate
haploid nuclei is similar to that of the diploid nucleus, if the two were
fused. Buller believes that the term "diploid cell" can apply equally
well to a cell containing two conjugate haploid nuclei and to a cell con-
taining one diploid nucleus. He prefers to use the terms "haploid" and
"diploid" in describing mycelium or cells to the terms "monocaryotic"
and "dicaryotic" which are also in use. We prefer to follow Buller in
the use of these terms.

In the higher Basidiomycetes, principally the Agaricales, genetic studies
have been chiefly limited to the inheritance of sex factors or compatibility
factors. Fewer studies have dealt with the more strictly physiological
characters. In a heterothallic species, two compatible haploid mycelia
unite to initiate the diploid mycelium, which in many species is recog-
nized by the presence of clamp connections. Usually, the formation of
the diploid mycelium is a prerequisite to the production of fruit bodies.

Exidia, in the Tremellales, Avill serve as an example of the higher
Basidiomycetes. In four species studied (Barnett, 1937) the single-

VARIATION AND INHERITANCE 409

spore haploid cultures from the same fruit body fell into two compatibility-
groups. Such a condition is described as bipolar. Diploid mycelium
was formed only when two haploid mycelia of different compatibility
groups were paired. If A and a represent the genes for compatibility,
the combination of Aa would be necessary for the formation of diploid
mycelium. AA and aa would be incompatible.

^\^lile the single-spore cultures of a single fruit body of Exidiaglandu-
losa give rise to two compatibility groups A and a, a second fruit body
collected at some distance away may give rise to haploid mycelia which
apparently fall into the same groups {A and a). Yet we may find that
all the haploid mycelia of the first fruit bod}^ are compatible with all the
haploid mycelia of the second fruit body. In other words, the two groups
of the second fruit body are slightly different from the two groups of the
first fruit body. It is, therefore, likely that numerous compatibility
groups exist, even though only two occur in any one fruit body. Com-
patibility, in this case, is apparently determined by multiple alleles. The
existence of geographic races has been described for a number of fungi
by Buller (1941) and others.

A somewhat different situation exists in Collybia velutipes and a number
of other species. It has been found that each spore on a basidium may
differ in its compatibility factors. Compatibility in this case is deter-
mined by two pairs of genes on different chromosomes. These groups
are usually designated as AB, Ah, aB, and ah. The combination of
AaBb is then necessary for compatibility and formation of diploid
mycelium.

Compatibility in itself does not necessarily indicate that fertile fruit
bodies will be formed. For instance, some of the single-spore isolates
of Lenzites trahea were found to produce fertile fruit bodies, while other
cultures failed to do so (Barnett and Lilly, 1949). By pairing compatible
fruiting isolates and also the compatible nonfruiting isolates, it was possi-
ble to establish a correlation between the fertility of the diploid mycelia
with that of the haploid "parents." It seems probable, therefore, that
the ability to produce fertile fruit bodies has a genetic basis, in addition
to that of compatibility.

In Schizophyllum commune the ability to produce normal fruit bodies is
dominant over the formation of abnormal, knot-like fruit bodies (Zatler,
in Buller, 1941). If G represents the factor for normal fruit bodies and
g the factor for knot-like fruit bodies, the results could be expressed as
follows: G crossed with G or G crossed with g gives normal fruit bodies,
while g crossed with g gives knot-like fruit bodies. Zatler also showed
that in Collybia velutipes inheritance of pigmentation of his cultures was
due to two genes located on different chromosomes. A combination of
the two dominant factors in the haploid mycelium resulted in a deep

410 PHYSIOLOGY OF THE FUNGI

brown color; one dominant and one recessive factor gave lighter shades
of brown ; while a combination of the two recessive factors gave pure white
mycelium.

The normal haplont of Peniophora allescheri is reported (Nobles, 1935)
as slow-growing with scant mycelium bearing conidia. A mutant grew
rapidly with abundant mycelium but bore no conidia. The combination
of normal haplont and mutant haplont yielded diploid mycelium which
grew rapidly and abundantly and produced conidia. Thus, rapid growth
and conidial production were dominant over slow growth and nonproduc-
tion of conidia.

Bioluminescence of the North American race of Panus stypticus was
found (Macrae, 1942) to be dominant over nonluminescence of the
European race when the two haplonts were crossed (Fig. 77).

The single-spore isolates from a single fruit body of Lenzites trahea were
found (Lilly and Barnett, 1948) to vary nearly fourfold in their ability to
synthesize thiamine. WTien a haplont of low synthetic ability was
crossed with one of high synthetic ability, no definite evidence of domi-
nance was observed. By making other types of crosses it was found that,
in general, the synthetic ability of the Fi haplonts was similar to that of
the "parent" haplonts. Yet, when the "parent" haplonts differed
widely in synthetic ability, the Fi haplonts did not segregate into the
1 to 1 ratio, as would be the case if inheritance were due to a single gene.
Single-spore cultures from haploid fruit bodies produced by certain
haplonts were more uniform in their ability to synthesize thiamine than
were single-spore cultures from diploid fruit bodies of known origin. It
was concluded that the ability to synthesize thiamine by L. trahea is
genetically controlled, and that the mode of inheritance is complex and
not due to a single gene.

The smuts have received much attention in genetic studies by Stakman,
Christensen, and their associates at the University of Minnesota. Such
characters as sex, pigmentation, pathogenicity, and morphological fea-
tures of the mycelium and spores have been included in the study. Little
is known regarding the factors governing the more strictly physiological
or nutritional processes. An excellent review of the genetics of the smuts
is given by Christensen and Rodenhiser (1940). These authors discuss
the work of Goldschmidt, who found that the diploid mycelium derived
from two haplonts of different races of Ustilago violaceae was able to
attack the hosts which were susceptible to each parent race. This
indicates that the diplont contained the combined pathogenic characters
of the two haplonts. Hanna (1932) made an interspecific cross between
Tilletia levis, with smooth spores and an odor of trimethylamine, and T.
tritici, which had rough spores and no odor of trimethylamine. The
Fi "chlamydospores" had smooth walls and emitted an odor of tri-
methylamine, showing that both characters were dominant.

VARIATION AND INHERITANCE 411

Numerous articles on the inheritance of the rusts may be cited. Among
the characters commonly studied are color of urediospores and patho-
genicity. One striking example of inheritance of pathogenicity of races
of Puccinia graminis tritici is reported by Johnson and Newton (1940).
Using pathogenically homozygous mycelia of race 9 and race 36, it was
found that Kanred wheat was not attacked by race 9 but was highly
susceptible to race 36. Urediospores were obtained from a hybrid of
these two races and were sown on different varieties of wheat. No infec-
tion occurred on Kanred, indicating that the nonpathogenicity of race 9
w^as dominant over the pathogenicity of race 36. In the r2 uredio-
spores the pathogenicity to Kanred wheat segregated in a 1 to 3 ratio,
indicating true ]\Iendelian inheritance.

The basis of variation in the imperfect fungi. Any change or variation
in the imperfect fungi may be either temporary or permanent. Tem-
porary variations do not involve gene changes, ^vhile the permanent
variations are believed to have their basis in the gene, or at least in the
nucleus. The Mendelian inheritance of these variations cannot be proved
in those fungi with no sexual stage.

Most investigators studying the permanent variations which arise in
culture or which are recognized in different isolates of many of the imper-
fect fungi would explain the origin of these variants as mutations. For
example, Dickinson (1932) studied "saltation" in the genera Fusarium
and Helminthosporium and noted frequent anastomoses between hyphae
of different "saltants." This author discussed the possibility of cyto-
plasmic inheritance but concluded that the permanent variations were
due to actual mutations.

Hansen (1938) would explain many such variations in the imperfect
fungi on a different basis and presents abundant evidence to substantiate
his argument. Only the essential features of Hansen's "dual phenom-
enon" will be presented below. The conidia and mycelial cells of many
of the imperfect fungi contain two or more nuclei. These nuclei may
not all be alike. Considering the nucleus rather than the cell as the
basic unit of the individual, an isolate may be composed of two culturally
distinct individuals. This condition is referred to as the dual phenom-
enon. A heterocaryotic fungus, when single-spored, would give rise to
homotypes of each of the individuals and the heterotype like the parent
isolate. One homotype is characterized by abundant mycelium and few
or no conidia and is called the M (mycelial) type. The other is charac-
terized by abundant conidia and often a lesser amount of mycelium and is
called the C (conidial) type. The heterotype is, in general, intermediate
between the M and C types and is called the MC type. Cultures of the
M and C types give only the parent type when single-spored. The
frequency w4th w^hich the dual phenomenon is encountered in the imper-
fect fungi suggests that this is the natural condition for many fungi.

412 PHYSIOLOGY OF THE FUNGI

In a later paper, Hansen and Snyder (1943) state that the change from
the C to the M type is a true mutation. In Hypomyces solani f . cucurhitae
this change also involved the change from the hermaphroditic phase to
the unisexual male phase. They conclude that the M and C genes are
alleles and are inherited independently of the factors for compatibility.
On the contrary, Robbins and McVeigh (1949) have presented evidence
that variants of Trichophyton mentagrophytes arise as mutations and that
the dual phenomenon does not exist in this fungus.

Nutritional adaptations. Most of our present knowledge regarding
nutritional adaptations has come from experiments with yeasts, and to a
lesser extent with Neurospora and bacteria. Leonian and Lilly (1942)
were able to "train" eight strains of Saccharomyces cerevisiae so that they
grew without the addition of one or more of the vitamins which they
formerly required. The techniciue employed consisted in increasing the
amount of initial inoculum, prolongation of the incubation period, and
repeated subculturing on media deficient for one of the necessary vita-
mins. Reversions occurred in most of the strains after being cultured
continuously on media containing all the vitamins. Such adaptations
as these may or may not involve gene changes.

Lindegren and his associates have written numerous articles on the
genetics and adaptations of yeasts. From over 400 isolates of Sac-
charomyces cerevisiae, Skoog and Lindegren (1947) found 12 which could
not utilize glucose. Eleven of these isolates reverted to glucose utiliza-
tion within a period of a few days. One isolate remained glucose-negative
for a period of 3 months when grown on lactate medium. They believe
that the reversion to glucose utilization involves more than a single-step
change.

Spiegelman (1950) points out that "a basic assumption of modern
biology is that genes function by controlling enzyme synthesis. From
this point of view it is obvious that enzymatic adaptation has profound
implications for one of the central themes of biological thinking." This
does not mean that the presence of the gene is always accompanied by
the presence of the enzyme in the cell, but merely that the potentialities
for the production of the enzyme are present. The synthesis of the
specific enzyme, as well as its subsequent activity, depends upon other
factors, a major one being the type of substrate. From numerous experi-
ments it is evident that the specific enzyme either is produced, or becomes
detectable, only when its corresponding substrate is present in the
medium. It appears, however, that the specific enzyme may be formed
even when the corresponding substrate is not present but that, under
such conditions, the adaptive enzymes are usually not detected. In the
course of a "long-term adaptation," there is not only synthesis of the
specific enzyme but also an increase in the rate of enzyme formation. In

VARIATION AND INHERITANCE 413

this respect, Spiegelman states, "In particular, the rate of formation
of a given enzyme is an autocatalytic function of the amount of that
particular enzyme present in the cytoplasm."

Ryan (1946) found that certain adaptations of the " prolineless " and
" thiamineless " mutants of Neurospora are not inherited. For example,
the "thiamineless" mutant may not grow for several days after being
placed on a thiamine-free agar medium and may finally begin to grow.
This is explained on the basis of adaptive enzyme formation. For further
discussion of adaptive enzymes, see Chap. 4. For a more complete
discussion of cytoplasmic inheritance and adaptive enzymes in yeast, the
reader is referred to Lindegren (1945, 1949) and Spiegelman (1950).

Back mutations. The mutations studied by Beadle and others were
those involving deficiencies for growth factors which the wild type was
able to synthesize. It has been shown that, under certain conditions,
there may be a reversion from the deficient type to the wild type. This
may be clue to a noninherited condition (an adaptation), such as that
described above, or it may involve a gene change (a back mutation).
Ryan (1946) has discussed at some length the topic of back mutation and
adaptation in certain organisms. Only a few of his ideas will be presented
here, omitting the detailed results of his experiments. He points out
that the change from a deficient to a self-sufficient habit for growth fac-
tors may be induced experimentally in both the fungi and the bacteria.
In some cases these nutritional changes are inherited, indicating gene
changes. In the case of the "leucineless" mutant of Neurospora the
adaptation back to the autotrophic habit was determined to be due to a
reverse mutation of the leucineless factor to the wild type. Ryan believes
that the ultraviolet rays, in causing the original "leucineless" mutation,
caused a change in the wild-type gene so that it still retained the ability
to reproduce but was unable to act in the synthesis of leucine. He found
that the "adaptation frequency" varied inversely with the amount of
leucine present in the medium. This theory assumes merely the inactiva-
tion and reactivation rather than the destruction of a gene by the ultra-
violet rays.

Lindegren (1949) found that a mutation from pantothenate deficiency
to pantothenate independence was at a different locus, and that the
synthesis of pantothenate by the mutant was by a different route than
in the original wild type.

Giles and Lederberg (1948) have recently studied the effects of various
mutagenic agents in inducing adaptations (reversions) of deficient
mutants of Neurospora crassa. They found that the frequency of adapta-
tion of certain mutants vaay be greatly increased by ultraviolet radiation.
This was true with the "inositolless," "cholineless," "methionineless,"
and ' ' ribofla vinless ' ' mutants. The ' ' pantothenicless ' ' mutants remained

414 PHYSIOLOGY OF THE FUNGI

unchanged by the same treatment. Indications are that these changes
represent mutations to the wild type. These adaptations may also be
initiated by X rays, nitrogen mustard, and radiophosphorus.

Chemically induced mutations. Nitrogen and sulfur mustard gases
have been used to induce mutations in various fungi. The method of
treatment is simple and consists in exposing spores or mycelium to a
buffered solution of the chemical for 30 min. or longer. The spores or
mycelium are then washed and plated out. The methods used in detect-
ing mutants are then the same as when X rays or ultraviolet irradiations
are used. Treatment of young conidia or germinating conidia of Neuros-
pora crassa with nitrogen mustard produced more mutants than treat-
ment of old or ungerminated conidia (McElroy et al., 1947). The tech-
nique of using the vapor of mustard gas to induce mutation is described
by Hockenhull (1948). Mustard gas in a buffered solution (pH 6.9 to
7.0) was used by Hockenhull (1949) to produce mutants of Aspergillus
nidulans.

The mustard gases, in common with nitrous acid, react with proteins.
For example, casein which has been treated with mustard gas no longer
supports the growth of the chick or rat. This is due to the inactivation
of certain essential amino acids (Kinsey and Grant, 1946).

Mutation-inducing chemicals may be encountered by fungi under
natural conditions. It was shown that toxic metabolic products of
Bacillus mesentericus affected the production of mutants by certain strains
of Helminthosporium sativum (Christensen and Davis, 1940). The
filtrate of B. mesentericus cultures induced sectoring of H. sativum. These
mutants differed from the parent in morphology, pathogenicity, and other
physiological characters.

Among the chemical compounds which induce mutations in fungi, the
action of nitrous acid has been especially studied (Thorn and Steinberg,
1939; Steinberg and Thom, 1940, 1942). Mutants of Aspergillus niger,
A. amstelodami, A. variecolor, A. fumigatus, A. fischeri, A. flavus, A.
alliaceus, and A. nidulans w^ere produced with ease by growing fungi on
mannitol-nitrite medium. Some of these mutants w^ere stable in culture
for over 20 years. In addition to morphological changes, these nitrous
acid-induced mutants were characterized by reduced ability to sporulate
and other physiological changes, especially ability to utilize certain amino
acids and a reduced rate of growth. It was postulated that nitrous acid
reacted with free amino groups of the proteins of the genes. Evidence
supporting this hypothesis was obtained when it w^as found that other
chemicals which also react with free amino groups (ninhydrin, chlora-
mine-T, potassium iodide, and hexamethylenetetramine) induced similar
mutations in A. niger.

Certain amino acids, when added to the medium on which these

VARIATION AND INHERITANCE 415

mutants were cultured, induced partial or complete reversion to the
morphology of the parent type. Of the single amino acids tested, only
lysine, cystine, /3-phenyl-/3-alanine, threonine, and vahne induced com-
plete reversion with the mutant of A. nigcr studied. Nicotinic acid,
lysine, and valine in combination gave the best results. Complete
reversion of a mutant of A. amstelodami was obtained only with a mixture
of lysine and threonine.

It should be pointed out that, although the mutants of Aspergillus
have been apparently stable for a long period of time, proof of the inherit-
ance of these characters by crossing the "mutants" with the parent type
is not available.

SUMMARY

Some knowledge of physiological variation and of the inheritance of
the underlying factors is of great importance to the investigator who
cultures fungi. The plant pathologist is greatly interested in knowing
the stability of the pathogenicity of the plant pathogenic fungi and in
learning whether the variations which he finds are apt to be permanent
or only temporary. Considerable effort has been made toward an under-
standing of the genetics of the smuts, the rusts, and the yeasts. A few
other fungi, such as Neurospora, Glomerella, and Phycomyces, have received
attention because they are particularly adapted to genetic studies.
However, the work up to the present leaves much to be desired regarding
the relation between genetics and physiology.

The genetics of sex and compatibility has been most frequently studied.
It is only natural that such is the case, for much of the understanding
of a fungus depends upon the completion of its sexual life cycle and a
clear knowledge of the various stages. Studies on the inheritance of
morphological characters are represented by numerous isolated reports
on characters more or less clearly defined. The reasons for the limited
number of investigations on the genetics of physiological characters are
numerous. Perhaps the principal reason is our meager knovvledge of the
intricate physiological processes of the fungi. The difficulty in obtaining
strains of the different sexes of suitable fungi which have clear-cut physio-
logical differences is great. The actual difficulty in carrying out the
physiological tests has no doubt discouraged work along this line.

Studies on the inheritance of induced deficiencies for a number of
vitamins and amino acids have indicated that such deficiencies may be
inherited in a simple Mendelian fashion. On the other hand, the inherit-
ance of partial thiamine deficiency in Lenzites trabea is complex and does
not follow the simple Mendelian pattern. Studies of the yeasts have
indicated that some physiological characters are inherited through, or
influenced by, the cytoplasm. This possibility should not be overlooked
in the filamentous fungi, where anastomoses of hyphae are common.

416 PHYSIOLOGY OF THE FUNGI

The basis for permanent variation in imperfect fungi (as well as in other
groups) is believed by many to be true mutation. There is circumstantial
evidence that many such sudden changes are mutations, but the inherit-
ance of such a change in the imperfect fungi cannot be proved. Other
variations in the imperfect fungi are explained on the basis of hetero-
caryosis. Separation of such nuclei into different spores would result in
the segregation into different mycelial or sporulating types. Thus, the
dual phenomenon would explain many of the variations in the imperfect
fungi.

The type of variation known as physiological specialization is of prac-
tical interest and importance among the pathogenic fungi. Pathogenicity
on a number of varieties of hosts offers a rigorous test to distinguish races
which differ perhaps only slightly in their physiology. The future of the
work dealing with physiology and genetics of the fungi lies principally
in the hands of competent, well-trained investigators who may be inter-
ested enough to spend much time and effort in this narrow field of investi-
gation. Many problems in this field are waiting to be solved.

