PHYSIOLOGKAL STUD-1E3 QN
CALVA‘EIA SPEQEES
M

Thesis ¥or the Degree 0* pk. D.
MICHNAN STATE UNWERSITY
Magdalena Sedfimayr
nee 3mm.

1%60

TH ESYS

This is to certify that the
thesis entitled

PhysiOIOgical Studies on Calvatia Species

presented by

Magdalena Sedlmayr nee Puzna

has been accepted towards fulfillment
of the requirements for

Ldegree tum
(Department of Botany & Plant Pathology)

< ,g‘j/l, 2,? 76,341? '~..£

Major professor \
E. S. Beneke

 

Date Feb. 1t; 1950

 

0-169

 

LIBRARY

Michigan State
University

 

PHYSIOLOGICAL STUDIES ON CALVATIA SPECIES

BY

‘Magdalena Sedlmayr
nee Buzna

A THESIS

Submitted to the School for Advanced Graduate Studies of
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1960

33%Qfl\
L-lfiv‘i

ACKNOWLEDGMENTS

The author wishes to express her sincere thanks to Dr. Everett
8. Beneke, under whose constant interest, helpful criticism and guidance
this investigation was undertaken. The writer is also greatly indebted
to Dr. G. B. Wilson for his valuable suggestions and kind help in the
preparation of this thesis. Sincere thanks are also due to Dr. Hans
A. Lillevik for his aid and criticisms in the chemical evaluations in
this thesis.

The author especially wishes to express her thanks and gratitude
to the late Dr. E.H. Lucas for his encouragement and assistance towards
her acceptance into Graduate School.

This work.was made possible by a grant from the National In-

stitute of Health for which the author expresses grateful appreciation.

PHYSIOLOGICAL STUDIES ON CALVATIA SPECIES

BY

3/
Magdalena Sedlmayr
nee Buzna

AN‘ABSTRACT

Submitted to the'School for Advanced Graduate Studies of
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1960

Approved W

 

ABSTRACT

The genus Calvatia has assumed a greater importance in recent
years following the discovery of tumor-retarding properties in some
of its species. A review of the literature revealed no detailed re-
ports on the nutritional-physiological requirements of these fungi.
Consequently, this was undertaken as the general purpose of this work.

The specific purpose of this investigation was to determine the
vitamin and carbon requirements of four strains of Calvatia. The
utilization of the nutrients were determined by the growth reSponse
of the organisms and measured on the basis of the average mycelial
dry weights of two or four replicates after four weeks of incubation.

The superiority of the submerged to the surface culture method,
as a technic for the study of the nutrition of the fungi, was demonstra-
ted. A synthetic medium was found available for culturing Calvatia
species. The optimum pH and temperature range for the mycelial growth
of the organism was determined.

The investigations of the vitamin requirements indicated that
Calvatia strains have in common a total deficiency for thiamine. The
capacity to synthesize the other vitamins studied, however, (biotin,
pyridoxine and inositol) appeared to be adequate. The action of thiamine
seemed not to be quantitative within certain limits because an increase
in the dosage caused no significant increases in the growth.

Different carbon sources and their utilization by the four strains
were investigated. The ability to utilize these carbon sources by the
Calvatia strains tested depended both upon the configuration of the

compounds and the particular abilities of the organisms.

 

 

 

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TABLE OF CONTENTS

INTRODUCTION

PRELIMINARY EXPERIMENTS

VITAMIN UTILIZATION BY CALVATIA SPECIES

DISCUSSION AND RESULTS

CARBON UTILIZATION BY CALVATIA SPECIES

SUMMARY

BIBLIOGRAPHY

18

34

48

78

8O

INTRODUCTION

Physiological studies of the higher Basidiomycetes have proved in
recent times to be an important field of investigation. These organisms
have not only been used for routine biological studies, but also for
bioassays, for determination of steps involved in the synthesis and
degradation of essential metabolic substances, as well as for investi-
gations which followed the discovery of several antibiotics produced by
them.

In the last 10 to 15 years several Basidiomycetes have been found
to produce antibiotics. Wilkins (1945) investigated the production of
bacteriostatic substances by certain Basidiomycetes. Hervey (1947) sur-
veyed 500 Basidiomycetes for antibacterial activity. Bose (1947) found
an antibiotic in Polyporus species; Kavanagh g£.gl.(l949) in Marasmius

coniggnus; Doery g£_§l.(l951) in Caprinus quadrifidus; Anchel g£_§l_(1952)

 

in Fames juniperus. Atkinson (1954) named and described Psalliotin, the

antibiotic produced by Psalliota xanthoderma.

 

In the Basidiomycetes the nutritional-physiological experiments are
limited mostly to studies on mycelial growth. The production of basidio-

carps by Collybia velutipes on synthetic medium was studied by Plunkett

 

(1953) and with Caprinus species by Bille-Hansen (1953). Ashan (1954)
reported on the influence of different culture conditions on the growth
of Collzbia velutipes, and Scheler-Correns (1957) studied the effect of

various sources of nitrogen on fruiting body production by Caprinus lagopus.

 

Recently Koch (1958) reported his investigations on the mycelial growth
and fruiting body formation of Polystictus versicolor, Polysporus annosus,

Pleurotus ostreatus and Psalliota bispgra. Lucas gg_gl.(1958) found,

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that Calvatia species have certain tumor-retarding properties. Con-
sequently there has arisen a requirement for physiological studies with
these organisms to provide a basis for further investigations of the
circumstances under which Calvatia species will be able to produce agents
active against tumors.

As far as Calvatia species are concerned, just a few reports dealing
primarily with the taxonomy and morphology of this fungus have been men-
tioned in the literature. One of the first comparative descriptions on
the Gasteromycetes has been reported by Hollos (1904). Since this publi-
cation, a number of reports have been published about the development of
Gasteromycetes in different geographical zones, by Fries (1919) for South
America, and by Garner (1956) for central America. Coker and Couch (1928)
described the Gasteromycetes of the eastern United States and Canada;
Cunningham (1944) the Gasteromycetes of Australia and New Zealand; Dennis
(1953) the West Indian Gasteromycetes.

Schwartz (1933) discussed the morphological and taxonomical characters
of several Lycoperdaceae. Ritchie (1948) investigated the develOpment of
the fruit body of Lycoperdon oblongispgrum.

Various synthetic or semi-synthetic media have been deve10ped for
growing Basidiomycetes, but the nutritional aspects of Lycoperdaceae have
not been studied extensively, and a chemically defined medium for their
growth has not been developed.

An excellent growth of Calvatia gigantea has been observed by Stevens
(1957) in modified Czapek-Dox medium. Successful attempts to germinate
the spores of Calvatia species have not yet been reported. This fact

eliminates the possible genetical approach to solve physiological problems

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in the organism. Results of nutritional experiments can best be expressed
by the mycelial growth of the fungus studied. Therefore, it seems to be
necessary to determine the meaning of growth. Growth is an increase in
‘mass or in number of cells, which requires time for its various manifesta-
tion. Cochrane (1957) claims that the precise definition of growth in
fungi depends on the method of measurement used.

The Calvatia species used in this investigation were supplied and
originally identified by Dr. J.A. Stevens of Michigan State University,
East Lansing, Michigan. The identification was confirmed by Dr. A. H.
Smith of the University of Michigan, Ann.Arbor, Michigan. The following
strains were employed in this study:

1. Calvatia gigantea (1018F) was obtained from an area five miles
west of Lansing. The spores average 3.7 u, are globose or sphaerical
with smooth walls and under high power minute echinulations can be observed.

2. Calvatia gigantea (1019B) was obtained from a woods on the
Michigan State campus. The spores average 4.0 u, are globose or sphaeri-
cal with smooth walls and under high power minute echinulations can be
observed.

3. Calvatia gigantea (766) was obtained in Bronson,'Michigan. It
is thought to be close to Calvatia gigantea because of the following
characteristics. The spores measure between 3.5-4.2 u, are nearly smooth
and globose or sphaerical. The capillitium is long, thin, 6 u in dia-
meter, and unbroken. It differs from the hundreds of other Calvatia
gigantea species in that the peridium measured minimally 3-4 mm thick,
which is at least twice the size of the normal Calvatia gigantea peridia.

Culturally, the growth of this organism is typically flat, sparse and

relatively fast growing. This may be a new Calvatia species. It appeared
to have characteristics midway between Calvatia gigantea and Calvatia
Bovista. The capillitium of the latter is thick, 17 u in diameter and
breaks up easily at maturity. In addition, the peridium is suggestive

of Calvatia pachyderma, however, the spores of the species are ellipsoid

 

and the capillitium curved like a boomerang.
4. Calvatia fragilis (1020) was obtained northwest of East Lansing.