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*GiLES, N. H., and E. Z. Lederberg: Induced reversions of biochemical mutants in

Neurospora crassa, Am. Jour. Botany 35: 150-157, 1948.
Hanna, W. F.: The odor of bunt spores. Phytopathology 22: 978-979, 1932.
Hansen, H. N.: The dual phenomenon in imperfect fungi, Mycologia 30: 442-455

1938.
*Hansen, H. N., and W. C. Snyder: The dual phenomenon and sex in Hypomyces

solanii. cucurbitae, Am. Jour. Botany 30: 419-422, 1943.
Harris, H. A.: Heterothallic antibiosis in Mucor racemosus, Mycologia 40: 347-351^

1948.
Hawker, L. E. : The nature of the accessory growth factors influencing growth and

fruiting of Melanospora destruens Shear and of some other fungi, Ann. Bot.

(N.S.) 3 : 657-676, 1939.
Hockenhull, D. J. D.: Mustard-gas mutations in Aspergillus nidulans. Nature 161 :

100, 1948.
Hockenhull, D. J. D.: The sulphur metaboHsm of mould fungi: The use of "bio-
chemical mutant" strains of Aspergillus nidulans in elucidating the biosynthesis

of cystine, Biochim. et Biophys. Acta 3: 326-335, 1949.
Houston, B. R.: Culture types and pathogenicity of isolates of Corticium solani,

Phytopathology 35: 371-393, 1945.
Johnson, T., and M. Newton: Mendelian inheritance of certain pathogenic charac-
ters of Puccinia graminis tritici, Can. Jour. Research 18: 599-611, 1940.

418 PHYSIOLOGY OF THE FUNGI

KiNSEY, V. E., and W. M. Grant: The reaction of mustard gas with proteins. II.

Biological assay of amino acids affected, Arch. Biochem. 10: 311-322, 1946.
Kreitlow, K. W.: Sclerotinia trifoliorum, a pathogen of ladino clover, Phytopath-

ologijZd: 158-166, 1949.
Leonian, L. H.: Studies in the variability and dissociations in the genus Fusarium,

Phijtopathologij 19: 753-868, 1929.
Leonian, L. H., and V. G. Lilly: The effects of vitamins on ten strains of Sac-

charoinyces cerevisiae, Am. Jour. Botany 29: 459-464, 1942.
Lilly, V. G., and H. L. Barnett: The inheritance of partial thiamine deficiency in

Lenzites trabea, Jour. Agr. Research 77: 287-300, 1948.
LiNDEGREN, C. C.: Mcndelian and cytoplasmic inheritance in yeasts, Ann. Missouri

Botan. Garden 32 : 107-123, 1945.
LiNDEGREN, C. C.'. Gcuetics of the fungi, Ann. Rev. Microbiol. 1948: 47-70.
LiNDEGREN, C. C.: The Yeast Cell, Its Genetics and Cytology, Educational Pub-
lishers, St. Louis, 1949.
McElroy, W. D., J. E. CusHiNG, and H. Miller: The induction of biochemical

mutations in Neurospora crassa by nitrogen mustard, Jour. Cellular Comp.

Physiol. 30: 331-346, 1947.
Macrae, R. : Interfertility studies and inheritance of luminosity in Panus stypticus,

Can. Jour. Research, Sec. C, 20: 411-434, 1942.
Martin, W. J.: Strains of Phytophthora infestans capable of surviving high tempera-
tures, Phytopathology 39: 14, 1949.
Nobles, M. K.: Conidial formation, mutation and hybridization in Peniophora

allescheri, Mycologia 27: 286-301, 1935.
RoBBiNS, W. J., and R. Ma: Biotin and the growth of Fusarium avenaceuni, Bull.

Torrey Botan. Club 68: 446-462, 1941.
RoBBiNS, W. J., and L McVeigh: The dual phenomenon and Trichophyton menta-

grophytes, Mycologia 41 : 128-140, 1949.
Ryan, F. J.: Back mutation and adaptation of nutritional mutants. Cold Spring

Harbor Symposia Quant. Biol. 11: 215-227, 1946.
Skoog, F. K., and C. C. Lindegren: Adaptation in a yeast unable to ferment

glucose. Jour. Bad. 53: 729-742, 1947.
Spiegelman, S. : in J. B. Sumner and K. Myrback (Editors), The Enzymes, Vol. I,

Academic Press, Inc., New York, 1950.
Spiegelman, S., C. C. Lindegren, and G. Lindegren: Maintenance and increase of

a genetic character by a substrate-cytoplasmic interaction in the absence of the

specific gene, Proc. Natl. Acad. Sci. U.S. 31 : 95-102, 1945.
Steinberg, R. A., and C. Thom: Mutations and reversions in reproductivity of

Aspergilli with nitrite, colchicine and d-lysine, Proc. Xatl. Acad. Sci. U.S. 26:

363-366, 1940.
Steinberg, R. A., and C. Thom: Reversions in morphology of nitrite-induced

"Mutants" of Aspergilli grown upon amino acids, Jour. Agr. Research 64:

645-652, 1942.
Thom, C, and K. B. Raper: A Manual of the Aspergilli, The WiUiams & Wilkins

Company, Baltimore, 1945.
Thom, C, and R. A. Steinberg: The chemical induction of genetic changes in fungi,

Proc. Natl. Acad. Sci. U.S. 25 : 329-335, 1939.
Thren, R.: Zur Entwicklungsphysiologie der Dikaryophase von Ustilago nuda

(Jensen) Kellerm. et Sw., Arch. Mikrobiol. 12 : 192-228, 1941.
Wolf, F. A.: A squash disease caused by Choanephora cucurbitarum, Jour. Agr.

Research B: 319-328, 1917.
Wolf, F. A., and F. T. Wolf: The Fungi, Vol, II, John Wiley & Sons, Inc., New

York, 1947.

SUGGESTED LABORATORY EXERCISES

Each of the laboratory exercises is designed to ilKistrate one or more
important principles regarding the phj'siological activities of the fungi.
Each represents a more or less complete unit or phase, but there is neces-
sarily some overlapping and duplication of the techniques. Laboratory
exercises requiring greenhouse space and living plants have been omitted.
This does not mean that an understanding of the cultural activities should
be the only aim of the laboratory work. Rather, it is believed that such
information regarding the nutritional and environmental requirements of
fungi in pure culture will aid in the better understanding of the behavior
of both parasitic and nonparasitic fungi in nature.

The laboratory exercises are planned so as to allow a high degree of
flexibility. The instructor may wish to omit, change, or add to some of
the suggested exercises to suit the equipment and other facilities available.
The exercises are outlined to require a minimum of laboratory equipment.
Any or all of the exercises may be expanded or shortened as desired for a
large or small number of students.

Each student should select one or more laboratory exercises in which
he has a particular interest. He may then be designated as the leader
of that exercise or exercises. His duties would include (1) the general
planning of the exercise in consultation ^^^th the instructor, (2) the direct-
ing of the execution of the exercise, (3) collecting and organization of the
data from other students, and (4) the writing of a comprehensive report
on the subject. References should be read and discussed in comparison
with the results of the experiments in the laboratory.

The supply of test fungi is almost unlimited. In most exercises each
student will use two or more species of fungi. The total number for the
class should be sufficiently large to emphasize the differences as well as
the similarities that exist among various fungi. In many instances our
knowledge is limited, and little or nothing has been published regarding
the physiology of some of the species used. In this sense, much of the
work done in the laboratory will be experimental and should be carried
out with all the care of a research problem. Fungi should be selected
so that both growth and sporulation may be studied in the same experi-
ment. In each exercise are listed a few fungi, some of which have been
used in our laboratory and have given quite satisfactory results. Other
species may be added or substituted.

Notes should be recorded in a full-sized permanent notebook reserved

419

420 PHYSIOLOGY OF THE FUNGI

only for the laboratory work in this course. The student should take
notes and record data so that anyone familiar with the subject could
organize the data and write an intelligent summary. Data taken during
the experiments are important and in themselves may be quite convincing
at that time, but data alone without organization and discussion of their
meaning are often sterile and soon forgotten.

The paper should be written as soon as possible after the termination
of the laboratory exercise. It is suggested that all students read the
excellent discussion of Riker (1946) on the preparation of manuscripts.

The student should become familiar with the microscopic character-
istics of the fungi used in the experiments. He should make frequent
microscopic observations during the experiment and look for microscopic
changes, such as abundance and maturity of spores. Contaminants are
often more easily recognized under the microscope. Drawings are often
desirable as records of differences in microscopic characteristics.

EXERCISE 1
General Laboratory Procedure

Exercise 1 is suitably carried out by the instructor as a demonstration
of general laboratory techniques. This affords the instructor an oppor-
tunity to discuss the details of various procedures and to acquaint the
students with the laboratory facilities. It is convenient to prepare suffi-
cient stock culture medium, e.g., medium 1 (Ex. 2), for growing the
inoculum required by the class for the next exercise.

General directions for preparing media. The culture medium should
be selected with the purpose of the experiment in mind. The precautions
to be observed may be elemental or elaborate, depending upon the pur-
pose for which the medium is to be used. The accuracy of one measure-
ment should be consistent with the accuracy of the others. The weights
of each constituent of a given lot of medium should be written in a note-
book. As each constituent is measured, make a check mark against this
constituent.

Never weigh chemicals directly on the balance pans. Use a clean piece
of paper or watch glass. The weights are placed on the right-hand pan
as you face the balance. Be sure the spatula is cleaned between weigh-
ings of different chemicals. If you remove more of a chemical than
necessary, discard the excess. (Material still on the spatula may be
returned to the stock bottle.) Keep the stock bottles closed. This
prevents the entrance of dust and atmospheric moisture. Malt extract,
yeast extract, and peptone quickly absorb water from the air, and when
these substances have done so, an intractable mass results.

For work of ordinary accuracy use a graduated cylinder for measuring
liquids. Volumetric glassware should be used for precise work. The

SUGGESTED LABORATORY EXERCISES 421

chemicals should be dissolved in less than the desired volume of water,
and after they are in solution, the medium should be made up to volume
by the addition of distilled water.

Constituents of media. The salts used in making media should be of
c.p. grade. These will generally contain enough of the micro essential
elements to satisfy the needs of most fungi. Iron, zinc, and manganese
should be added routinely to all synthetic media. It is convenient to
make up a solution containing the above micro essential elements in such
strength that 1 ml. of the master solution will contain 0.1 mg. Fe+ + +,
0.1 mg. Zn+ +, and 0.05 mg. Mn+ +. The following amounts of the given
salts have been found convenient to use: Fe(N03)3-9H20, 723.5 mg. ;
ZnS04-7H20, 439.8 mg.; MnS04-4H20, 203.0 mg. Dissolve these three
salts in 600 ml. of distilled water, add sufficient c.p. sulfuric acid to yield
a clear solution, and make up the volume to 1 liter with distilled water.
Use 2 ml. of this solution per liter of medium. Add the source of carbon
at the rate of 10 to 25 g. per liter, depending on the use of the medium.
Add the nitrogen source at the rate of 2 g. per liter, or the amount which
will give an amount of nitrogen equivalent to that furnished by 2g . of
anhydrous asparagine (0.425 g. of N).

The activated carbon used in Ex. 9 to absorb residual traces of vitamins
is a commercial preparation, Norit A (pharmaceutical grade). Use at
the rate of 5 g. per liter, or more if necessary. The vitamins used rou-
tinely are thiamine and biotin. Inositol and pyridoxine are also used in
Ex. 9. These vitamins may be purchased from JMerck and Co., Rahway,
N.J., and many other pharmaceutical houses. It is convenient to make
up master solutions of these four vitamins. Make the master solutions
in 20 per cent alcohol, store in 100-ml. volumetric flasks, and keep in a
refrigerator when not in use. It is convenient to make the master solu-
tions of thiamine and pyridoxine to contain 100 /xg per ml. (10 mg. per
100 ml.). The master solution of inositol contains 5 mg. per ml. Biotin
is used at the rate of 5 Mg per liter. Either the contents of an ampule may
be made up, or a microbalance used to weigh this vitamin. A master
solution containing either 1 or 5 jug per ml. is convenient.

For a semisolid medium, agar is commonly used at the rate of 20 g.
per liter. Agar is not a pure compound, and its use introduces various
unsuspected constituents into media. No medium may be said to be of
known composition if agar is used, although such media are very useful.
Some of the impurities in agar may be removed by leaching with aqueous
pyridine solution. Place 1 lb. of agar in a 6-liter flask, add 5 liters of
distilled water and 500 ml. of pyridine. Allow to stand 24 hr. Insert
a piece of 6-mm. glass tubing of sufficient length to admit air to
the flask, tie a piece of cheesecloth over the neck of the flask, invert the
flask, and allow the pyridine solution to drain. Wash the agar three

422 PHYSIOLOGY OF THE FUNGI

times with distilled water. Wash the agar twice with 95 per cent alcohol,
allowing the alcohol to stand on agar overnight before draining. Dry
the agar in thin layers between cheesecloth. This procedure takes about
10 days (see Robbins, 1939). In some instances the agar and other con-
stituents of the medium may be autoclaved separately and the two solu-
tions mixed, using antiseptic precautions. This should be done when it
is required to have a very acidic agar medium. A known amount of
sterile acid may be added to the agar medium after sterilization.

pH or reaction of the medium. See Chap. 8 for a discussion of pH.
An approximate method of determining pH is sufficiently accurate for
many purposes. On a white porcelain spot plate place one drop of
Hellige (or other) wide-range indicator in each depression. Have the
drops of indicator of equal size. Then add one drop of the medium to
a drop of indicator in one of the depressions of the spot plate. The color
of the mixed drops indicates the pH of the medium. Thus, red indicates
a pH in the neighborhood of 4, light green 7, purple 10. Standard buffers
(solutions of known pH) may be provided so that the student may have
standards with which to make comparisons.

The buffers used in testing pH are most conveniently made from buffer
tablets (Coleman). Dissolve one tablet in 100 ml. of distilled water.
Add a crystal of thymol as a preservative. Thymol aids in preventing
contamination and does not appreciably affect the pH of the buffer. It
is convenient to store the buffers in brown-glass dropping bottles fitted
with pipettes.

Unless otherwise specified, media used in the laboratory should be
adjusted to pH 6 before autoclaving. This may be done by the use of
the spot plate, adding a drop of pH 6 buffer to a drop of indicator. This
is the standard color to which the media should be adjusted. Add either
QN NaOH or QN HCl to the medium until the color produced by one drop
of medium matches the color produced by the standard buffer. Always
agitate the medium after each addition of acid or base and then test the
pH. The use of concentrated acid and alkali is recommended so that
dilution of the medium may be avoided. More precise methods of
measuring pH may be used if desired.

Autoclaving usually lowers the pH of a medium. In general, this
change will not be great, but the student should never assume that the
pH will remain unchanged in autoclaving.

Sterilization of media and glassware. Except in special instances, the
autoclave may be used to sterilize both media and glassware. Fifteen
minutes at 15 lb. steam pressure is adequate for test tubes and flasks
which do not contain over 150 ml. of medium. Larger lots of media
should be autoclaved 20 min. at 15 lb. steam pressure. Petri dishes may
be sterilized 20 min. in the autoclave. It is convenient to wrap two Petri

SUGGESTED LABORATORY EXERCISES 423

dishes in a paper towel or several in a paper bag before placing in the
autoclave. This wrapping should remain on the Petri dishes until they
are used, to prevent contamination. Do not remove wrapped glassware
from the autoclave until several minutes after the pressure is down.
Pipettes should be wrapped and placed in pipette cases. Sterilize in the
same way as Petri dishes.

Use either a water bath or the autoclave for melting agar. Never melt
agar in or sterilize flasks which are more than half full. Test tubes should
not be more than one-fourth full. The reason for this lies in the fact
that the autoclave cools quicker than the medium. This leaves the
medium superheated, and under this condition it is likely to boil violently.
Never remove a flask of melted agar from the autoclave as soon as the
pressure is down. Agitation may cause violent boiling. Your instructor
will give you full instructions for operating the autoclave. Follow these
instructions carefully.

On handling cultures. It will be necessary for each student to main-
tain the identity of his cultures. This may be done by name or by stock-
culture number. Each medium used in the course will receive a number.
If a medium is used more than once, it will be given another number.
The composition of each medium should be entered in the laboratory
notebook. The name of the fungus (or stock-culture number) and the
number of the medium should be also written on each culture vessel.
The date of inoculation and the kind of inoculum used should be entered
in the notebook. It is convenient to fasten together duplicate or tripli-
cate cultures in test tubes with a rubber band.

Preservation of stock cultures. The maintenance of a stock culture
collection of filamentous fungi for class use is highly desirable. Such a
collection need not be extensive but should include a sufficient number of
selected species of known phj^siological reaction and any others which
may be desired for general use. The method of preserving cultures in
our laboratory has proved quite satisfactory when frequent transfers are
needed for research or class use. Test tubes with constricted tops and
plastic screw caps are used. Malt extract or any other suitable agar may
be used. After inoculation the tubes are allowed to remain at room tem-
perature for a few days until the inoculum starts to grow. Then the caps
are screwed down tightly and the cultures stored at 5 to 10°C. Most
species continue to grow slowly, and under these conditions the tube?
remain free from contamination and the agar dries out very slowly. Thi.=
method also excludes mites. Some vegetative cultures have remained
viable for a period of more than 2 years without being transferred. How-
ever, it is suggested that all cultures be transferred every year, and the
entire stock should be looked over carefully every few months, as some
species may require more frequent transfers. The first transfer from

424 PHYSIOLOGY OF THE FUNGI

stock culture should be to another stock-culture tube, and the old tube
should be kept until the new culture begins to grow free from contamina-
tion. Other methods of storing stock cultures of fungi are described by
Greene and Fred (1934), Thom and Raper (1945), Fennell et al. (1950),
and Buell and Weston (1947).

Methods of inoculation. It is customary to use a bit of mycelium from
a growing culture to inoculate fresh media. For ordinary uses this is
satisfactory, if only a few cultures are to inoculated at a time and no
special precautions need be taken. Some fungi produce a tough mat of
mycelium difficult to cut with a needle. Often this can be overcome by
growing the mycelium for inoculum on an agar medium quite low in
sugar. A small cork borer may be used for cutting out uniform disks of
mycelium and agar from Petri dishes.

Spores alone may be transferred by a dry needle, or they may be
suspended in water and inoculated by use of a loop or a sterile pipette
with a cotton plug at the upper end. The use of a pipette fitted w^ith a
small rubber bulb greatly decreases the inoculation time when many
cultures of the same fungus are made. It is preferable to use spores as
inoculum in studies of vitamins or micro elements, where none of the
previous medium should be added.

Nonsporulating mycelium may be fragmented by placing it with about
50 ml. water in a sterile Waring Blendor jar for about 30 sec. Either
agar or liquid medium may be used if the addition of the medium is of no
consequence. In vitamin studies the mycelium may be grown in liquid
medium and, w^hen ready for use, w'ashed in sterile distilled water and
fragmented in the Blendor. Either a loop or a pipette may be used to
dispense the mycelial suspension.