This organism is considered to be Calvatia fragilis based on strictly its

 

morphological characteristics. The culture was isolated from an immature
spor0phore, which had the typical "top" shape of the species. Since no
spores were available, its tentative identification was made on the basis
of information obtained from the owner on whose property it was found;
namely that in previous years spor0phores arose in the same location and
never grew larger than 4 cm high.

These four strains were chosen for this investigation because of
their different types of growth and their responses to the Sarcoma 180
test as performed by the Sloan-Kettering Institute for Cancer Research.
The photographs of the four strains of Calvatia illustrate the types of
growth and the different growth rates. The cultural characteristics of
these four strains are described below:

Strain 1018F. The mycelimm-growth in the colony is a dense tan
mat which shows submerged growth with furrows and cracks in the medium.
The mycelial mat exhibits a colored zonation. The spores are smooth and
spherical, with a diameter of 3.7 u. The mycelial growth at 24°C is 8 mm
after 7 days; 21 mm after 14 days; 28 mm after 21 days and 44 mm after

28 days. It is a relatively slow growing strain. The sporophore extract

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“I‘Thfee weeks old culture of Calvatia gigantea (1019 B) I“"

 

 

 

Three weeks old culture of Calvatia gigantea (766)

 

 

 

Three weeks old culture of Calvatia fragilis (1020)

for this strain did not inhibit Sarcoma 180 in mice at a dilution of
1:25.

Strain 1019B produces a dense, tan matted colony which does not have
any cracks in the agar. The diameter of the spores are 3.7 u, smooth
and spherical. The mycelial growth at 24°C is 10 mm after 7 days; 22 mm
after 14 days; 31 mm after 21 days; and 48 mm in 28 days. This strain
is relatively slow growing. The aporophore extract for this strain
inhibited Q ') Sarcoma 180 in mice at a dilution of 1:100.

Strain Z§§.has a thin, flat type of growth during the first 10
days, later developing aerial hyphae which are never so dense as the
other strains. The mycelial growth at 24°C is 23 mm after 7 days, 51
mm after 14 days, 80 mm after 21 days and 90 mm after 28 days. It
is considered a relatively fast grower. The sporophore extract from
this strain has shown a strong inhibition 6:43 of Sarcoma 180 in mice
at a dilution of 1:120.

Strain lggg_exhibits a thin flat type of colony growth in 10 days.
Toward the end of 18 days, the colony becomes cottony and granular. In
addition, a water soluble brown pigment is produced. Mycelial growth
at 24°C is 20 mm after 7 days; 49 mm after 14 days; 77 mm after 21 days;
and 90 an after 28 days. The rate of growth for this strain can be
considered as relatively fast. The sporophore extract for this strain
inhibited (t ‘9) Sarcoma 180 in mice at a dilution of 1:10.

This work is limited to the physiology of Calvatia species. It
deals with the effect of various growth promoting substances and carbon

sources on the metabolism of the four strains.

PRELIMINARY EXPERIMENTS

In order to obtain certain necessary information to perform the
investigation on the effects of the different growth factors and carbon
sources, some preliminary experiments were made.

It is well known that internal and external environmental factors
may affect the mycelial growth of a fungus. In the case of Calvatia
species there is not much information about the internal factors as
far as genetical constitution is concerned; but the age, source and
kind of inoculum can be controlled. Among the external environmental
factors, temperature, pH, and the type of nutrient in the medium.have
the most influence on growth of Calvatia. Therefore, the first two
external factors were investigated at this time.

Temperature

Temperature affects every function of the organism, and has a very
important effect upon enzyme systems. It is known, that the rate of
enzyme activity, enzyme production and enzyme destruction are all in-
creased by a rise in temperature up to a point. Slight changes in
temperature may markedly alter the rate at which a certain nutrient
is utilized.

For each fungus there is a temperature below which it will not
grow - the minimum temperature. Likewise there is a temperature above
which growth ceases - the maximum.temperature. The two extremes in
temperatures indicate the temperature range for the organism, which
varies to some extant with the various species. ‘Most fungi do not
grow or grow‘very slowly at 0°C, and they are usually unable to grow

at temperatures above 30°~35°C. The optimum temperature for growth is

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usually between 20°C and 30°C. There are, however, certain very striking
exceptions to this generalization, in both directions.

It has to be considered that the reported optimum temperatures for
the fungi are valid only under specified conditions of time, medium and
method of measurement. That there is no single temperature optimum for
growth has been stated by Cochrane (1958). A.given metabolic process,
respiration, antibiotic production or vitamin synthesis does not necessarily
respond to temperature in the same way as does the process of growth.
Fries (1953) mentioned an example of the dependence of temperature
characteristics on other factors. He reported that Caprinus fimetarius
grows poorly at 44°C because of the failure of methionine biosynthesis;
however, if exogenuous methionine is supplied, growth at this temperature
is normal.

Physiological experiments with Basidiomycetes are most often per-
formed at 25°C. Optimum growth of some of the mycorrhiza forming §2l2££.
has been found byVMelin (1925) to occur at 25°C. Nikola (1948) found
the same true for Cenoccocum.species. Norkrans (1950) reported similar
results with Tricholoma species. Certain other species preferred a
slightly lower temperature. Several‘gygggg_species showed the best
growth at 20°C, according to Fries (1949). The coprophilic Psalliota
bispgra showed a good growth in a temperature range between 20°C and
27°C, with the Optimum at 24°C as observed by Treschow (1944). The
influence of temperature on the growth of seventeen different Coprinus
species has been investigated by Fries (1956). About half the tested
species showed optimum growth at 25°C. Two species preferred temperature

as low as 15-250C. All of the species which grew at higher temperatures

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were coprophilic.

To determine the optimum.temperature of Calvatia species studied,
a simple agar plate method was used. To get comparable replicates and
to avoid the drying out of the agar, equal amounts of 40 ml of modified
Czapek-Dox agar (medium.A), (see experimental), was poured into sterilized
flat bottom Petri dishes. ‘Mycelial plugs measuring 3.5mm were used as
the inoculum. ‘These were obtained from the growing edge of a 14-day old
culture, using a sterile cork-borer. The excess agar was removed and
the plugs were then placed in the center of a Petri dish, containing
medium A agar. Quadruplicate plates were incubated at 8, 12, 16, 20,
22, 24, 26, 28, 30 and 37°C. The recording of the diameter of each
growing colony at each temperature was made after 7, 14, 21 and 28 days.

The average resultant values of a typical fast growing-and a typical
slow growing strain are shown diagrammatically. (Figure 1, 2). It was
established from the results of the temperature investigations, that
Calvatia species grew at temperatures ranging from 8°C to 28° C, with
the optimum temperature range between 20°C and 24°C.

Stevens (1957) investigated the elaboration of the tumor retarding
material of Calvatia maxing.(gigantes) #642 by using shake alltures at
five temperature levels: 16, 19, 22, 25 and 28°C. The result was that
the active principle reached a maximum concentration on the 24th day
of submerged culture at 19°C.

pH of the Medium

Most fungi will tolerate a wide range of hydrogen-ion concentration

of the medium. Inhibition of growth is usually rather sharply defined

at the limits of this range. Neutral or slightly acid reaction of the

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FIGURE 1
28 days
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The effect of temperature on the growth of Calvatia gigantea (766)

12

FIGURE 2
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The effect of temperature on the growth of Calvatia W (10131?)

13

medium has been found the best pH for growth of most of the fungi.
Growth is usually stapped on the acid side at pH 3 and on the alkaline
side at pH 8-9. There are, however, exceptions to these generalizations.

Fungi, as a result of their metabolic activities, ordinarily change
the pH of the media in which they grow. The pH is raised by absorption
of anions or production of ammonia from nitrogenous compounds, and lowered
by formation of organic acids or absorption of cations. These metabolic
products of growth complicate pH experimental results, particularly in
the poorly buffered media comonly employed. Since fungi differ in their
metabolic activities and their rates of growth, the pH changes produced
in the culture medium will differ too. The pattern of pH changes for
the same fungus will depend upon the composition and concentration of
the media used.

The changes in environmental factors, which affect the rate of
growth of the organism such as temperature, time of harvest, gross changes
in medium, growth factor supply, etc. , may also affect the changes of
pH of the culture medium.

As far as the Basidiomycetes are concerned, earlier investigations
has shown that most species prefer an acid or neutral reaction of the
medium as smarized by Wolpert (1924).