Methods of obtainmg single-spore cultures. In certain physiological
studies it is desirable to use single-spore cultures. These may be obtained
by a number of different methods. A review of the literature on these
techniques has been given by Hildebrand (1938). Other modified tech-
niques are described by Georg (1947) and Thom and Raper (1945).
Still another modification may be worthy of brief mention. In this
laboratory we have used a specially prepared small sewing needle as a
tool for picking out single germinated spores. The eye of the needle is
rounded and the thick metal portion filed down, making a rather thin
edge for cutting agar. The pointed end is fastened in a convenient
holder, and the needle bent in such a way that, when held over a Petri
dish, the eye portion will be parallel with the surface of the medium so
that it can be pushed straight down into the agar. An isolated, germi-
nated spore is located on a dilution plate by use of a microscope. The
needle is then held in place under the objective so that the spore is visible
through the eye of the needle. The eye is pressed down around the spore

SUGGESTED LABORATORY EXERCISES 425

and is lifted up with a bit of agar and the germinated spore. Another
needle may then be used to transfer the bit of agar and spore to a tube or
plate. This method requires a steady hand but has the advantage of
being rapid and simple. It may be employed with high magnification
of the stereoscopic microscope or the low-power objective of the com-
pound microscope.

EXERCISE 2

The Influence of Temperature on Growth and Sporulation

This exercise is outlined to illustrate three main points: (1) the general
effects of different temperatures upon growth; (2) the approximate
optimum temperature for growth of a few fungi ; (3) that the temperature
limits for sporulation are narrower than those for vegetative growth.
Inasmuch as the composition of the medium may influence temperature
limits, only one medium should be used in this experiment.

Mediimi

1. Malt extract, 20 g.; j^east extract, 2 g. ; agar, 20 g.; distilled water, 1,000 ml.

Adjust the pH, if necessary, to approximately 6, and autoclave. Pour
into sterile Petri dishes, about 20 ml. per plate, and inoculate them at the
center. Inoculate plates in duplicate or in triplicate for each condition.
Incubate the plates at a range of temperatures with con^^enient intervals.
The range and the exact temperatures used will depend upon the facilities
available. Suggested temperatures are 10, 15, 20, 25, 30, 35, and 40°C.
Since light affects some of the species listed below, it is desirable to have
the cultures illuminated for a part of each day.

Records. Observations of growth should be made daily or at least
every 2 days. For the purpose of this exercise the radial extension of the
mycelium may be used as a measurement of growth. This usually can be
easily measured by placing a rule (preferable one calibrated in millimeters)
on the bottom of the Petri dish and looking through it towai'd a light. In
order to have an accurate measure of the effect of different temperatures,
the cultures must be compared before the mycelium reaches the edge of
the Petri dish. A more accurate measure of growth may be obtained by
determining the dry weight of mycelium grown in liquid medium. Rec-
ords may be made in table form for each fungus, giving the days of incuba-
tion and the diameter of the colony. The average daily radial extension
of the mycelium may then be calculated for each temperature. The
amount of sporulation should likewise be recorded each time.

List of test fungi: Alternaria sp., Aspergillus rugidosus, Botryis cinerea,
Cephalothecium roseum., Ceratosiomella fimhriala, Choanephora cucur-
hitarum, Glomerella cingulata, Guignardia hidwellii, Monilinia fructicola,
Penicillium expansum, Phytophthora infestans, Sclerotinia sclcrotiorum,
Septoria nodorum.

426 PHYSIOLOGY OF THE FUNGI

EXERCISE 3
The Influence of Light on Growth and Sporulation

This experiment is designed to illustrate the variable effects of visible
and ultraviolet light, particularly upon reproduction of some common
fungi. Exposure to light is essential to spore formation in some fungi,
while other fungi may sporulate abundantly in total darkness. For a
more complete discussion, see Chaps. 3 and 15.

Media

2. Same as medium 1

3. .\ny semisynthetic medium, such as the basal medium in Ex. 4

Adjust the pH of the media to approximately 6.0, autoclave, and pour
into sterile Petri dishes. Inoculate plates in triplicate for each condition.
Place the inoculated plates under the following conditions:

A. Continuous total darkness at 25°C.

B. Continuous artificial light at 25°C.

C. Alternating light and darkness at 25°C.

D. Same as C, but expose to ultraviolet three times for 2 min each time, at intervals
of 2 days

The exposure to ultraviolet light should be made in an inoculating
chamber, with the lids of the dishes removed, at a distance of approxi-
mately 10 to 12 in. from the source (the G.E. germicidal lamp is quite
satisfactory). Wearing of spectacles or sunglasses will protect the eyes
from the ultraviolet rays. The first exposure should be made when the
fungus colony is approximately 1 in. in diameter. Subsequent exposures
should be timed so that the last one is made before the mycelium reaches
the edge of the plate. With rapidly growing species, the interval between
exposures may be shortened. At the time of each exposure, use a wax
pencil to outline the extent of the mycelium by marking the bottom of the
Petri dish. The plates kept in total darkness should be wrapped in paper
or stored in a lighttight cardboard box. Examination of most fungi
should be made after about 7 days. The rapidly growing species should
be placed in a separate box which can be opened earlier.

List of test fungi: Botrytis cinerea, Cephalothecium roseum, Cerato-
stomella fimbriata, Choanephora cucurhitarum, Dendrophoma obscurans,
Endothia parasitica, Moniliniafructicola, Neocosmopara vasinfecta,Penicil-
lium expansum, Septoria nodorum, Trichoderma lignorum.

EXERCISE 4
The Effect of the Carbon Source on Growth and Sporulation

This exercise is designed to show that fungi differ in their ability to
utilize certain compounds as a source of carbon. For discussion of this

SUGGESTED LABORATORY EXERCISES 427

topic see Chaps. 7 and 14. In this study, the nitrogen source in the
media should be simple and available to as many fungi as possible. For
this purpose, asparagine is quite satisfactory.

Malt extract-yeast extract agar may be used as a standard controi
medium, since this is an excellent natural medium for most fungi. If
desired, any other standard natural medium may be used as a control
medium. All other media used in the exercise will have the same basal
composition, with the carbon source as the only variable. Adjust the
pH of all media to approximately 6.0 before autoclaving.

Basal Semisynthetic Medium

Carbon source 10 g.

Asparagine 2 g.

KH2PO4 1 g.

MgSOrTH.0 0.5 g.

Fe+ + + 0.2 mg.

Zn++ 0.2 mg.

Mn++ 0.1 mg.

Biotin 5 Mg

Thiamine 100 ^g

Distilled water to make 1 liter

Agar (for solid media) 20 g.

For the most accurate measure of growth, liquid media should be used,
so that the mycelium may be filtered off, washed, dried, and weighed.
However, agar media are often more satisfactory for reproduction. It is
suggested that this exercise be carried out on agar slants in test tubes.

Media

4. Same as medium 1

5. Glucose. Media 5 to 13 will all contain the basal medium above.

6. Sucrose 7. Sorbose
8. Lactose 9. Maltose

10. Galactose 11. Starch

12. Cellulosei 13. No sugar

Each student should select two or more species of fungi and inoculate
with each fungus three tubes of each of the 10 media listed above. Incu-
bate at 25°C., or at room temperature. Use for inoculum spores or small
bits of mycelium with as little agar as possible.

Records. The student will be responsible for taking notes or data on
the growth and sporulation of the fungi he selects, but he should follow
the form suggested by the leader of the exercise. He should also observe
the results of students who use other species. Records on growth may be
made at the end of 3 to 7 days, depending upon the fungus, while a greater
time should be allowed before making final records on sporulation. For
most purposes a record of the relative amount of growth or sporulation,

' Use good grade of filter paper, add water, and cut to a pulp in a Waring Blendor.

428 PHYSIOLOGY OF THE FUNGI

when compared with that on a control medium, is cjuite satisfactory.
Thus, if the growth and sporulation (if any) of each fungus on medium 4
are arbitrarily given the values of 4 + , the estimated abundance on other
media may be designated as greater or less than 4, as the case may be.
Such a rough method has been found satisfactory for illustrating principles
and determining the availability of carbon sources utilized by the fungi.
If a more accurate measurement is desired, liquid media should be used
and dry weights of the mycelia obtained. Observations and records
should be made on any other characteristics which are affected by changes
in carbon source.

List of test fungi: Aspergillus rugulosus, Ceratostomella fimbriata,
Dendrophoma obsmrans, Endothia parasitica, Glomerella cingulata, Guig-
nardia bidwellii, Melanospora sp., Monilinia friicticola, Phycomyces
blakesleeanus, Pleurage curvicolla, Sordaria Jimicola, Sphaeropsis malonan,

Ustilago striiformis.

EXERCISE 5

The Effect of the Nitrogen Source on Growth and Sporulation

The purpose of this exercise is to illustrate the utilization of different
sources of nitrogen by different fungi. This exercise should follow soon
after Ex. 4, and the procedure should be the same. The use of some of
the same test fungi in this exercise should emphasize the importance of a
suitable semisynthetic medium for growth and reproduction. In this
case the carbon source (glucose) shall be kept constant and the nitrogen
source varied with each medium. The other constituents of the basal
medium will be the same as listed under Ex. 4. Medium 1 may again
be used as a control, but if a different lot is made, it must carry a new
number.

Media

14.

Malt extract-yeast

extract

15.

Potassium nitrate

16.

Ammonium sulfate

17.

Ammonium tartrate

18.

Asparagine

19.

Glutamic acid

20.

Glycine

21.

Urea

22.

Casein hydrolysate

23.

No nitrogen

The amount of nitrogen should be kept constant. The weights of the
compounds used should be calculated to contain a weight of nitrogen
equivalent to that in 2 g. of asparagine.

EXERCISE 6
Special Nutritional Conditions Which Influence Growth and Sporulation

The two previous exercises have dealt mainly with the effect of the
constituents of the medium on a qualitative basis (see Chaps. 3 and 14).
This exercise is outlined to emphasize some of the effects of quantitative

SUGGESTED LABORATORY EXERCISES 429

differences in media. This ma}^ be illustrated by altering the concentra-
tion of one or more components of the medium. It is suggested that this
exercise be carried out in Petri dishes, or in flasks if liquid media are used.
Media: This exercise may be divided into four parts based upon the
variations in media.

A. Dilutions of the entire medium. Either liquid or agar media may be
used.

24. Basal medium, containing asparagine and 20 g. glucose per liter

25. Medium 24 diluted to one-half strength

26. Medium 24 diluted to one-fourth strength

27. Medium 24 diluted to one-sixteenth strength

28. Medium 24 diluted to one sixty-fourth strength

B. Varying concentrations of sugar. Use either liquid or agar media.
The same controls as in A may be used, if the same fungi are tested.

29. Basal medium, with 40 g. glucose

30. Basal medium, with 10 g. glucose

31. Basal medium, with 5 g. glucose

32. Basal medium, with 2 g. glucose

C. Change in medium during incubation. Use liquid media in flasks
(15 ml. in 125-ml. Erlenmeyer flasks, or 25 ml. in 250-ml. flasks).

33. Basal medium, same as medium 24 above. Inoculate 10 flasks; after growth is

near maximum (see instructor), separate the flasks into three groups.
a. Replace old medium with fresh medium
h. Replace medium with sterile distilled water
c. Leave as control

D. Different natural products as media. Use as agar media.

34. V-8 juice (diluted to one-half strength)

35. Potato extract (200 g. potatoes per liter)

36. Malt extract (20 g. per liter)

37. Stems of bean, pea, etc., in water agar

Each student should select one fungus for use in this exercise. It is
suggested that the fungi used should ordinarily produce considerable
mycelium before fruiting.

Records; Careful notes must be taken regarding time of appearance
of fruit bodies and spores. The amount of growth and sporulation may
be compared to that on medium 24. Consult the leader or your instruc-
tor for further details on recording data.

List of test fungi: Aspergillus riigidosus, Ceratostomella Jimhriata,
Choanephora cucurhitarum, Endothia parasitica, Glomerclla cingulata,
Guignardia hidwellii, Helminthosporium victoriae, Melanospora sp., Moni-
linia fructicola, N eocosmopara vasinfecta, Phoma betae, Sordaria fimicola,
and Sphaeropsis malorum.

430 PHYSIOLOGY OF THE FUNGI

EXERCISE 7
The Influence of Hydrogen-ion Concentration on Growth and Sporulation

This experiment is outlined to demonstrate (1) that the pH require-
ments for optimum growth and reproduction vary with the different
species of fungi, (2) that the pH of the culture medium changes during the
growth of the fungus, (3) the techniques by which pH changes may be
followed during growth, (4) that the pH at the time of sporulation may be
considerably different from that during most rapid growth, (5) that the
pH changes are also influenced by the composition of the medium. For a
discussion of pH, see Chap. 8. The pH of liquid media is more easily
tested than that of solid media by colorimetric methods; hence liquid
media should be used in this exercise. Twenty-five milliliters of medium
in a 250-ml. Erlenmeyer flask gives rapid, even growth of many fungi and
has been found to be quite satisfactory. Two different media are given
below, each set at four different pH values. For convenience each is
given a separate number.

Media

38. Glucose-asparagine (otherwise, basal medium as given in Ex. 4), pH 3.0

39. As above, pH 4.0

40. As above, pH 6.0

41. As above, pH 8.0

42. Sucrose-nitrate (otherwise, basal medium as given in Ex. 4), pH 3.0

43. As above, pH 4.0

44. As above, pH 6.0

45. As above, pH 8.0

Prepare these media, adjust the pH of each, and distribute to flasks
before autoclaving. Then use one flask of each medium to determine the
pH after autoclaving. This value should be considered the "initial pH."
Each student should select one fungus and inoculate eight flasks of each
medium listed above. Incubate the flasks at 25°C. or at room tempera-
ture. The pH of the culture filtrate should be determined at three differ-
ent times during the period of active vegetative growth (about the fourth
and eighth days) and at about the time or shortly after maximum growth
is reached (sporulation of some fungi will occur at this time). Duplicate
cultures should be used for each determination. See your instructor
regarding the method of determining pH of culture medium. The
sucrose-nitrate media may not be favorable for the growth of some of the
fvmgi selected for use in this exercise.

Records. The relative amounts of growth should be recorded at the
time of each pH determination. If more accurate growth measurements
are desired, the mycelium can be dried and weighed. Also record the
time of the earliest sporulation and the amount at subsequent intervals.

SUGGESTED LABORATORY EXERCISES 431

Consult the leader of the exercise or the instructor as to when the experi-
ment should be terminated. A portion of these data may be presented
in the form of a graph, plotting changes in pH against time for each
fungus and each medium.

List of test fungi: Aspergillus rugulosus, Cephalofhecium roseum, Cerato-
stomella fimhriata, Glomerella cingulata, Monilinia fructicola, Neocosmo-
para vasinfecta, Penicillium spiculosporum, Penicillium expansum , Sordaria
fimicola, Sphaeropsis malorum, Phycomyces blakesleeanus (plus and
minus).

EXERCISE 8

Methods of Inoculating Agar Media and Their Effect upon Growth

and Sporulation

This is a brief and simple exercise, but it is outlined to demonstrate a
principle which seems to be fundamental, at least for certain fungi. The
most common way of inoculating agar media is to place a bit of actively
growing mycelium or a few spores at the center of the medium surface.
For most purposes this is entirely satisfactory, but in special cases other
methods may be used. A drop of spore suspension or of finely cut
mycelium may be placed at the center of the agar plate, or the entire
surface may be flooded with heavy spore suspension or suspension of cut
mycelium. The mycelium may be fragmented by cutting in a Waring
Blendor in 50 ml. of sterile water for 30 sec. to 1 min.

For this exercise it is suggested that Petri dishes containing glucose-
asparagine-sucrose agar (such as the basal medium in Ex. 4) be used.
Fungi which ordinarily produce considerable mycelial growth before
fruiting abundantly may give the best results and will provide spore-free
inoculum when cultures are young. Some pycnidium-producing species
should be included.

Each student should choose one or more fungi and inoculate plates in
triplicate by the following methods:

A. A bit of mycelium placed at the center

B. A few spores transferred by a needle placed at the center

C. A drop of suspension of cut mycelium placed at the center.

D. A drop of spore suspension placed at the center.

E. Flooding the entire plate with suspension of cut mycelium

F. Flooding the entire plate with a heavy spore suspension

G. Diluting the spore suspension 1/1,000 and flooding the plate

Observe the cultures daily and note the abundance of vegetative growth
and the time and abundance of sporulation in each case.

List of test fungi: Alternaria sp., Ceratostomella fimhriata, Dendrophoma
ohscurans, Endothia parasitica, Fusarium sp., Glomerella cingulata, Guig-
nardia bidwellii, Helminthosporium sativum, Monilinia fructicola, Neo-

432 PHYSIOLOGY OF THE FUNGI

cosmopara vasinfecta, Phoma bctae, Septoria nodorum, Sordaria fimicoia,
Sphaeropsis malorum.

EXERCISE 9
Vitamin Deficiencies in the Fungi

This exercise is oiithned to demonstrate (a) vitamin deficiencies in the
filamentous fungi, ih) the differences in the needs of the different species
of fungi, (c) the techniques used to determine these deficiencies. For a
discussion of vitamin deficiencies and lists of vitamin-deficient fungi, see
Chap. 9 and Robbins and Kavanagh (1942).

In studying the vitamin requirements of the fungi, great care must be
taken to use glassware and chemicals which are free from vitamins. The
glucose-asparagine medium has the advantage of being a suitable source
of carbon and nitrogen for most fungi. Casein hydrolj'sate may be used
in the place of asparagine. In its preparation, the medium should be
boiled with activated charcoal (Norit, 5 g. per liter), to remove any
vitamins present, and filtered. Thus, the medium is "essentially free"
of vitamins. Contamination may occur from dust, cotton fibers from the
plug, dirty glassware, etc. Micro essential elements and vitamins must
be added after this treatment.

Media. It is best to use litiuid media for this exercise so the mycelium
can be dried and weighed.

46. Basal glucose-asparagine (vitamin-free)

47. As above, with thiamine (100 yug per liter)

48. As above, with biotin (5 ng per liter)

49. As above, with thiamine and biotin

50. As above, with thiamine, biotin, inositol (5 mg. per liter), and pyridoxine (100 pg
per liter)

Adjust the pH to 6.0 and distribute to tubes or flasks (25 ml. per 250-ml.
flasks or 15 ml. per 125-ml. flasks) before autoclaving.

A. Screening test for vitamin deficiencies. Simple screening tests to
determine roughly the deficiencies of fungi may be carried out in either
liquid or purified-agar (see Ex. 1) media. Test tubes may be used for
agar media, but Erlenmeyer flasks are suggested for liquid media. The
student should select four or five species from the stock-culture collection,
and inoculate tubes or flasks of each of the above media, in triplicate,
with each species. Either spores or a small bit of mycelium may be used
as inoculum. Daily observations and records of growth should be made.
Visual estimates of relative amounts of growth are sufficiently accurate
to detect most deficiencies. If liquid media are used, the mycelium may
be weighed.

B. Growth curves of vitamin-deficient fungi. Inoculate 10 flasks each
of media 46 to 50 with a filamentous fungus shown in part ^4. to be defi-

SUGGESTED LABORATORY EXERCISES 433

cient. The fungus will be harvested at intervals and the amount of
growth determined by obtaining the dry weight of the mycelium. If
there is sufficient growth in the flasks, the first harvest should be made
after 3 or 4 days.

Harvesting of the mycelium is accomplished by filtering the medium
through a fine cloth and washing the mycelium with distilled water.
Harvest duplicate cultures at each time. The mycelium is then trans-
ferred to small aluminum pans of known weight, dried for 12 to 24 hr. at
90°C., and weighed. The subsequent harvests should be made at inter-
vals of 2 to 4 days, depending upon the growth rate of the fungus used.
A convenient method of presenting the results is in the form of a graph,
ploting time against weight of mycelium for each of the four media used.

EXERCISE 10
The Influence of Vitamin Concentration on Growth and Sporulation

This exercise is designed to illustrate a few important principles regard-
ing the need for an adequate supply of vitamins in the medium for vita-
min-deficient fungi. Some of these points are (1) that vegetative growth
may be limited by an inadequate supply of the needed vitamins ; (2) that
higher concentrations of vitamins are needed for reproduction than for
vegetative growth; (3) that the absolute amount of a vitamin necessary
to induce reproduction varies with the amount of sugar in the medium;
(4) that the number of perithecia (or other reproductive structures) is
partiality dependent upon the concentration of the vitamins in the
medium ; (5) that fungi may be used in bioassays for the vitamin content
of various products.

The following experiments are suggested (these may be conducted as
demonstrations before the whole class, if desired) :

A. Thiamine concentration and growth; thiamine assay

Media

51. Basal glucose-asparagine medium, no vitamins

52. As above, but with 100 ng thiamine per liter

53. As above, 25 fig thiamine per liter

54. As above, 12.5 ^g thiamine per liter

55. As above, 6.25 Mg thiamine per liter

56. As above, 3.12 ng thiamine per liter

57. As above, 1.56 ng thiamine per liter

58. As above, add 0.5 g. cake flour per flask

59. As above, add 0.5 g. whole-wheat flour per flask

Other amounts of cake and whole-wheat flour may be used, or polished
and brown rice may be used instead.

Adiust the pH of the above media to 6.0 and distribute 25 ml. each to

434 PHYSIOLOGY OF THE FUNGI

250-ml. flasks. Inoculate four flasks of each medium with one strain of
Phycomyces blakesleeanus or Ceratostomella Jlmbriata.

B. Thiamine concentration and sporulation. Use media 51 to 53, 55,
and 57, but sohdify with 20 g. purified, vitamin-free agar (see Ex. 1) per
Hter. Pour into sterile Petri dishes (about 20 ml. each). Inoculate four
plates with Ceratostomella fimhriata (be sure to use ascospores or mycelium
producing perithecia), Phycomyces blakesleeanus (plus and minus strains
on opposite sides of plate), Choanephora ciicurhitarum, and Dendrophoma
obscurans. Incubate Phycomyces at 20 to 22°C., the others near 25°C.
Cultures of Choanephora must be adequately aerated and must receive
alternate light and darkness. Observe cultures of Ceratostomella for
production of perithecia, Phycomyces for zygospores, Choanephora for
conidial heads, and Dendrophoma for pycnidia.

Allow sufficient time for the above cultures to grow; then add one or
two drops of sterile (autoclaved) solution of thiamine to some of the
thiamine-starved, nonsporulating cultures (leave controls). Observe the
effects.

C. Effects of added thiamine on thiamine-starved mycelium

Media

60. Distilled water and purified agar

61. Distilled water, 100 ^g thiamine per liter, purified agar
Note: The addition of agar in media 60 and 61 is not essential.

From a thiamine-starved culture of C. fimbriata which has produced
no perithecia cut quarter-inch disks with a cork borer and place them in
tubes of media 60 and 61. If liquid media are used, make sure that the
disks of inoculum float on the surface. Observe the results after a few
days.

D. Relation of required thiamine to sugar in medium. To show that the
concentration of thiamine required for the production of perithecia
depends upon the amount of sugar in the medium, this short experiment
may be performed.

Media. Liquid glucose-asparagine

62. Glucose 25 g., thiamine 1 ng per liter

63. Glucose 2.5 g., thiamine 1 fig per liter

64. Glucose 0.25 g., thiamine 1 ng. per liter

Distribute the media in 250-ml. Erlenmeyer flasks, inoculate with C.
fimbriata, and incubate at 25°C. Observe the rate of growth, time of
perithecium formation, and relative number of perithecia formed in each
medium. Harvest and weigh the mycelium of each culture after peri-
thecia have formed.

SUGGESTED LABORATORY EXERCISES 435

E. Effects of hiotin starvation on a biotinr-deficient fungus

Media

65. Glucose-asparagine, purified agar; no bio tin

66. As above, 5 ng biotin per liter

67. As above, 1 ng. biotin per liter

68. As above, 0.5 Mg biotin per liter

69. As above, 0.1 ng biotin per liter

Pour into Petri dishes and inoculate each medium in tripHcate with
Sordaria fimicola. Observe the results after about 6, 8, and 10 days.
Note the amount of growth, the time of perithecium formation, and the
relative numbers of perithecia. Examine microscopically the perithecia
formed in the low concentrations of biotin, and look for deformed asci
and ascospores. Add a drop or two of sterile biotin solution to some of
the nonfruiting, biotin-starved cultures. Observe the effects in a few
days.

Records. Take full notes on all observations of the above experiments.
Write out a full explanation of the results with interpretations based
upon physiological processes in fungi (see Chap. 14).

EXERCISE 11
Factors Affecting Spore Germination

This exercise demonstrates the effects of nutrients, humidity, pH, and
temperature upon the time and percentage of germination of the spores
of some common fungi. While the germination of fungus spores may be
influenced by a number of factors, only a few of them can be easily studied
in the laboratory. See Chap. 16 for a discussion of factors which influ-
ence spore germination.

A. Effects of nutrients upon spore germination. Place filter paper in the
bottoms of Petri dishes. Cut two holes in the filter paper about }^ in.
in diameter and place a glass slide over these. Add water to moisten
the paper, and autoclave. Make up a spore suspension in media 70 and
71, and place drops of this suspension on the slide over the holes in the
paper, which must be kept moist w^ith sterile water. Incubate at 25°C.
After incubation the slide may be examined by placing the Petri dish on
the microscope and removing the lid.

Media

70. Distilled water

71. Distilled water plus 2 g. yeast extract per liter

72. Same as medium 71 but solidified with agar; adjust to pH 8.0

73. Same as medium 71, but pH 7.0

74. Same as medium 71, but pH 6.0

75. Same as medium 71, but pH 5.0

76. Same as medium 71, but pH 4.0

436 PHYSIOLOGY OF THE FUNGI

B. Effect of acidity. This experiment may be carried out in liquid
media 72 to 76, using drops of spore suspension as described above in
part A, or the media may be solidified with agar and Petri dishes used.
Observe the results at intervals up to 48 hr. Your records should include
the approximate time required for germination of the first spores and the
percentage of germination at each examination. The first appearance of
a germ tube may be considered as germination.

C. Effect of relative humidity. Place drops of spore suspension in dis-
tilled water on sterile glass slides to serve as controls. On three other
dry glass slides, place dry spores. Place these in desiccators as follows:
(1) spores in water and in desiccator which will maintain saturated atmos-
phere; (2) spores on dry slide in saturated atmosphere; (3) spores on dry
slide in desiccator with relative humidity at approximately 98 per cent;
(4) spores on dry slide in desiccator with relative humidity at approxi-
mately 92 per cent. To maintain 98 per cent humidity, use a 1.00 molal
solution of sucrose; for 92 per cent humidity use a saturated solution
of K2HPO4 (see Clayton, 1942). Open the desiccators after 24 to 30 hr.
and examine the spores for germination.

D. Effect of temperature. Use four Petri dishes with medium 74 or 75.
On each place three or four drops of spore suspension and mark these
spots on the bottom of the dish. Incubate these as follows: (1) in an
incubator at 30°C.; (2) at 25°C.; (3) in a refrigerator at 18°C.; (4) in a
refrigerator at 10°C.

Examination of spores. The time required for spores of the various
species to germinate under the usual conditions varies from 2 to 24 hr. or
more. For some fungi all the experiments in the exercise may be exam-
ined and compared after 12, 18, or 24 hr. One examination should be
made after 48 hr. For most species the experiments may be concluded
at this time.

List of test fungi: Alternaria sp., Cephaloihecium roseum, Choanephora
cucurhitarum, Glomerella cingulata, Guignardia hidwellii, Helminiho-
sporium sativum, Monilinia fructicola, Penicillium sp., Phytophthora
infestans.

EXERCISE 12
The Associative Effects among Fungi

Pure cultures of a single organism seldom exist in nature. Instead,
each organism is constantly exposed to a biotic as well as a physical
environment. As a result, there is usually competition between different
fungi and between fungi and other organisms in the same substrate,
particularly the soil. On the other hand, many organisms are benefited
by their association with others. Often the metabolic products of one
favor the growth of another.

SUGGESTED LABORATORY EXERCISES 437

If one desires to demonstrate these principles in the laboratory, pure
cultures must be used, and by combining two or more of these species in a
culture vessel, the associative effects may be studied. This exercise is
outlined to demonstrate the main types of associative reactions between
species of fungi in the laboratory under controlled conditions and to
show that a species may react differently in its association with different
fungi.

Media

77. Malt extract-yeast extract agar

78. Glucose-asparagine purified agar (vitamin-free)

79. Glucose-asparagine liquid (vitamin-free)

A. Each student should select three pairs of the fungi and test their
interaction on agar plates of media 77 and 78. Duplicate plates should
be inoculated for each condition. It is suggested that the two pairs of
fungi be inoculated on opposite halves of the agar plates 1 to 2 in. apart.
This will allow both fungi to make some growth before they come in
contact. Incubate all cultures at 25°C. Notes should be taken on about
the fifth day and the tenth day, and for most cultures the final observa-
tions may be made after 2 weeks. Carefully made sketches may add
greatly to the value and clarity of your notes.

The types of reactions may be grouped under (1) none, (2) stimulation,
(3) symbiotic, (4) antagonistic. Each type of reaction should be
explained on the basis of the present experiments.

B. Using vitamin-free liquid medium 79, inoculate flasks with Phyco-
viyces blakesleeanus and Sordaria fimicola separately and with both species
together. After a few days observe the results. This part of the exercise
may be conducted as a demonstration for the entire class.

List of test fungi: Actinomyces sp., Alternaria sp., Aspergillus rugulosus,
Botrytis sp., Cephalotheciimi roseum, Guignardia hidwellii, Helmintho-
sporium sativum, Monilia sp., Monilinia fructicola, Penicillium, chryso-
genum, Phycomyces blakesleeanus, Sordaria fimicola, Trichodermalignorum.

Suggested Demonstrations

In addition to the experiments outlined in the above exercises, the
following are suggested as demonstrations for the entire class. These
may be expanded into complete exercises for individual student
participation.

1. Need for micro elements for growth and sporulation. Steinberg (1919)
describes the procedure for preparing a medium essentially free of micro
elements to which the desired elements may be added. Use Aspergillus
niger as a test species. See Chap. 5 for other methods of removing micro
elements from media.

438 PHYSIOLOGY OF THE FUNGI

2. Influence of light on spore discharge. This experiment demonstrates
the phototrophic response of sporangiophores and perithecial beaks and
the discharge of the spores toward the source of Hght. For a discussion
of this subject, see Buller (1934), Use species of Piloholus, Sordaria, or
Pleurage.

3. Influence of aeration on sporulation. Choanephora cucurbitarum is
an excellent species to use in demonstrating the need for adequate aeration
for the production of conidia. Grow the fungus on agar in Petri dishes.
Some of the dishes may be sealed with Scotch tape, w^hile the lids of others
may be raised to permit free exchange of gases.

4. Longevity of spores. This may be designed as a long-time experi-
ment to determine the longevity of spores of several fungi under different
conditions of storage. It may be continued from year to year, tests for
the ability to germinate being made every few months.

5. Action of fungicides and fungistatic agents. These experiments
should be outlined to show the effectiveness of various agents in prevent-
ing spore germination. For references, see Chap. 11 and Horsfall (1945).
These agents include the action of sulfur, copper, 8-hydroxyquinoline, anti-
vitamins, and ultraviolet radiation.

6. Action of antibiotics against fungi. The specificity of the action of
some antibiotics is easily tested against growth or spore germination of
some common fungi by the use of penicylinders in agar plates flooded
with spores.

7. Inheritance of physiologic characters. (A) The inheritance of vita-
min or amino-acid deficiency may be demonstrated by crossing deficient
mutants of Neurospora with a self-sufficient strain (see Beadle, 1946, and
Chap. 18). {B) The inheritance of bioluminescence may be demon-
strated by crossing the North American and European strains of Panus
stypticus (see Macrae, 1942).

REFERENCES

Beadle, G. W.: Genes and the chemistry of the organism, Am. Scientist 34: 31-53,

1946.
BuELL, C, and W. H. Weston: Application of the mineral oil conservation method

to maintaining collections of fungus cultures, Am. Jour. Botany 34 : 555-561, 1947.
Buller, A. H. R.: Researches on Fungi, Vol. VI, Longmans, Roberts and Green,

London, 1934.
Clayton, C. N.: The germination of fungous spores in relation to controlled humidity,

Phytopathology 32: 921-943, 1942.
Fennell, D. I., K. B. Rapeb, and M. H. Flickinger: Further investigations on the

preservation of mold cultures, Mycologia 42 : 135-147, 1950.
Georg, L. K. : a simple and rapid method for obtaining monospore cultures of fungi,

Mycologia 39: 368-371, 1947.
Greene, H. C., and E. B. Fred: Maintenance of vigorous mold stock cultures, Ind.

Eng. Chem. 26: 1297-1298, 1934.

SUGGESTED LABORATORY EXERCISES 439

HiLDEBRAND, E. M.: Techniques for the isolation of single microorganisms, Bolan.

Rev. 4: 627-664, 1938.
HoRSFALL, J. G.: Fungicides and Their Action, Chronica Botanica Co., Waltham,

1945.
Macrae, R.: Interfertility studies and inheritance of luminosity in Panus stypticus,

Can. Jour. Research, Sec. C, 20: 411-434, 1942.
RiKER, A. J.: The preparation of manuscripts for Phytopathology, Phytopathology

36:953-977, 1946.
RoBBiNs, W. J.: Growth substances in agar, Am. Jour. Botany 26: 772-778, 1939.
RoBBiNs, W. J., and V. Kavanagh: Vitamin deficiencies of the filamentous fungi,

Botan. Rev. 8: 411-471, 1942.
Steinberg, R. A.: A study of some factors in the chemical stimulation of the growth

of Aspergillus niger, Am. Jour. Botany 6: 330-372, 1919.
Thom, C, and K. B. Raper: Manual of the Aspergilli, The Wilhams & Wilkins Com-
pany, Baltimore, 1945.

INDEX

Abrams, E., 222, 223
Absidia coerulea, 100

cylindrospora, 70, 100

dubia, 100

glauca, 100

orchidis, 100
Acetohader stiboxydans, 119
Achlya bisexualis, 326

conspicna, 92, 93

oblongata, 93

polyandra, 93

prolifera, 93
Aconiturn velatum, 159
Actinomyces, 286, 386, 437

lavendulae, 287

scabies, 373, 387
Adaptations, nutritional, 412-413
Adaptive enzymes, 59, 60, 412, 413
Adermin (see Pyridoxine)
Aegerita icebberi, 384
Aerobacter aerogenes, 138
Agar, chemical composition of, 14

purification of, 14, 421

vitamins in, 15
Agaricus campestris, 269, 318, 348

{See also Psalliota campestris)
Ajl, S. J., 138, 146
/3-Alanine, 198, 234, 239
.Albrecht, H., 104, 112
Albugo Candida, 360, 373
Aldoses, 117
Aleuria repanda, 346

vesiculosa, 346
Alexander, D. F., 284, 301
Allen, R. F., 373, 392, 395
Allomyces, 295, 327

carbon sources used by, 121
Allomyces arbiiscida, 121, 159, 308

cystogenus, 121
javanicus, 121

moniliformis, 121

441

Alternaria, 319, 327, 425, 431, 436, 437

oleracea, 261

solani, 127, 247, 248, 262, 319, 320, 356

tenuis, 69
Amanita, 292

chlorinosma, 292

mappa, 366

muscaria, 81, 292

pantherina, 292

phalloides, 292

porphyria, 292, 366

radicata, 292

rubescens, 366

spreta, 292

strobiliformis, 292

verna, 292

virosa, 292

toxin (see Phalloidin)
Ames, L. M., 222, 224, 404, 416
Amino acids, 105-110

antagonists of, 236-239

as carbon sources, 127

deamination of, 108

list of, 106
p-Aminobenzoic acid, 201-202

antagonists of, 227, 229-236

formula of p-aminobenzoate ion, 227
Ammonium nitrogen, list of fungi uti-
lizing, 100
Amylase, 47, 48, 50, 274
Andersen, A. L., 163, 169, 308, 309, 317,

335, 351, 353
Andersen, E. N., 348, 354
Anderson, A. K., 158, 169, 308, 335
Anderson, D. B., 37, 44
Anderson, H. W., 291, 302
-4nderson, P. J., 368, 369
Aneurine (see Thiamine)
Antagonists, metabolite, 226-240
Antibiotics, control of plant diseases by,
288
detection of, 284-285
as factor in resistance, 390
production of, 283-291

442

PHYSIOLOGY OF THE FUNGI

Antimetabolite, definition of, 22G
Anti vitamins, 229-23(3
Anwar, A. A., 286, 296
Aphanornyces, 93

camptostylus, 92
Apium graveolens, 377
Apodachyla brachynema, 127
Apoenzymes, 53, 228
Appling, J. W., 219, 223
Appressoria, 373
Arabinose, formula of, 123
Arcyria denudata, 367
Area Leao, A. E., 197, 203
Armillaria mellea, 99, 134, 160, 374, 387

shii-take, 269
Armstrong, E. F., 129, 146
Armstrong, J. I., 167, 168
Arnon, D. I., 21, 23, 73, 86
Arsenic, 110
Arthur, J. C, 372, 395
Aschersonia aleyrodis, 384
Ascobolus, 345, 346, 349, 353, 357

denudata, 99

leveillei, 99

rnagnificus, 346
Ascochyta nymphaeae, 311, 317

pisi, 99
Ascophanus carnens, 88, 319
Ashbya gossypii, 273
Ashley, J. N., 295, 296
Aspergillus, 9, 94, 99, 172, 271-275, 283,
288, 385, 401

alliaceus, 414

amstelodami, 414, 415

aureus, 279

awaniori, 279

carbonarius, 279

cinnamomeus, 279, 280

clavatus, 279, 286, 288

fischeri, 414

flavus, 70, 76, 78, 414

metabolic products, 272, 275, 283,

287, 288
spore germination of, 361, 362, 366

fumaricus, 279

jumigatus, 34, 76, 414

fuscus, 280

giganteus, 283

glaums, 37, 275, 279

herbariorurn, 318

itaconicus, 281

Aspergillus, nidulans, 93, 283, 414

niger, 10, 55, 92, 98, 103, 136, 140, 167,
194, 229-231, 248, 258, 260, 261,
294, 325-327, 360-362, 366, 414,
415, 437
nutrition of, carbon, 121-129
metallic-element, 65-83
nitrogen, 105
phosphorus, 94-96
organic acids produced by, 279, 280
resistance of onion to, 390, 391
spore germination of, 358, 359
use of, in assays, 217-222
vitamin synthesis by, 171, 172
oryzae, 74, 76, 88, 89, 103, 159, 274,
280, 283, 368
carbon sources utiUzed by, 121, 123-
125, 127, 129, 132, 134, 136
repens, 307, 308
rugulosus, 28, 220, 328, 329, 425, 428,