Modess (1941) surmised that the acidity of the habitat might be a
guide to finding a most suitable pH range for cultivation of Coprinus
species and, therefore, some samples of substrate from the habitats
were collected to determine the required acidity of the fungi. His
findings sensed to prove his hypothesis. In culture many of the larger

Basidiomycetes are often unable to grow at an initial pH above 7.0.

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Melin (1925) found that pH 5.0 was the Optimal hydrogen-ion con-
centration for Boletus variegatus. Norkrans (1950) determined the optimum
pH for Tricholoma nuggg_to be between 5.0-6.0. The Optimal pH range was
even narrower in case of Fomes annosus; it was between 4.6 and 4.9, as
was observed by Etheridge (1955). Lindeberg (1944) reported the pH
range of 5.7-6.4 as the Optimal for most Of the'Marasmius species. How-
ever, he also reported that Marasmius Eggglg_grew in culture and has been
found in nature on substrates of a widely different pH. Several other
fleshy Basidiomycetes require alkaline conditions for best growth. In
nature Cgprinus species grow on manure or soil rich in humus, which may
indicate special demands for a less pronounced acid reaction of the en-
vironment, as was noted by Johnson and Jones (1941). They also found
that Coprinus cubensis could grow at pH 5.9-9.2 on potato agar.

Fries (1956) investigated the pH requirements for different Coprinus
species. She frequently found two pH Optima for certain Organisms,
and between these two Optima a depression at pH 7.5-8.0. This minimum
‘merely reflects pH dependent on unavailability of one or more inorganic
elements. The provision of iron, zinc and calcium in available forms '
eliminates this double Optimum and replaces it with a broad single
Optimum zone. Fries concluded, from her results, that the cOprOphilic
Coprinus species are extremely basiphilic and even other species of this
genus prefer a more alkaline pH range than most other fungi.

The initial pH resulting in Optimum growth of Calvatia species was
detenmined by agar plate method. The initial pH was adjusted to varying
values from pH 3.5-pH 7.0. The technic employed was the same as with

the temperature experiments, but only one temperature,’24°c}'was used.

 

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The mycelial growth was measured after 7, 14, 21 and 28 days.

Figure 3 shows diagrammatically that the best mycelial growth was
attained at initial pH 5.5, which was the unadjusted (natural) pH Of
both medium employed in this work on the physiology of Calvatia species.
In most physiological studies it is necessary to have the pH controlled.
In the case Of Lindeberg's medium, M/25 phOSphate buffer (KH2P04) was
employed. Lindeberg (1944) Observed that a concentration up to 0.04 M
phosphate had a sufficient buffering capacity and just a slight in-
hibitory effect for the higher Basidiomycetes. The use of calcium was
found to reduce this slight toxic effect of phosphate.

Nitrogen Requirement

In this physiological study Of Calvatia species, the nitrogen re-
quirement Of these fungi was not investigated extensively. Therefore,
Lindeberg's conclusion that ammonium-tartarate and asparagine are the
best nitrogen sources to use in chemically defined medium for studying
the higher Basidiomycetes has been assumed.

Later publications have confirmed this assumption. For example,
Fries (1955) reported that ammonium-tartarate and asparagine gave the
widest range Of growth and the smallest change Of pH in case of CoErinus
species.

Lamprecht (1957) investigated the physiological influence of pH,
concentration of constituents of the medium and the isoelectric point
in the growth Of Marasmius species. He considered ammonium-tartarate
the best nitrogen source for these fungi. However, NH4, N03 and KCN
uptake was not dependent on the isoelectric point. He also found
that the N03-uptake was better in lower pH and the pH close to neutral

was more suitable for the NH4-uptake.

80 90 100 110 120 mm
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70

50

40

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FIGURE 3

 

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7 14

The effect of various pH levels on the growth of Calvatia gigantea

(766) at 24°C

3
21

16

pH 5.5

pH 6.5

'pH 4.5

pH 7.0

pH 3.5

28 days

17

To find out how the Calvatia species were able to utilize ammonium,
taitrate and organic nitrogen, the growth response on four nitrogen sources
were tested: ammonium-tartarate, asparagine, NH4Cl, and KN03. In this
experiment a quantity of nitrogen identical to that employed by Lindeberg
(1944), 0.28 gram.per liter was used for each source Of nitrOgen investi-
gated. Ammonium-tartarate turned out to be the best Of the four nitrogen

sources tested as observed by growth studies on Calvatia species.