429, 431, 437
terreus, 70, 222, 282, 287, 401
variecolor, 414
wentii, 278
Assays, microbiological, for amino acids,
216-217
certain media used in, 210, 211
for essential elements, 217-219
general discussion of, 208-209
procedure, 209-214
for vitamins, 214-216
Assimilation, definition of, 87
Association, effect of, on growth, 219, 220
on sporulation, 327-328, 332-333
laboratory exercise, 436-437
Astbury, W. T., 110, 112
Atkin, L., 198, 204, 215, 223
Auernheimer, A. H., 219, 223
Aureomycin, 291

Autoclaving, effect of, on media, 16
Axelrod, A. E., 194, 204
Ayres, T. T., 328, 334
Azotobader, 98

chroococcum, 79, 218
vinlandii, 98

Bacillus mesentericus, 414
subtilis, 286
typhosujn, 229
weidmaniensis, 327

INDEX

443

BaUey, A., 316, 336
Bailey, J. H., 290, 296
Barker, B. T. B., 248, 263
Barnes, T. C, 88, 112
Barnett, H. L., 34, 38, 43, 44, 167, 169,
178, 181-184, 196, 204, 205, 232, 242,
307, 309, 310, 312, 313, 317, 319,
329-331, 334, 336, 400, 401, 408-410,
416, 418
Barratt, R. W., 259, 263
Barron, E. S. G., 57, 62, 96, 112, 251,

263, 264
Basidiohlus, 343, 353

ranarum, 100, 342
Basisporium gallarum, 27, 172, 365, 388
Basu, S. N., 329, 335
Battarrea, 307

Baumberger, J. P., 60, 61, 64
Bayliss, W. M., 54, 62
Beadle, G. W., 36, 161, 196, 197, 199,
202, 204, 207, 211, 215, 217, 223,
224, 401, 405-408, 416, 438
Behr, G., 68, 69, 85
Behrens, O. K., 289, 296
Bellamy, W. D., 290, 296
Benedek, T., 327, 334
Bennett, C. W., 79, 86, 100, 115
Bennett, I. G., 36, 43
1,4-Benzoqiunone formula, 257
Berberis, 387

thunbergii, 387
Berger, J., 51, 63
Bergmann, M., 51, 62
Bernhard, K., 104, 112
Bernhauer, K., 279, 296
Bertrand, D., 81, 83
Bertrand, G., 119, 146
Bessey, E. A., 375, 395
Biological substitution of elements, 68
Bioluminescence, 402, 403, 410
Biotin, 192-195

antagonists of, 233

formula of, 192

fungi deficient for, 192, 193

mode of action of, 194, 195

specificity of, 193, 194
Birkinshaw, J. H., 94, 112, 276, 283, 296
Bisby, G. R., 313, 334
Black, A., 212, 224

Blakeslea trispora, 102, 122, 124, 125, 133,
190

Blank, L. M., 76, 83

Blastocladia pringsheimii, 93, 102, 137,
156, 159, 197, 281

production of resistant sporangia by,
319

vitamin deficiencies of, 180
Blastodadiella simplex, 159
Blastomyces brasiliensis, 40

dermatitidis, 40
Block, R. J., 268, 296
Bock, H., 50, 63
Boletus, 293, 367

bovinus, 367

jdulis, 267

elegans, 367

flavidus, 367

granulatus, 367

luridus, 52, 292

hiteus, 367

miniato-olivaceus var. sensibilis, 293

satanus, 292

variegatus, 367

viscidus, 367
Boiling, D., 268, 296
Bonar, L., 309, 336
Bonner, D., 93, 112, 197, 204, 217, 223,

401, 408, 416
Bonner, J., 50, 62
Bonner, J. T., 358, 359, 369
Booer, J. R., 250, 263
Borchers, R., 273, 300
Bordeaux mixture, 247-248
Bortels, H., 79, 84
Bose, S. R., 319, 335
Botryotinia convoluta, 99, 311
Botrytis, 253-255, 314, 437

alia, 99, 172, 250, 261, 285

production of pectinase by, 376
resistance of onion to, 390, 391

cinerea, 37, 69, 99, 373, 425, 426
parasitism of, 376, 377
production of pectinase by, 377, 378
spore germination of, 360, 364-366

paeoniae, 247, 248
Bourquelot, E., 47, 62
Bovarnick, M. R., 197, 204
Boyle, A. M., 288, 296, 389, 395
Brand, E., 216, 223, 225
Brassica, 377
Bray, C. W., 269, 296

444

PHYSIOLOGY OF THE FUNGI

Brefeld, O., 6, 7, 9, 11, 22, 266, 296, 310,

335, 339, 353, 362, 370
biographical note, 6
Brenner, W., 107, 112
Brian, P. W., 97, 103, 104, 108, 112, 165,

168, 285, 287, 290
Briarea, 35

Brink, N. G., 273, 297
Brodie, H. J., 324, 335, 358, 370, 402, 416
Brooks, F. T., 382, 395
Brown, W., 364, 365, 370, 372, 373, 376,

387, 390, 395
Brown rot, fungi causing, 134, 135
Buchanan, R. E., 17, 22, 25, 43, 65, 84,

99, 112
Buchner, E., 46, 62, 277, 297
Buell, C, 424, 438
Buffer capacity of media, 155
Buffers, 152-156
Bull, H. B., 110, 112
Buller, A. H. R., 6, 7, 267, 297, 339, 341,

345-349, 351, 353, 408, 409, 416, 438
biographical note, 6
Bunker, H. J., 49, 64
Blinning, E., 55, 62, 167, 168
Burgert, I. A., 362, 370
Burk, D., 108, 112, 186, 204, 233, 241
Burkholder, P. R., 42, 43, 104, 112, 174,
180, 181, 186, 190, 192, 195, 197-199,

204, 272, 297, 400, 416
Buston, H. W., 329, 335
Butler, E. J., 381, 387-389, 395

Calam, C. T., 282, 297
Calcium, 79-81
Caldwell, R. M., 373, 395
Calfee, R. K., 77, 78, 85
Camp, W. G., 320, 335
Campbell, W. G., 134, 146
Canavanine, 239
Candida albicans, 42, 186, 287

flareri, 272

guilliermondi, 219, 272
Cantherella cibarius, 267
Cantino, E. C, 93, 102, 112, 137, 146,
156, 159, 168, 169, 180, 197, 204,
319, 335
Caputto, R., 140, 146

Carbon dioxide, effect of, on spore germi-
nation, 365
as metabolite, 136-138
Carbon sources, effect of, on sporulation,
321-325
laboratory exercise, 426-428
utilized by fungi, 116-138
Carbon utilization, 138-144
Carboxylase, 52
Caroselli, N. E., 379, 396
Carroll, W. R., 91, 92, 114
Carter, H. E., 291, 297
Catechol, 390

Cavallito, C. J., 290, 296, 297
Cellobiose formula, 130
Cellulase, 47

Cellulolytic fungi, 221-222
Cellulose, 134-135
Cephalosporium, 378
Cephalotheciuni roseum, 99, 425, 426, 431,

436, 437
Ceratostomella, 200, 293, 352, 378
anipullacea, 344

fimbriata, 31, 37, 38, 100, 101, 175, 178,
181, 190, 232, 345, 425, 426, 428,
429, 431, 434
factors affecting sporulation, 307,

329, 330
methods of measuring growth of, 30
ips, 34, 180, 190, 194, 200
inicrospora, 200
montium, 190, 200
multiannulata, 200

(See also Ophiostoma multiannula-
tum)
penicillata, 232
piceaperda, 200
pilifera, 34, 35, 200
pint, 190, 200, 229
pluriannulata, 200, 329
pseudotsugae, 200

ulmi, 100, 175, 200, 234, 260, 314, 345,
375
causes of wilting by, 378, 379
{See also Ophiostoma ulmi)
Cercospora apii, 99, 172
beiicola, 99, 172, 373
nicotianae, 80
Chaetocladium, 385
Chaetomium, 222, 345

INDEX

445

Chaetomium, cochlioides, 99

convolutum, 38, 99, 178, 190, 193, 232
dilution of medium and growtli of, 38

funicola, 96, 222

globosum, 99, 127, 172, 173, 222, 318
growth curve of, 173
Chain, E., 290, 297
Chalaropsis thielavioides, 232
Challenger, F., 111-113
Chan, S. Y., 319, 320, 336
Charles, J. H. V., 294, 297
CheldeKn, V. H., 199, 207, 232, 243
Cheo, P. C, 120, 146, 362, 370, 401, 416
Chilean Nitrate Educational Bureau, 71,

84
Chilton, S. J. P., 404, 417
ChIai7iydopus, 307
Chloromycetin, 291
Chlorosplenium aeruginosum, 293
Choanephora cucurbitarum, 33, 100, 190,
232, 401, 425, 426, 429, 434, 436, 438

factors affecting sporulation of, 309,
310, 312, 313, 321, 331, 332
Chona, B. L., 376, 377, 395
Christenberry, G. A., 312, 335, 401, 417
Christensen, J. J., 293, 297, 402, 410,

414, 417
Chrysophlydis endohiotica (see Synchy-

triuin endohioticum)
Ciboria acerina, 27
Cicinnobolus cesatii, 385
Citric acid, production of, 279-280
Citromyces, 275, 279

citricus, 279

glaber, 279

pfefferianus, 279

{See also Penicillium)
Cladosporium cucumerinum, 288

fulvum, 390
Clarke, H. T., 290, 297
Clausson-Kaas, N., 378, 397
Claviceps, 332

paspali, 293

purpurea, 291-293, 296, 304, 352
Clayton, C. N., 355, 358, 370, 436, 438
Clitocybe illudens, 293
Clostridium, 376

acetobutylicum, 201, 272, 273

pasteurianum, 79

septicum, 198
Cobalt, 81, 273

Cocarboxylase (see Thiamine pyrophos-
phate)
Coccomyces hiemalis, 304
Codehydrogenase, 197
Coemansia interrupta, 178, 190, 193
Coenzyme, 53
Coghill, R. D., 282, 284, 289, 297, 300,

301
Colletolrichum, 368

circinans, 390, 391

falcatum, 388

gossypii, 360

lagenarium, 99, 288, 356

lindemuthianum, 99, 160, 186, 196, 307,
309, 332, 374
resistance to, 391
spore germination, 364-366
CoUybia tuberosa, 99

velutipes, 99, 409
Colwell, C. A., 258, 263
Committee on the Standardization of

Fungicidal Tests, 262, 263
Conant, N. F., 333, 335, 386, 395
Concentration of nutrients, 17-20

effect of, on growth, 38, 39
on sporulation, 320-321, 330
Coniophora cerebeUa, 159
Coniothyrium, 316
Cook, E. S., 251, 263
Cook, M. T., 388, 390, 395
Coons, G. H., 307, 310, 311, 313, 317,

335
Copper, 76-77, 217
Coprinus, 310

comatus, 362

jimetarius, 362

lagopus, 102

micaceus, 362
Cordyceps militaris, 99, 172, 332, 384
Coriicium solani, 386, 401, 402

(See also Rhizodonia solani)
Coryell, C. D., 60, 62
Couch, J. N., 352, 353, 384, 385, 396
Craigie, J. H., 387, 397
Craterellus liitescens, 367
Cristol, S. J., 238, 241
Cronartiurn ribicola, 356, 368, 395
Crosier, W., 34, 43
Cryptococcus neoformans, 260, 287
Crystal violet, 259

446

PHYSIOLOGY OF THE FUNGI

Cultures, preservation of, 423-424
single-spore isolation, 424

Cunninghamella elegans, 70

Currie, J. N., 278, 283, 297

Curtin, T., 294, 297

Cury, A., 197, 203

Cyanide, effect of, on respiration, 56

Cyathus, 353
siercorius, 324
striatus, 100, 348

Cylindrosporium scoparium, 358

Cystine, biosynthesis of, 93

Cytophaga, 16

Cytospora, 368

Cytosporella mendax, 311

Daedalea quercina, 134

tinicolor, 351
Daldinia concenirica, 172
Dalphin, C, 191, 204
Darluca filum, 385
Das Gupta, S. N., 402, 417
Davis, A. R., 98, 113
Davis, F. R., 414, 417
Davis, J. B., 137, 146, 282, 298
Dawson, C. R., 52, 63, 77, 85
Day, D., 14, 22, 29, 43
Deamination, 107, 108
De Bary, A., 6, 7, 283, 343, 354, 376, 396

biographical note, 6
Debar ijomyces, 309
Decker, P., 386, 398
Dendrophoma obscurans, 99, 190, 426,

428, 431, 434
Denny, F. E., 317, 335
Desoxypyridoxine formula, 234
Desthiobiotin, antagonists of, 233

formula of, 193
Dianthus, 377
Diaporthe phaseolorum var. batatatis, 312,

316, 321, 324
Diastase (see Amylase)
Dichtyuchus monosporus, 93
Dickinson, S., 402, 411, 417
Dickson, J. G., 393, 396, 402, 417
Dicranophora juiva, 295
Didydium cancellatum, 367
Dillon- Weston, W. A. R., 250, 263
Dimond, A. E., 164, 168, 378, 396

Dinitrophenol, 61, 91

Diplodia macrospora, 17, 120-122, 193
natalensis, 28, 34, 122
tubericola, 37

Disaccharides, 130-131

Dissimilation, definition of, 87

Dittmer, K., 233, 238, 241

Dixon, M., 45, 62, 90, 113

Dodge, B. O., 1, 7, 348, 354, 357, 370,
382, 396, 404, 417

Dodge, C. W., 386, 396

Doermann, A. H., 216, 224

Dole, M., 158, 168

Donovick, R., 290, 301

Doran, W. L., 356, 358, 360, 361, 368, 370

Dormancy of spores, 355, 357

Dorrell, W. W., 91, 113

Dothidella quercus, 99

Doudoroff, M., 324, 335

Dowding, E. S., 401, 417

Dox, A. W., 95, 113, 129, 146

Drayton, F. L., 311, 335

Drosophila, 405

Dual phenomenon, 411-412

Dubos, R. J., 6, 7

Dufrenoy, J., 290, 300, 390, 396

Duggar, B. M., 98, 113, 270, 291, 297,
361, 362, 366, 370

Dulaney, E. L., 127, 146, 290, 297

Dung infusion, effect of, on spore germi-
nation, 362
as medium, 9

Dunn, C. G., 142, 147, 266, 277, 278, 300

Dunn, M. S., 216, 224

Durrell, L. W., 365, 370

Duschinsky, R., 233, 241

Duthie, E. S., 290, 297

Du Vigneaud, V., 192, 194, 204, 233, 237,
241

Dyctidiurn cancellatum, 367

Eakin, E. A., 194, 199, 204
Eakin, R. E., 194, 204, 233, 241
Eastcott, E. v., 195, 204
Echinodontium tinctorium, 293
Edgerton, C. W., 404, 417
Edwards, G. A., 40, 44
Ehrlich, J., 291, 297
Elfving, F., 35, 43, 314, 335

INDEX

447

Elliott, E. S., 184, 204

Elvehjem, C. A., 51, 62

Emerson, M. A., 363, 370

Emerson, R., 159, 169, 295, 297, 319, 327,

335
Emerson, S., 26, 43
Emmons, C. W., 386, 396
Enantiomorphs of sugars, 117, 119
Endoconidiophora monilifonnis, 283
Endomyces vernalis, 231, 232, 235, 239,

240, 271
Endothia -parasitica, 100, 180, 190, 193,
368, 375, 426, 428, 429, 431
ascospore discharge by, 344
factors affecting sporulation by, 311,
314, 321
Energy utilization, 60
heat evolved in, 61
Englis, D. T., 16, 22
Enteridium rozeanum, 357, 367
Entomophthora, 343, 353, 384

sphaerosperma, 343
Enzymes, 45-62
activators of, 55
adaptive, 59, 60, 412-413
chemical nature of, 52, 53
classification of, 47-51
effect of radiation on, 57, 58
factors affecting activity of, 53-58
inhibition of, 55-57, 227, 228, 377-378
lock-and-key relationship of, 228
mechanism of action of, 58
naming of, 47

pH and rate of reaction of, 55, 161
production of, 274-275
role of, in parasitism, 376, 377, 382
suberin-dissolving, 374
temperature and rate of reaction of, 53
toxicity of, 376, 382
Epichloe typhina, 196
Epidermophyton floccosum, 70, 238
Epstein, S., 291, 297
Erb, N. M., 274, 297
Eremothecium ashbyi, 273
Ergot, 291

Erwinia carnegieana, 288, 289
Erysiphe, 359
graminis, 358
graminis hordei, 358
polygoni, 311, 358, 377

Escherichia coli, 166, 201, 234, 238, 240,
251, 285

Essential elements, functions of, 66
list of, 67, 82

Esters, production of, 283

Ethyl pyridoxine formula, 234

Eucalyptus, 365

Exidia, 408

glandulosa, 409

Experimental results, presentation of,
31-32

External factors, effect of, on mor-
phology, 39-42

Ezekiel, W. N., 391, 396

Fastness, 240

Fat production, 270-272

Fawcett, H. S., 28, 33, 34, 43, 384, 396

Feldman, A. W., 378, 379, 396

Felix, E. L., 235, 243, 257, 265

Fellows, H., 12, 22, 89, 113

Fennell, D. I., 424, 438

Ferger, M. F., 237, 241

Fermentation, 89, 142, 275-277

Fieser, L. F., 235, 241

Fildes, P., 57, 63, 230, 241, 251, 263

Fischer, E., 219, 224

Fischer, O. E., 292, 293, 298

Fitzpatrick, H. M., 343, 354, 385, 396

Fitzpatrick, W. H., 267, 298

Fleming A., 220, 224, 290, 298

Fleury, C, 261, 263
Florey, H. W., 290, 297
Fomes applanatus, 41

roseus, 159
Fontaine, T. D., 390, 396
Food from fungi, 267-270
Foote, M. W., 258, 263
Formaldehyde, 256
Foster, A. A., 249, 263
Foster, J. W., 39, 43, 65, 71, 76, 84, 86,
103, 113, 136-138, 146, 156, 169, 266,
272, 273, 277, 278, 280-283, 290, 298,
302
Fourneau, E., 231, 241
Fox, D. L., 295, 297, 327, 335
Frear, D. E. H., 246, 247, 256, 263
Fred, E. B., 424, 438
Frey-WyssUng, A., 82, 84

448

PHYSIOLOGY OF THE FUNGI

Friedemunn, T. E., 142, 146
Fries, N., 29, 31, 42, 43, i)3, 113, 139,
146, 190, 193, 195, 204, 205, 219,
224, 366, 367, 370, 401, 417
Fromageot, C, 184, 204
Fructose formula, 119
Fudge, J. F., 391, 396
Full go se plica, 361, 367, 368
Fuller, W. H., 49, 63, 134, 147
Fulmer, E. I., 17, 22, 25, 43, 65, 84, 99,

112
Fumago vagans, 280
Fumaric acid, production of, 282-283

scheme for biosynthesis of, 137
Fungi, as food, 267-270

list of vitamin-sufficient, 172
role of, in nature, 1, 266, 267
Fungicide, meaning of, 245, 246
Fungicides, copper, 246-249
dithiocarbamate, 259
evaluation of, 261-262
mercury, 249-251
mode of action of, 245, 249
organic, 256-261
quinone, 257-258
Fungistatic agent, definition of, 245
Fusarium, 99, 140, 141, 172, 271, 273,
275, 293, 313, 360, 375, 378, 402,
411, 431
avenaceum, 184, 400
cepae, 316
coeruleurn, 70
discolor sulphureum, 313
eumartii, 89, 316
graminearum, 91, 261, 295
{See also Gibberella zeae)
lini, 96, 102, 103, 108, 138, 141, 186,
191, 192, 270, 275, 295, 374, 387,
390
lycopersici, 122, 133, 160, 378, 390
{See also oxysporum var. lycopersici,
below)
moniUforme, 127
niveum, 188, 189
orthoceras, 287
oxysporum, 80, 88, 89, 222
var. lycopersici, 127, 260

{See also lycopersici, above)
var. nicotianae, 80
sambucinum, 139
solani, 295

Fusarium, trichothecioides, 96
vasinfectum, 380

Galactitol formula, 124
Galactose formula, 1 19
Gallegly, M. E., 392, 396
Gallium, 81

Ganoderma lobatum, 134
Gardner, M. W., 356, 370
Gastrock, E. A., 280, 298
Gaumann, E., 372, 378, 396
Geiger, W. B., 258, 260, 263
Georg, L. K., 424, 438
Gerhardt, P., 279, 298
Gibberella saubinetti, 159, 391

{See also zeae, below; Fusarium grami-
nearum)
zeae, 293, 393, 402

{See also saubineiii, above; F usarium
graminearum)
Giles, N. H., 413, 417
Gillespie, J. M., 201, 206, 230, 242
Gilman, H., 117, 130, 146
Gimingham, C. T., 248, 263
Gingrich, W., 197, 204
Gliomastix convoluta. Til
Gloeosporium, 368
Glomerella, 404, 415

cingulata, 36, 99, 172, 247, 248, 254,
255, 261, 262, 402, 425, 428, 429,
431, 436
nutrients, and spore germination of,
362, 363
and sporulation of, 322, 324, 325
ultraviolet light and sporulation of,
315
gossypii, 98
Gluconic-acid production, 280-281
Glucose, fermentation of, 141, 142
formula of, 119
a-D-gkicose, 128
|3-D-glucose, 128
fungi not utiUzing, 120
Glutathione, 93
Glycolysis, 141, 142
Glycosides, 128-129
Glynne, M. D., 253, 264
Gnomonia ulmi, 304, 344
Goddard, D. R., 357, 370

INDEX

449

Goepp, R. M., Jr., 14, 22, 117, 119, 128,

133, 147
Goering, H. L., 238, 241
Goldberg, M. W., 194, 204
Goldsworthy, M. C, 247-249, 2G3
Gonatorrhodiella highlei, 328
Goodman, I., 238, 241
Gorcica, H. J., 268, 298
Gordon, M. A., 283, 298
Gortner, R. A., 37, 43, 47, 51, 63, 70,

84, 88, 105, 113, 154, 169
Gottlieb, D., 127, 146, 286, 298, 356, 370
Gould, B. S., 96, 113
Graham, T. W., 402, 417
Grant, W. M., 414, 418
Grassmann, W., 134, 146
Greathouse, G. A., 222, 224
Green, E. L., 247-249, 263
Greene, H. C, 424, 438
Greene, R. D., 212, 224
Gregory, P. H., 350, 351, 354
Gries, G. A., 257, 263
Grob, E. C, 236, 243
Grossbard, E., 286, 298
Growth, abnormal, 39-41
definition of, 24

effect of depth of medium on, 38
factors affecting, 32-38
measurement of, 27-31
phases of, 25-26

of filamentous fungi, 26
of unicellular organisms, 25
rate of, 26
tube, 29
Growth factors {see Vitamins)
Guignardia bidwellii, 304, 344, 389, 425,

428, 429, 431, 436, 437
Guilloud, M., 186, 196, 206, 219, 225
Guirard, B. M., 201, 206
Gunness, M., 193, 207
Gupta, B. M., 288, 298
Gustafson, F. G., 70, 84
Gymnoascits setosus, 99
Gymnoconia peckiana, 348
Gymnosporangiurn juniperi-virginianae,

358, 368
Gyromitra esculenta, 293

Haag, E., 191, 204
Haagen-Smit, A. J., 326, 337

Haehn, H., 108, 113

Haenseler, C. M., 32, 43

Haldane, J. B. S., 55, 63, 161, 169

Hamilton, E., 327, 336

Hanahan, D., 16, 22

Hanna, W. F., 410, 417

Hansen, H. N., 17, 22, 305, 313, 332, 335,

337, 411, 412, 417
Hansenida suaveolens, 219
Hao, L. C., 274, 298
Harden, A., 46, 63, 96, 113, 277, 298
Harris, E. E., 270, 298
Harris, G. C. M., 200, 205, 285, 303
Harris, H. A., 402, 417
Harris, S. A., 192, 205, 232, 233, 243
Hart, H., 373, 388, 396
Hartelius, V., 229-231, 239, 242
Harter, L. L., 50, 63
Hartree, E. F., 281, 299
Harvey, L. B., 374, 396
Haskins, R. H., 35, 43
Hatfield, W. C., 390, 396
Haugen, G. E., 142, 146
Haustoria, 380-381
Hawker, L. E., 90, 113, 133, 147, 321,

325, 329, 335, 400, 417
Hawkins, L. A., 37, 43, 374, 396
Haymaker, H. H., 378, 396
Hazen, E. L., 286, 301, 327, 334, 335
Heald, F. D., 344, 354
Helicostylum pyrifonne, 122
Helminthosponum, 99, 294, 327, 402, 411
cynodontis, 294
euchlaenae, 294
gramineinn, 172, 294, 311
ravenellii, 294
sacchari, 379
sativum, 101, 122, 127, 221, 285, 286,

414, 431, 436, 437
turcicum, 294
vidoriae, 172, 379, 429
Helvellic acid, 293
Hemitrichia clavata, 368
Hemming, H. G., 285, 296
Hemophilus parainjluenzae, 197
Render sonia, 311
Hendlin, D., 273, 298
Henry, A. W., 286, 298
Henry, B. W., 163, 169, 308, 309, 317.

335
Herbst, R. M., 109, 113

450

PHYSIOLOGY OF THE FUNGI

Herrick, J. A., 121, 123, 124, 147

Hertz, J., 219, 224

Hervey, A., 29, 43, 77, 78, 85

Hesse, A., 270, 272, 298

Hestrin, S., 46, 49, 63

0-Heteribiotin {see Oxybiotin)

Heuser, E., 135, 147

Hevesy, G., 87, 113

Hexoses, 119-122

Hickey, R. J., 73, 84, 272, 298

Higgins, B. B., 376, 396

Hildebrand, E. M., 424, 439

Hill, E. G., 16, 22

Hirneola polytricha, 269

Hitchens, A. P., 14, 22

HockenhuU, D. J. D., 93, 113, 414, 417

Hodgson, R., 378, 396

Hok, K. A., 32, 44

Hollis, J. P., 88, 113

Holoenzymes, 53

Holzappfel, H. H., 139, 147

Hopkins, R. H., 50, 63

Hormones, effect of, on reproduction, 326

Horn, M. J., 216, 224

Horner, C. K., 108, 112

Horowitz, N. H., 211, 216, 217, 224, 225,

239, 242
Horr, W. H., 125, 126, 147
Horsfall, J. G., 235, 242, 246, 257, 259,

260, 262-264, 438, 439
Host-parasite relationship, 372
Host penetration, 372-375
Houlahan, M. B., 182, 202, 205
Houston, B. R., 311, 335, 386, 396, 401,

402, 417
Howard, B. H., 294, 299
Howard, F. L., 379, 396
Humfield, H., 270, 299
Hurd, A. M., 388, 396
Hutchings, B. L., 216, 224
Hydnum imbricatum, 367
repandum, 367
Hydrogen, 87
Hydrogen-ion concentration, changes in

culture media of, 160, 162-164
effect of, on cell morphology, 42

on enzymes, 55, 161, 162

on growth, 158-161

on medium composition, 164

on oxygen supply, 165

on spore germination, 360-361

Hydrogen-ion concentration, effect of,
on sporulation, 318-319
on utilization of nutrients, 165-167

as factor in resistance, 388-389

laboratory exercise, 430-431

relation of, to pH, 151, 152

{See also pH)
Hydrogen sulfide, production of, by
spores, 254, 255

toxicity of, 254-256
Hydrolases, 47
Hydroxyproline formula, 238
8-Hydroxyquinoline, 260
Hygrophorus conicus, 292
Hyper parasites, 385
Hypersensitiveness, 382
Hypomyces, 385

solani f. cucurbitae, 412
Hypoxanthine, effect of, on spore germi-
nation, 364, 365
Hypoxylon pruinatum, 190, 193

Imbibition, 37

Impatiens balsaminia, 257, 390

Indicators, pH, 157

Ingold, C. T., 342, 344, 354

Inheritance, 404-411

in Ascomycetes, 404-408

in Basidiomycetes, 408-411

basis of, 404

cytoplasmic, 413
Inhibition analysis, 299
Inoculation, effect of, on sporulation, 332

laboratory exercise, 431-432

methods of, 424
Inocybe infelix, 293

infida, 293
Inositol, 195-196

Internal factors, effect of, on growth, 32
Invertase {see Sucrase)
Ion antagonism, 70, 75, 77
Ionization, 149, 150
Ions as enzyme activators, 55, 68, 69, 76,

79
Iron, 71, 74-76
Isoachlya monilifera, 92, 93
Isomers of sugars, 117
Itaconic-acid production, 281-282

INDEX

451

Jaag, O., 378, 396

Jarvis, F. G., 68, 84

Javillier, M., 72, 77, 84

Jillson, O. F., 42, 43

Johnson, H. W., 159, 169

Johnson, J., 288, 302

Johnson, M. J., 51, 63, 68, 73, 84, 86, 280,

302
Johnson, T., 411, 417
Jones, E. S., 360, 370
Jones, F. R., 391, 396
Jones, M. J., 228, 242
Jones, R. C, 308, 335
Juglone, 257

Kakeura, M., 136, 147
Karhng, J. S., 384, 385, 396
Karlingia rosea, 35

{See also Rhizophlyctis rosea)
Karow, E. O., 278, 279, 299
Kauffman, C. H., 6, 7, 305, 335
Kauffman, F. H. O., 356, 361, 370
Kavanagh, F., 182, 190, 206
Kavanagh, V., 100, 172, 181, 206, 432,

439
Keilin, D., 281, 299
Keitt, G. W., 261, 263, 288, 299
Kellerniania yuccagena, 311
Kelner, A., 58, 63
Kern, F. D., 381, 397
Kernkamp, H. C. H., 293, 297
Ketoses, 117
Kinsel, K., 120, 147
Kinsey, V. E., 414, 418
Kirkwood, S., 236, 242
Klebs, G., 6, 7, 305-308, 320, 336

biographical note, 6
Klebs's laws of growth and reproduction,

306
Klimek, J. W., 290, 296
Kloeckera brevis, 181
Ivlotz, L. J., 377, 397
Knaudt, J. H., 291, 300
Knight, B. C. J. G., 196, 205
Knobloch, H., 279, 296
Koch, R. S., 289, 297
Koffler, H., 75, 84

Kogl, F., 192, 193, 195, 205, 209, 219, 224

Kolthoff, I. M., 157, 169

Koser, S. A., 194, 205

Krampitz, L. O., 231, 242

Krebs citric acid cycle, 143, 144

Krehl, W. A., 215, 224

Kreitlow, K. W., 402, 418

Krieger, L. C. C., 292, 293, 299

Kroemer, K., 37, 43

Krumbholz, G., 37, 43

Kubowitz, F., 52, 63

Kuhn, R., 292, 299

Kunkel, L. 0., 319, 336, 374, 397

Laboratory demonstrations, 437-438
Laboratory exercises, 419-428
Laborey, F., 69, 70, 84
Laccaria amythestina, 293
Lachaux, M., 273, 301
Lachnea scidellata, 346
Ladarius piper at us, 52

torminosus, 293
Lactase, 49, 133
Lactic-acid production, 281
LadobaciUus, 202

arabinosus, 193, 194, 215, 216, 232

casei, 193, 194, 216, 218, 229, 232, 233
Lactose formula, 131
La Far, F., 39, 44, 111, 113
Lambertella corni-maris, 99

pruni, 179, 180, 184, 190, 193
Lampen, J. 0 , 228, 242
Landerkin, G. B., 199, 205
Large, E. C., 245, 252, 263
Larsh, H. W., 120, 148
Lavollay, J., 69, 70, 84
Leach, J. G., 248, 249, 264, 269, 299, 352,
354, 362, 364, 365, 370, 383, 391,
392, 397
Leaver, F. W., 331, 336
Leben, C., 261, 263, 288, 299
Lederberg, E. Z., 413, 417
Lee, A., 379, 397
Lee, S. B., 277, 289, 290, 299
Leibowitz, J., 46, 63
Leikind, M. C., 14, 22
Le Mense, E. H., 274, 299
Lemieux, R. U., 290, 299
Lentinus lepideus, 123, 134, 135

tigrinus, 99

452

PHYSIOLOGY OF THE FUNGI

Lenzites hetulinus, 134, 351

saepiaria, 135, 159, 300, 363, 364
trabea, 100, 135, 191, 401, 402, 415
inheritance by, of fruiting ability,
409
of thiamine deficiency, 410
thiamine deficiency of, 177, 178

Leonian, L. H., 7, 14, 22, 42, 44, 60, 63,
74, 84, 92, 102-105, 107, 113, 164,
169, 183, 188, 189, 191-193, 195-199,
205, 211, 214, 215, 224, 229, 233,
242, 258, 264, 310, 317, 319, 321,
326, 336, 400, 402, 412, 418
biographical note, 7

Leopold, H., 108, 113

Leptomitus lactens, 120, 127, 145

Letcher, H., 275, 299

Levan, A., 39, 44

Levine, H., 268, 272, 298, 299

Lewis, J. C, 75, 84

Lewis, R. W., 144, 147

Light, effect of, on growth, 35
on irradiated spores, 58
on spore discharge, 339-341, 344, 346
laboratory exercise, 426

Lillie, R. J., 273, 299

Lilly, V. G., 14, 22, 34, 38, 43, 44, 60,
63, 74, 84, 92, 102-105, 107, 113,
164, 167, 169, 178, 181-184, 188, 189,
191-193, 195-199, 204, 205, 211, 214,
215, 224, 229, 232, 233, 242, 307,
309, 310, 312, 313, 317, 319, 326,
329-331, 334, 336, 400, 401, 409,
410, 412, 416, 418

Lime sulfur as fungicide, 256

Liming, O. N., 253, 264

Lin, C. K., 363, 370

Lindeberg, G., 80, 84, 100, 113, 154, 159,
169

Lindegren, C. C., 30, 44, 60, 63, 120, 148,
212, 224, 327, 336, 404, 412, 413, 418

Lindegren, G., 327, 336

Lii der, P., 271, 299

Lindgren, R. M., 35, 44

Ling, L., 361, 366, 370

Lingane, J. J., 157, 169

Link, K. P., 390, 391, 397, 398

Lintzel, W., 267, 299

Lipmann, F., 61, 63, 199, 205

Little, J. E., 257, 264, 390, 397

Livingstone, B. E., 89, 114

Lockhead, A. C, 199, 205

Lockwood, L. B., 70, 84, 271, 281, 282,

299, 318, 326, 336
Lohrmann, W., 70, 84
Longevity of spores, 368
Lophodermium pinastri, 190, 195, 196
Lowther, C. V., 368, 370
Lundeg&rdh, H., 159, 169
Lycogola epidendrnm, 367
Lycoperdon echinatum, 367

nigrescens, 367

perlatum, 350, 351

pratense, 367

pyriforme, 367

umbrinum, 367
Lyxose, 123

Ma, R., 180, 194, 200, 201, 206, 234, 242,

329, 337, 400, 418
McCall, M., 258, 263
McCalla, T. M., 166, 169, 251, 264
McCallan, S. E. A., 245, 247, 248, 252-

255, 261, 264, 265, 319, 320, 336
McCrea, A., 368, 370
McElroy, W. D., 226, 227, 242, 414, 418
McGowan, J. C., 261, 264
McHargue, J. S., 77, 78, 85
Mcllwain, H., 226, 236, 242
MacLeod, R. A., 74, 85
McNew, G. L., 261, 264
Macow, J., 229, 243

Macrae, R., 402, 403, 410, 418, 438, 439
Macrosporium commune, 98

sarcinaeforme, 99, 250, 253, 255, 262

tomato, 316
McVeigh, I., 104, 112, 238, 242, 412, 418
Mader, E. O., 318, 336
Magnesium, 68-70, 218
Maillard, L. C., 16, 22
Mains, E. B., 381, 397
Malachite green, 258
Maltose formula, 130
Mandels, G. R., 91, 113, 114
Manganese, 77-79, 80
Mann, T., 95, 114
Mannitol formula, 124
Mannose formula, 119
Marasmius, 80, 100

alliaceus, 81, 100

I
I

INDEX

453

Marasviius, androeceus, 100

chordalis, 95, 100, 132

epiphyllHs, 80, 81, 100

foetidis, 100

fulvobulbillosus, 99

graminum, 100, 159

perforrnis, 100

personatus, 100

putillus, 100

rainealis, 100

rotula, 100

scorodoniiis, 100
Margolin, A. S., 16, 17, 22, 121-126, 136,

147
Marryat, D. C, 392, 397
Marsh, P. B., 77, 85
Marten, E. A., 248, 249, 264
Martin, G. J., 235, 242
Martin, W. J., 401, 418
Mass, J. M., 291, 299
Massospora cicadina, 384
Mathur, R. S., 160, 169, 186, 205, 309,

332. 336
Meacham, M. R., 159, 169
Media, autoclaving of, 16, 17, 422, 423

basal semisynthetic, 427

choice of, 13

comparison of, 20, 21

concentration of, effect of, on growth,
38

constituents of, 12-14, 421-422

formula of, ghicose-asparagine, 210
glucose-casein hydrolysate, 211
sucrose-ammonium tartrate-ammo-
nium nitrate, 211

kinds of, 9-11

liquid, 14

naming of, 14

natural, 9, 11

pH of, 422

preparation of, 17, 420-421

removal from, of metallic ions, 72
of vitamins, 210, 432

semisynthetic, 10, 427

solid, 14

specific metabolites in, 12

sterilization of, 16, 17, 422, 423

synthetic, 10-11, 20, 421

units of measure, 18, 19
Meehan, F., 379, 397
Melanconium betulinnm, 190, 196, 311

Melander, L. W., 387, 397
Melanospora, 323, 325, 428, 429

destruens, 91, 133, 190, 193, 324, 329
Melibiosc, 132

Memnoniella echinata, 193, 195, 222, 329
Mercury-toxicity theory, 251
Merulius lacrymans, 95, 135, 159
Metabolic products, effect of, on sporula-

tion, 327-329
tests for presence of, 219-221
variation in, 401-402
Metabolism, intermediary, 139-144
Metabolite antagonists, theory of action

of, 226-229
Metabolites, 12
specific (see Amino acids; Growth,
factors affecting; Vitamins)
Metarrhizium glutinosum, 103

{See also Myrothecium verrucaria)
2-Methoxy-l ,4-naphthoquinone formula,

257
a-Methyl-D-glucoside formula, 129
j3-Methyl-D-glucoside formula, 129
Methylpentoses, 124
Metz, O., 294, 299
Meyer, B. S., 37, 44
Meyerhof, O., 51, 63, 142, 147, 277, 299,

300
Michaelis, L., 51, 63
Michener, H. D., 292, 300
Microbiological assays, standard curve,

214
Microsphaera alni, 358
Microsporum audouini, 197, 327

canis, 238
Milhngton, R. H., 143, 148
Mirsky, A. E., 97, 114
Mitchell, H. K., 182, 199, 202, 205,

237, 242, 325, 326, 337
Mix, A. J., 321, 324, 336
Moisture requirements for growth, 35
Molliard, M., 68, 85
Molybdenum, 79, 218, 219
Monascus purpurea, 27, 172
Monilia Candida, 39
fructigena, 362
tarnari, 281
Monilinia fructicola, 27, 100, 118, 172,

304, 321, 325, 375, 402, 425, 426,

428, 429, 431, 436, 437
(See also Sclerotinia frurficola)

454

PHYSIOLOGY OF THE FUNGI

Monocotyledons, immunity of, to cotton

root rot, 391
Monosaccharides, 116-126
Morchella esculenta, 267
Morphology, effect of external factors on,
39-42, 89
of yeast, 39
Mortierella rhizogena, 100
Morton, H. E., 283, 300
Moyer, A. J., 280, 282, 300
Moyer, D., 186, 195, 204, 400, 416
Mrak, E. M., 309, 336
Mucor, 9, 39, 271
flavus, 100
hiemalis, 100, 295
mucedo, 295
nodosus, 100
pusilhis, 70
pyriformis, 100, 279
racemosus, 275, 402
raviannianus, 103, 133, 231, 232

carbon nutrition of, 118, 122, 124,

125
vitamin requirements of, 188-191
rouxii, 281
saturninus, 100
spinosus, 362
stolonifer, 100
stridus, 100
Mudd, S., 231, 242
Mueller, J. H., 194, 196, 205
Mulder, E. G. 14, 22, 73, 79, 85, 217-

219, 224, 225, 294, 300
Mull, R. P., 96, 114, 140, 147, 277, 300
Muntz, J. A., 68, 85
Murphy, H. E., 379, 397
Muscarin, 292
Mutations, back, 413
chemically induced, 414-415
natural, 93

radiation-induced, 93, 282, 401, 405,
408
Mutualistic symbiosis, example of, 173,

384
Mycobacterium tuberculosis, 240, 290
Mycoderma cerevisiae, 39
valida, 176, 199
vini, 186
Mycogone, 385

MycosphaereUa citrullina, 319
Mycoiorula lactis, 197

Myrback, K., 50, 63, 135, 147, 275, 300
M yrophagus ucrainicus, 384
Myrothecium verrucaria, 104, 109, 222
(See also Metarrhizium glutinosum)

Naemosphaera, 311
1,4-Naphthoquinones, antibiotic activity

of, 390
Nectria cinnabarina, 328
coccinia, 190, 328
coryli, 328
cucurbitula, 328
galligena, 328
Neidig, R. E., 129, 146
Neisseria gonorrhoeae, 290
Nelson, J. M., 52, 63, 77, 85
Nematospora gossypii, 193, 195, 196, 219
Neocosmopara vasinfecta, 99, 172, 321,

325, 426, 429, 431, 432
Neopyrithiamine, 232
Neuberger, A., 10, 22
Neufeld, C. C, 358, 370
Neurospora, 29, 102, 144, 216, 223, 238,
401, 408, 415, 438
amino acids and spore germination of,

364
back mutations of, 413
genetics of, 404-407
mutants of, 182, 183, 202, 215-217,

405-408
nutritional adaptations of, 412, 413
temperature and riboflavin deficiency

of, 182
vitamin deficiencies of, 193, 197, 199,
202
Neurospora crassa, 26, 36, 144, 167, 196,
202, 228, 232, 237, 363, 405, 413, 414
pH, and p-aminobenzoic-acid defi-
ciency of, 183, 184
and growth of, 161, 162
production of perithecia by, 325, 326
use of, in assays, 211, 215
Neurospora sitophila, 29, 34, 184, 193,
215, 248, 317, 405
tetrasperma, 357
Newton, M., 411, 417
Nickerson, W. J., 39, 40, 42-44, 70, 85,
91, 92, 114, 327, 336, 385, 386, 397
Nicotinic acid, 196-198
antagonists of, 236

INDEX

455

Nicotinic acid amide, 196

Niemann, C, 228, 237, 242

Niklas, H., 218, 224

Nitrate utilization, effect of molybdenum

on, 79
Nitrite as source of nitrogen, 102
Nitrogen, 97-110

fixation of, by fungi, 98
nitrate, list of fungi utilizing, 99
sources of, effect of, on sporulation, 321

laboratory exercise, 428
utilization of, as basis of classification,
97
effect of organic acids on, 103, 104,

107
organic, 101, 105
Noble, R. J., 366, 371
Nobles, M. K., 134, 147, 410, 418
Nocardia gardneri, 287
Nord, F. F., 96, 102, 108, 114, 115, 123,
135, 140, 141, 147, 148, 186, 191, 192,
207, 270, 295, 300, 303
Norman, A. G., 49, 63, 134, 147
North, H. E., Ill, 113
Novelli, G. D., 199, 205
Nutrition, of host, effect of, on disease
development, 392-393
special conditions of, laboratory exer-
cise, 429-430
Nutritional requirements, variation of,

within species, 400-401
Nydalis, 385

Oldium lactis, 271
Oligosaccharides, 129-133
Ophiobolus graminis, 12, 97, 99, 180, 190,
193
miyabeanus, 99
oryzinus, 193
Ophiostoma, 401

catonianum, 193, 200
muUiannulahun , 42, 93

{See also Ceratostomella multian-
nulata)
ulmi, 31

{See also Ceratostomella ulmi)
Organic acids, as carbon sources, 126-128
as factor in resistance, 390
production of, 277-283

Orton, C. R., 7, 381, 383, 397
Osmotic pressure, 36, 37

in host-parasite relation, 376, 377, 382
Oswald, J. W., 311, 335
Owen, W. L., 274, 300
Owens, H. S., 136, 147
Oxidases, 51
Oxybiotin, 194
Oxygen, 88-92, 359, 360

Paneolus, 293
Pantothenic acid, 198-199

antagonists of, 233-234

moieties of, 198
Pantoyltaurine formula, 234
Panus stijpticus, 402, 403, 410, 438
Parasitella simplex, 188
Parasitism, 375-386

action in advance, 376

by balanced parasites, 375, 380-383

by destructive parasites, 375-378

by fungi, of fungi, 385
of insects, 383-385
of man, 385

types of, 375

by wilt-producing fungi, 378-380
Parker-Rhodes, A. F., 250, 264
Pasteur, L., 6, 7, 39, 44, 45, 63, 117, 147,
219, 224, 275, 276, 300

biographical note, 6
Pathogenicity, inheritance of, 411

variation in, 402
Patton, A. R., 16, 22
Payne, E. H., 291, 300
Pectinase, 47, 50

production of, 376-378

specificity of, 377-378
Pehrson, S. O., 34, 44, 160, 169, 380, 397
Peltier, G. L., 164, 168, 273, 300
Penetration, 372-375

direct, 373-375

into immune plants, 374, 391

through stomata, 372-373

through wounds, 375
Penicillin, 288-290

influence of, on cell morphology, 39

type formula of, 289
Penicillium, 93, 94, 99, 172, 197, 222,
260, 271, 272, 274, 275, 283, 286,
385, 401, 436

456

PHYSIOLOGY OF THE FUNGI

Penicilliurn, arenarium, 297

brevicaule, 110, 111

hrevicom-pactum, 287

chnjsogenum, 68, 94, 280-282, 402, 408,
437
ion antagonism in, 75
penicillin production by, 287-289

crustaceum, 280

cyclopium, 360

digitatum, 98, 193, 232, 261, 283, 375

divaricatum, 279

ex-pansum, 98, 262, 375, 425, 426, 431

flavo-cinereum, 212

glabrum, 280

glaucum, 35, 69, 70, 117, 219, 275, 279,
280, 358, 362

griseofulvum, 287

islandicum, 294

italicum, 360, 375

janczewskii, 287

javanicum, 271, 272, 318

luteum-purpiirogenuri} , 287

notatxivi, 164, 284, 288, 408

olivaceum, 279

oxalicum, 272

patulum, 287

phoeniceum, 294

piscarium, 272

purpurogenum var. rubrisderotiurn, 280

roquejorti, 121 , 272

rubrurn, 294

sanguifluus, 279

spiculisporurn, 431

variable, 159
Peniophora allescheri, 410
Pentoses, 122-124
Perlman, D., 71, 73, 75, 76, 85, 195, 205,

279, 300
Permeability, change of, as factor in re-
sistance, 382
in host cells, 376, 377, 382
Peronospora, 353

effusa, 343

geranii, 343

halstedii, 343

parasitica, 343

pygrnaea, 358

tabacina, 343
Pestalotia guepinia, 311

stellata, 255
Peterson. M. S.. 202, 205

Peterson, W. H., 139, 147, 202, 205, 216,
224

Peziza badia, 346
pH, definition of, 150, 151
equation for, 151
indicators for, 157
method of determining, 156-158
Phacidium infestans, 34, 160
Phalloidin, 292
PheUorina, 307

Phenolic compounds, toxicity of, 389-391
PhiUips, P. H., 236, 242
Pholiota autumnalis, 292
Phoma, 99
apiicola, 99

betoe, 98, 100, 172, 321, 325, 429, 432
causarina, 99
lingam, 377
terrestris, 402
urens, 311, 317
Phomopsis californica, 311

citri, 28, 34
Phosphorus, 94-97

in carbohydrate dissimilation, 96, 142
Phycomyces, 364, 415

blakesleeanus, 15, 17, 32, 35, 100, 164,

172, 173, 178, 181, 187, 191, 212,

220, 232, 295, 310, 314, 380, 428,

431, 434, 437

pH and formation of zygospores, 318

stimulants and spore germination,

364-365
thiamine deficiency of, 174, 177,

188-190
use of, in thiamine assay, 211, 215
utihzation of sources by, carbon, 122,

124-126, 133
nitrogen, 100, 103, 104, 107, 108
nitens, 91, 362
Phyllachora graminis, 383
Phylostida antirrhini, 356, 358
opuntiae, 311, 317
soUtaria, 321, 324, 362
Phymototrichum omnivorum, 76, 375, 391
Physarum cinereum, 367, 368

polycephalum, 320, 367
Phytophthora, 33, 42, 188, 190, 258, 402
boehtneriae, 327

cadorum, 17, 122, 132, 326, 327
cinnamomi, 232

INDEX

457

Phytophthora, colocasiae, 258, 360
cry throne ptica, 16, 17, 285, 327

carbon sources utilized by, 122, 124,

125
thiamine deficiency of, 188, 189, 191
fagopyri, 122, 136
hydrophila, 258

infestans, 34, 343, 352, 360, 373, 425,
436
parasitism of, 376, 377
temperature and germination of,

356-357
variation of, 401
megasperma, 327
melongenae, 258
palmivora, 132, 360
parasitica, 132, 360
richardiae, 258
terrestris, 28, 34
Pigman, W. W., 14, 22, 117, 119, 128, 133,

147
Pigments, production of, by fungi, 293-
295
relation to, of copper, 77
of iron, 76
Pilaira anomala, 295

moreaui, 122
Pilgrim, F. J., 194, 206
Pilobolus, 338, 342-345, 349, 353, 438
kleinii, 339, 340
longipes, 339
microsporus, 307
Pimelic acid, 194
Pinkard, J. A., 343, 354
Piptocephalis, 385
Piricularia oryzae, 163, 190, 193, 331, 351

sporulation of, 308, 309, 317
Pirschle, K., 102, 114
Pisum, 382

sativum, 377
pKa, 153, 154, 167
pKft, 153, 154

Plasmodiophora brassicae, 307
Plasmopara viticola, 358, 360
Piatt, B. S., 269, 300
Plattner, P. A., 378, 397
Platz, G. A., 360, 365, 371
Plenodomus destruens, 37, 311, 317

fuscomaculans, 307, 311, 313, 317
Pleurage, 344, 353, 438
anserina, 404

Pleurage, curvicoUa, 100, 175, 178, 190,

193, 322, 323, 325, 344, 428
Pleurotus corticatus, 102

ostreatus, 100, 134
Plumlee, C. H., 94, 114
Podaxis, 307

Podospora curvula, 190, 193
Pollard, A. L., 94, 114
Polyporus abietinus. 134
adustus, 219
betulinus, 135
cinnabarinus, 134
hirsutus, 351
pargamenus, 134, 135
squamosus, 238
versicolor, 351
Polysaccharides, 133-136

as wilt inducers, 378
Porges, N., 280, 281, 300
Porta vaillantii, 135
Porter, C. L., 327, 336
Potassium, 68, 218
Prasard, N., 380, 397
Pratt, E. F., 199, 206
Pratt, R., 32, 44, 290, 300
Prescott, S. C, 142, 147, 266, 277, 300
Preuss, L. M., 271, 300
Prevost, B., 246, 264
Price, W. C, 288, 298
Pritham, G. H., 158, 169
Proactinomyces cyaneus, 287
Proline, formula of, 238
Protectants, fabric, tests for, 221-222
Proteins, amino-acid composition of, 268

synthesis of, .110
Protocatechuic acid, 390
Psalliota campestris, 52, 267, 270
{See also Agaricus campestris)
Pseudomonas saccharophila, 324
Pseudopeziza ribis, 193
Puccinia antirrhini, 254, 255
coronata, 348
glumarum, 358

graminis, 348, 349, 356, 360, 373, 377
parasitism of, 382-383
resistance to, 392
graminis iritici, 9, 382, 392, 395, 402
411
inheritance in, 411
graminis tritici-compacti, 392
podophylli, 348

458

PHYSIOLOGY OF THE FUNGI

Puccinia, rubigo-vera tritici, 402
(See also triticina, below)

sorghi, 381, 383, 392

triticina, 373

{See a/.so nihicjo-vera tritici, above)
Pyridoxal formula, 200
Pyridoxamine formula, 200
Pyridoxine, 199-201

antagonists of, 234-235
Pyrithianiine, 232
Pyronema confluens, 100
Pyruvic acid, action of carboxylase on, 52

effect of thiamine concentration on,
191, 192

transformations of, 140-143
Pythiacystis citrophthora, 28, 34, 377
Pythiornorpha gonapodyoides, 77, 78, 100,

103, 122, 124-126, 188-190, 232
Pythium, 33, 327, 374, 377, 378

arrheno manes, 190

ascophallon, 103, 122, 136, 190

butleri, 182, 190

debaryanum, 100, 132, 249, 374

intermedium, 100

irregulare, 80, 100

oligandrum, 190

Raaf, H., 271, 300

Rabinovitz-Sereni, D., 69, 85

Raciborski, M., 37, 44

Radiation effects on fungi, 57, 58

Raffinose formula, 132

Rahn, O., 24, 25, 32, 44, 165, 166, 169

Raistrick, H., 94, 114, 139, 147, 275, 276,

294, 296, 299-301
Rake, G., 290, 301
Ramsey, G. B., 316, 336
Rands, R. D., 319, 336
Rankin, W. H., 368, 369
Rannefelt, A. N., 201, 206
Raper, J. R., 326, 337
Raper, K. B., 34, 44, 282, 284, 285, 301,

385, 398, 401, 418, 424, 439
Ratajak, E. J., 136, 147
Raulin, J., 11, 22, 66, 67, 71, 76, 85, 94,
114
synthetic medium of, 1 1
Raut, C., 30, 44, 60, 63, 212, 224
Ravel, J. M., 234, 242

Rawlins, T. E., 17, 22
Reddy, C. S., 388, 389, 397
Reeves, M. D., 70, 84
Reilly, H. C., 288, 301
Rcnaud, J., 273, 301
Resistance, 386-393

in colored onions, 390

due to, acidity, 388-389
antibiotics, 390
organic acids, 390
phenolic compounds, 390, 391

effect on, of environment, 393
of host metabolism, 393
of host nutrition, 392-393

functional, 388

mechanical, 387

physiological, 388-393

starvation theory of, 383, 391, 392

toxin theory of, 382, 392
Respiration, 89-92

effect of cyanide on, 55-57
Respirometer, 91
Reticularia lycoperdon, 367
Reynolds, E. S., 390, 397
Rhizobium, 98, 192

trifolii, 75, 164, 193, 208
Rhizoctonia solani, 79, 80, 100, 127
Rhizophlyctis rosea, 100

(See also Karlingia rosea)
Rhizopus, 49, 76, 274, 281, 319

arrhizus, 281

autocar pi, 50

chinensis, 50, 281

delemar, 274

elegans, 281

japonicus, 281

niicrosporus, 50

nigricans, 37, 50, 70, 76, 78, 100, 122,
160, 262, 375
carbon sources utilized by, 133, 136,

137
production of organic acids by, 282,
283

nodosus, 281

oryzae, 100, 274, 281, 326

pseudochinensis, 281

salebrosa, 281

shanghaiensis, 281

stolonifer, 281

suinus, 17, 122, 133, 186, 196

tritici, 281

(

INDEX

459

Rhodoiorula aurantica, 201, 231
(jlulinus, 271
rubra, 188
sanniei, 184

Rihes, 395

Riboflavin, 202

production of, 272-273

Ribose, 97, 123

Rice, M. A., 381, 382, 397

Rich, S., 2G0, 264

Richards, M. C, 319, 337

Richards, O. W., 66, 85

Richardson, G. L., 165, 166, 169

Rickes, E. L., 82, 85, 273, 301

Rickettsia, 291

Riker, A. J., 17, 22, 305, 337, 420, 439

Riker, R. S., 17, 22, 305, 337

Rippel, A., 68, 69, 85, 103, 114

Roach, W. A., 253, 264

Roark, G. W., Jr., 129, 146

Robbins, W. J., 14, 17, 22, 77, 78, 85, 97,
99, 100, 114, 155, 160, 164, 169, 172,
180-182, 190, 194, 196, 200, 201, 206,
212, 224, 232, 234, 238, 242, 285,
301, 310, 318, 329, 337, 364, 371, 400,
412, 418, 422, 432, 439

Roberg, M., 76, 85

Roberts, C., 76, 85

Roberts, E. C., 216, 225

Roberts, M., 274, 301

Roblin, R. O., Jr., 226, 227, 234, 242

RochUn, E. J., 390, 397

Rodenhiser, H. A., 410, 417

Rogosa, M., 196, 197, 206, 218, 225

Roholt, K., 231, 242

Roine, P., 109, 114

Rolfe, F. W., 266, 267, 301

Rolfe, R. T., 266, 267, 301

Rose, W. C, 106, 114, 267, 301

Rosellinia arcuata, 122, 193

Rosen, H. R., 380, 398

Rosenberg, H. R., 187, 206

Rosenblum, C, 157, 169

Rubbo, S. D., 201, 206, 230, 242

Rubin, S. H., 194, 206, 233, 241

Rubus, 382

Ruger, M. L., 273, 298

Russula emetica, 292
foetens, 52
niger, 52

Ruta, 365

Ryan, F. J., 28, 29, 36, 44, 161, 162, 169,
198, 206, 216, 225, 363; 371, 413, 418
Rydon, H. N., 229, 242

Sabouraud, R., 386, 398
Saccharase {see Sucrase)
Saccharomyces, 198

anamensis, 197

carlsbergensis, 59, 200, 215, 219

carlsbergensis var. mandshuricus, 199,
219

cerevisiae, 49, 60, 76, 78, 105, 120, 193,
194, 229, 251, 327, 400, 402
effect of penicilhn on, 39, 40
growth antagonists of, 232-239
induced vitamin synthesis by, 60
nutritional adaptations of, 412
use of, in assays, 213-215
vitamin content of, 269
vitamin deficiencies of, 176, 197, 198

chodati, 199

fragilis, 140, 197

ladis, 197

ludwigii, 181, 199

macedoniensis, 180

oviformis, 180, 199

uvarurn, 195
Saito, K., 281, 301
Salmon, E. S., 391, 398
Saltation, 411
Sandholm, N. K., 261, 264
Sanger, F., 10, 22
Saprolegnia, 349

jnixta, 92, 307, 320

parasitica, 93
Sarcoscypha protracta, 345
Sarett, H. P., 232, 243
Sarver, L. A., 73, 86, 260, 265
Sawyer, W. H., 343, 354
Scandium, 81

Schade, A. L., 120, 127, 148
Schatz, A., 286, 301
Scheffer, T. C, 89, 114
Schizophyllum commune, 94, 351, 409
Schizosaccharomyces, 25

octosporus, 49

pombe, 195
Schleef, M. L., 270, 301
Schlenk, F., 197, 204

460

PHYSIOLOGY OF THE FUNGI

Schmidt, M., 390, 398

Schmitt, M. B., 17, 22, 154, 164, 169, 310,

318, 337
Schneider, G. G., 50, 63
Schoenbach, E. B., 291, 301
Schomer, H. A., 57, 63
Schopfer, W. H., 2, 7, 32, 44, 172, 174,
177, 186, 187, 192, 196, 206, 211, 214,
215, 219, 220, 225, 236, 243, 295, 301
Schubert, W. J., 135, 148
Schultz, A. S., 127, 148, 215, 225
Sciarini, L. J., 295, 301
Scleroderma aurantirion, 367
Sclerospora graminicola, 360, 381
Sclerotinia, 254

americana, 252, 253, 255, 261
cameUiae, 34, 190, 193, 196

temperature and inositol deficiency

of, 182, 184, 185
vitamin deficiencies of, 178, 179
frudicola, 77, 247-249, 258, 262, 288,
358
(See also Monilinia frudicola)
Ubertiana, 136

(See also sclerotiorum, below)
minor, 100, 190, 400
sclerotiorum, 100, 172, 374, 376, 377,

402, 425
trifoliorum, 402
Sclerotium bataticola, 100
delphinii, 76
rolfsii, 80, 376
Scott, I. T., 160, 169
Sealock, R. R., 231, 243
Seaver, F. J., 346, 354
Seifriz, W., 37, 44
Semeniuk, G., 96, 114
Septobasidium, 352, 353, 384

burtii, 385
Septoria nodorum, 100, 172, 314, 321, 425,

426, 432
Septosporium acerinum, 317
Sevag, M. G., 231, 243
Shankman, S., 216, 225
Shanor, L., 332, 337
Sharvelle, E. J., 393, 398
Shive, W., 229, 234, 242, 243
Shoup, C. S., 121, 148
Shu, P. 73, 86, 280, 302
Siminoff, P., 286, 298
Singer, T. P., 226, 243, 251, 263, 264

Sinnott, E. W., 42, 43

Siu, R. G. H., 91, 113, 114

Skoog, F. K., 120, 148, 412, 418

Smart, R. F., 357, 361, 362, 367, 371

Smit, J., 218, 225

Smith, E. C., 316, 337, 368, 371

Smith, E. L., 273, 302

Smith, F. G., 389, 398

Smith, L. D. S., 376, 398

Smith, P. E., 357, 370

Smith, R. M., 291, 302

Smith, V. M., 95, 114, 132, 148

Snell, C. T., 156, 157, 169

Snell, E. E., 74, 85, 200, 201, 206, 211,

215, 216, 225, 234, 239, 243
Snell, F. D., 156, 157, 169
Snell, N., 292, 300
Snyder, W. C, 17, 22, 305, 313, 332, 335,

337, 412, 417
Solanine, 390
Solanum, 390

tuberosum, 377
Somers, G. F., 47, 51, 64, 70, 79, 86, 96,

115, 142, 148, 277, 302
Sorbitol formula, 124
Sorbose formula, 119
Sordaria, 344, 345, 349, 353, 438
fimicola, 39, 40, 100, 122, 164, 173, 175,
178, 190, 191, 193, 208, 220, 328,
400, 428, 429, 431, 432, 435, 437
biotin and sporulation of, 330-331
carbon sources utiUzed by, 122
depth of medium and growth of, 38
pH, and growth of, 160, 162, 167

and sporulation of, 319
spore discharge by, 344
vitamin deficiency of, 167, 181
induced thiamine, 182, 183
Spergon, 257
Sphaerobolus, 347, 349, 353

stellatus, 100, 347, 348
Sphaerographium fraxini, 310, 311
Sphaeronema pruinostim, 311
Sphaeropsis rnalorum, 100, 172, 314, 428,

429, 431, 432
Spiegehnan, S., 59, 63, 412, 413, 418
Spies, T. D., 187, 207
SpineUus, 385

Spongospora subterranea, 374
Spore discharge, of aeciospores, 348-349
of ascospores, 343-346

INDEX

461

Spore discharge, of basidiospores, 348-351
effect on, of humidity, 343
of light, 339-341, 344, 346
of temperature, 351
methods of, 338-351
of peridioles, 347-348
by rain, 350-351
of sporangia, 338-343
Spore dissemination, agents of, 351, 352
Spore germination, effect on, of moisture,
357-359
of nutrients, 361-364
of pH, 360-361
of stimulants, 363-367
of temperature, 356-357
laboratory exercise, 435-436
requirements for, 355
in slime molds, 367, 368
Sporodinia grandis, 100, 307
Sporormia intermedia, 193
Sporotrichum, 132

Sporulation, effect on, of associated
organisms, 327-328
of carbon sources, 321-325
of light, 310-314
of method, of inoculation, 332

of sterilization, 332
of nitrogen source, 321
of nutritional factors, 320-333
of pH, 318-319
of temperature, 307-310
of ultraviolet light, 314-316
of vitamins, 329-332
methods of inducing, 333-334
need for aeration in, 316-318
Srb, A. M., 217, 225, 239, 242
Stachybotrys atra, 193, 195
Stakman, E. C, 382, 391, 392, 398, 410
Standard curve, 214
Stanier, R. Y., 16, 22
Staphylococcus, 284

aureus, 285
Starch, 135

scheme of utilization of, 48
Stark, W. H., 71, 86
Starkey, R. L., 159, 169, 246, 264, 271,

302
Steinberg, R. A., 10, 22, 23, 67-69, 71-74,
76, 78-82, 86, 92, 97, 105, 114, 115,
121, 123-127, 139, 140, 148, 326, 414,
418, 437, 439

Steinhaus, E. A., 383, 398
Stemonitis axifera, 367

ferruginea, 368

fusca, 367
Stephenson, M., 46, 63
Stereum frustulosum, 188

gausapatum, 121, 123, 124
Stern, K. G., 58, 63
Stevens, F. L., 315, 316, 337
Stevens, N. E., 120, 148
Stoddard, E. M., 231, 243
Stokes, J. L., 105, 115, 184, 193, 195,

207
Stomata, formation of, as result of
parasitism, 382

penetration through, 372-373
Stone, G. M., 373, 395
Stotz, E., 143, 148
Stout, P. R., 21, 23, 73, 86
Strauss, B. S., 184, 207
Streptococcus faecalis, 16, 200, 201, 228
Streptomyces, 285, 288

aureofaciens, 291

griseus, 58, 82, 127, 273, 286, 287, 290,
291

venezuelae, 287, 291
Streptomycin, 290-291
Sucrase, 49, 133
Sucrose, effect of, on sporulation, 324

formula of, 131
Sugar acids, 125
Sugar alcohols, 124
Sulfanilamide, 227, 229-231
Sulfur, as fungicide, 251-256

as metabolite, 92-94
Sumner, J. B., 47, 51, 52, 64, 70, 79, 86,

96, 115, 142, 148, 277, 302
Sure, B., 268, 302
Syncephalastrum racemosum, 16, 17, 122,

132
Syncephalis, 385
Synchytrium endobioticum, 253, 381

Takamine, J. 274, 302

Tamiya, H., 89, 115, 121, 123-125, 127,

129, 132, 136, 148, 158, 169
Tanner, F. W., Jr., 272, 273, 302
Taphrina, 375
dejonnans, 311

462

PHYSIOLOGY OF THE FUNGI

Tapke, V. F., 388, 398

Tatum, E. L., 36, 161, 199, 202, 207, 215,

225
Taubenhaus, J. J., 388, 390, 395
Tauber, H., 96, 115, 142, 148, 277, 302
Taylor, C. F., 386, 398
Temperature, effect of, on growth, 34
on resistance, 393
on sporulation, 307-310

laboratory exercise, 425
Temperature coefficient, 53
Temperatures, cardinal, 34
Tenebris moHtor, 58
Ter Horst, W. P., 235, 243, 257, 265
Ternetz, C, 88, 99, 115, 319, 337
Thatcher, F. S., 37, 44, 376, 377, 382,

398
Thatcher, R. W., 66, 86
Thaysen, A. C, 49, 64, 267, 302
Theis, E. H., 258, 265
Thiamine, antagonists of, 231-233

formula of, 187

fungi, deficient for, 190

mode of action of, 190, 191

moieties of, 187-189

specificity of, 191, 192
Thiamine pyrimidine formula, 187
Thiamine pyrophosphate formula, 191
Thiamine thiazole formula, 187
Thielavia basicola, 122

sepedonium, 111
Thielaviopsis basicola, 80, 190, 387
Thimann, K. V., 120, 148, 327, 336
Thorn, C, 34, 44, 385, 398, 401, 414, 418,

424, 439
Thomas, H. E., 374, 387, 398
Thorn, R. S. W., 108, 115
Thornberry, H. H., 262, 265, 291, 302
Thraustotheca clavata, 193
Thren, R., 400, 418
Tilletia, 250

levis, 410

tritici, 410
Timnick, M. B., 312, 316, 324, 337
Tomatin, 390

Tonnis, B., 19?, 205, 209, 224
Torula, 37

cremoris, 194, 197

lactosa, 197

sphaerica, 197

uiilis, 269, 271

Torulopsis kefyr, 197

pulcherrima, 76

sanguinea, 366, 367

tdilis, 75, 109
Toursel, O., 218, 224
Tove, S. R., 99, 115
Toxins, as pathogenic agents, 378-380

produced by fungi, 292-293, 378-379

in relation to enzymes, 376, 382
Trametes americana, 135
Transamination, 109
Trehalose, 131, 132
Treponema pallidum, 290
Trichodernia koningii, 260

lignorum, 100, 286, 314, 426, 437

viride, 222, 287
Tricholoma, 366

personatum, 293
Trichophyton, 259

discoides, 196, 200

gypseum, 238, 287

inter digitate, 76

mentagrophytes, 238, 260, 288, 412

purpureum, 238

rubrum, 42, 287
Trichotheciuni roseuni, 288

(See also Cephalothecium roseum)
Troutman, M. C, 66, 85
Tryptophane, biosynthesis of, 229
Tschang, J. L., 184, 204
Typhida variabilis, 122
Tyrosinase, 52

Ultraviolet Hght, effect of, on sporulation,
314-316

inducing mutations by, 401, 405
Umbreit, W. W., 90, 115, 154, 169
Underkofler, L. A., 274, 275, 302
Units of measure, 18
Uppal, B. N., 257, 265, 360, 371
Urease, 52
Urocystis cepulae, 307, 356

occulta, 361, 366

tritici, 366
Uromyces caryophyllinus, 247, 248, 253-
255, 362, 377

fabae, 377, 393

pisi, 348

INDEX

463

Ustilago avenae, 360

nuda, 358, 400

striiformis, 118, 120, 172, 362, 380,
401, 402, 428

tritici, 388

violaceae, 410

zeae, 360, 365
Utech, N. M., 288, 302
Utilization, definition of, 87

Valsa leucostoma, 321

pini, 190, 196
Vanadium, 81

Vandecaveye, S. C, 219, 225
Van Lanen, J. M., 272, 273, 302
Van Niel, C. B., 132, 148
Van Rij, N. J. W., 39, 40, 44
Van Slyke, D. D., 58, 64, 161, 162, 170
Variation, basis of, in imperfect fungi,
411-412

physiological, 400-403
Vasudeva, R. S., 376, 398
Vaughan, J. R., 288, 302
Venturia inaequalis, 254-256, 288, 304,

356, 358
VerticiUium, 378

alho-atrum, 100
Vincent, J. M., 94, 114
Vitamers, 186
Vitamin Bi {see Thiamine)
Vitamin Be (see Pyridoxine)
Vitamin B12, 82, 273
Vitamin deficiencies, 172-186

absolute, 181, 182

conditioned, 181-184

methods of detecting, 174-176

multiple, 178-181

partial, 174, 177, 179

single, 178

total, 174, 177, 179
Vitamin H {see Biotin)
Vitamin K, antagonists of, 235-236
Vitamin K2 formula, 235
Vitamins, antagonists of, 229-236

characteristics ot, 171

concentration of, laboratory exercise,
433-435

deficiencies for, laboratory exercise,
432-433

Vitamins, effect of, on sporulation, 329-
332
fungi self-sufficient for, 172
inhibitory effects of, 184-186
production of, by fungi, 272-273
synthesis of, by fungi, 171, 172, 272,
273

Vitucci, J. C, 123, 135, 147

Volcani, B. E., 239, 243

Volkonsky, M., 93, 115, 132, 135, 148

Von Loesecke, H. W., 269, 279, 302

Waksman, S. A., 76, 84, 86, 159, 169, 246,

264, 266, 267, 278, 279, 281-283, 285,

286, 290, 298, 299, 302
Walker, J. C, 356, 371, 390-392, 397, 398
Walker, L. B., 347, 348, 354
Walker, T. K., 278, 302
Wallerstein, J. S., 267, 303
Wallerstein, L., 275, 303
Walton, R. C, 344, 354
Warburg, O., 58, 64
Ward, G. E., 271, 272, 281, 282, 299, 303,

326, 336
Ward, M., 392, 398
Waring, W. S., 73, 86
Water, 87, 88, 149
Waters, C. W., 393, 398
Webb, R. A., 269, 300
Webb, R. W., 360, 361, 363, 364, 371
Weedon, F. R., 253, 264
Weimer, J. L., 50, 63, 344, 354
Weinhouse, S., 143, 148
Weinstock, H. H., 198, 207
Weiss, S., 295, 303
Welch, A. D., 226, 243
Wellensiek, S. J., 383, 393, 398
Wellman, F. L., 356, 371
Wellman, R. H., 245, 261, 264, 265
Wells, P. A., 280, 303
Werkman, C. H., 73, 86, 128, 136, 138,

146, 148
West, R., 273, 303
Westergaard, M., 325, 326, 337
Weston, W. H., Jr., 35, 43, 424, 438
Weswig, P. H., 231, 243
Wheeler, H. E., 404, 417
Whiffin, A. J., 288, 303
White, A. G. C., 127, 148, 232, 233, 244

464

PHYSIOLOGY OF THE FUNOI

White, M. G., 138, 139, 148

White, N. H., 12, 23

White, W. L., 49, 64, 221, 222, 225

Wieland, H., 292, 303

Wilcoxon, F., 247, 248, 252-255, 264, 265

Wilkins, W. H., 285, 303

Willainan, J. J., 138, 139, 148, 275, 299

Williams, B., 291, 297

Williams, R. J., 198, 199, 204, 206, 207

Williams, R. R., 187, 207

Wilson, A. N., 232, 233, 243

Wilson, P. W., 51, 62, 99, 115

Wilson, W. E., 121, 148

Wingard, S. A., 393, 398

Winzler, R. J., 56, 60, 61, 64, 194, 207,

233, 241
Wirth, J. C, 102, 108, 115, 141, 148, 186,

191, 192, 207
Witkop, B., 292, 303
Wohler, F., 277, 303
Wolf, F. A., 35, 44, 99, 115, 293, 303, 347,

354, 371, 386, 399, 401, 402, 418
Wolf, F. T., 35, 44, 99, 115, 121, 148, 231,
243, 293, 303, 347, 354, 371, 386, 399,
402, 418
Wolfrom, M. L., 290, 299
Wolpert, F. S., 159, 160, 170
Wood, H. G., 136, 148
Wood, W. A., 201, 207
Woods, D. D., 230, 243
Woolley, D. W., 195, 207, 226, 229, 231-
233, 235, 236, 237, 239, 240, 242-244,
378, 399
Wooster, R. C, 199, 207
Worley, C. L., 28, 44
Wright, L. D., 215, 225, 226, 244
Wright, L. T., 291, 303
Wyss, O., 167, 170, 183, 184, 202, 207,
215, 225

Xylaria hypoxylon, 190

rnali, 100
Xylose formula, 123
Xylulose formula, 123

Yarwood, C. E., 311, 337, 358, 359, 371,

393, 399
Yaw, K., 121, 148
Yeager, C. C, 384, 399
Yeast, film formation by, 39

heat production by, 61

measuring growth of, 30

morphology of, 39

protein of, 268

respiration of, effect of cyanide on, 56

use of, for food, 268, 269

vitamin content of, 269
Yegian, D., 240, 244
Yoe, J. H., 73, 86, 260, 265
Yoshimura, F., 74, 86
Young, H. C., 79, 86, 100, 115, 253, 265
Yuill, J. L., 275, 303

Z factors, effect of, on spore germination,

364-365
Zentmeyer, G. A., 260, 265
Zikes, H., 33, 39, 44
Zinc, 76, 282

Zygorhynchus moeUeri, 100
Zygosaccharomyces, 199, 327

acidifaciens, 91, 92

ladis, 197

marxianus, 215

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