18

VITAMIN UTILIZATION BY CALVATIA SPECIES
Review of Literature

A11 living organisms, including fungi, require minute amounts of
specific organic compounds for normal growth, reproduction and other
vital processes, in addition to those which yield energy or are used
for structural purposes. The cell may synthesize its own supply of one
of these growth factors, i.e. vitamins, or it may be dependent in whole
or in part on an exogeneous supply. So, organisms differ widely in
their synthetic capacities for the various growth factors, i.e., vitamins.

Some fungi are self-sufficient with respect to growth factors.

They are able to synthesize their vitamins from pure inorganic chemicals
of a synthetic medium, autotrophic fungi. Others lack the ability to
synthesize sufficient quantities of one or more growth factors and are
called vitamin deficient fungi, heterotrophic fungi.

Both terms, growth factors and vitamins, will be applied to the
same compounds, although the terms are not synonymous. The term growth
factor has a broader meaning than.vitamin. It includes the components
and derivatives of some vitamins as well as other compounds which can
not be classified otherwise at present. Many Of the known vitamins have
a catalytic function in the cell as coenzymes or constituent parts of
coenzymes.

Scthfer (1934) was one of the first investigators who studied
extensively the vitamin requirements Of the filamentous fungi. He re-
cognized the thiamin deficiency of Phycomyces blakesleeanus. There
are circumstances, when the cell depends absolutely On an external supply

and at least over a certain range, growth will be proportional to the

 

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supply of the required factor or factors. In this case, the deficiency
is total, as it was reported for thiamine in Ehyggmy§g§_b1akesleeanus
by Sch3pfer (1934).

There are certain other fungi able to synthesize vitamins, but so
slowly that under the usual condition of culture, the rate of all other
processes is potentially faster than vitamin synthesis. In this case
the organism.grows slowly in the absence of exogeneous vitamin, but
responds to an external supply by a faster rate of growth. Such partial
deficiencies are also common, perhaps even more so than complete de-
ficiencies, according to Lindeberg (1944) who observed partial deficien-
cies for thiamine in species of‘Maggsmius. The degree of partial de-
ficiency may vary widely from slight to nearly total and it is more
pronounced during the early stages of growth.

Multiple requirements are especially common in the yeast, however,
it can be found among the filamentous fungi also. The multiple deficien-
cies may be total or partial. Lindeberg (1946) found that Collybia
dryophila was not able to assimilate the nutrient solution in the
presence of either thiamine or biotin. But if thiamine and biotin were
both added, a satisfactory growth was Obtained. Absolute deficiencies
are not known to be influenced by environment, while conditioned de-
ficiencies may be affected by nutritional factors or by factors of the
physical environment. According to Cochrane (1958) the known types of
conditioned deficiency in fungi may be classified as follows:

1. The deficiency is apparent or more severe at particular tem-
peratures, pH levels or salt concentrations.

2. The requirement for a vitamin is reduced, or more rarely,

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20

eliminated by provision in the medium of a precursor or of a metabolite
for the synthesis Of which the vitamin is essential.

3. The deficiency is limited to, or more acute, at a particular
stage of develOpment.

Since temperature, vitamin supply and pH are closely associated with
activities Of enzyme systems, it seems logical that these factors might
be closely associated in their effects upon growth. It is a possibility
that fungi change in their synthetic capacity and in their needs for
certain.vitamins during develOpment. It is quite possible that spore
germination may require factors which mature mycelium can synthesize for
itself.

In fungi the relative effect of the presence of vitamins in the
medium usually is measured by the resultant vegetative growth, although
vitamins are known to affect reproduction and other processes also. To
find out the vitamin deficiency of a fungus, we must consider two im-
portant features as noted by Lilly and Barnett (1951):

1. The effect of different amounts of vitamin in the medium.

2. The response of the fungus over a period of sufficient duration
to allow maximum growth.

Vitamin deficiencies among the fungi have been detected only for
certain members of the water soluble B complex group. The most common
vitamins are: thiamine, biotin, pyridoxine and inositol.

Thiamine

Thiamine was the first vitamin to have been studied as a known entity
in the nutrition of fungi. ‘Most of the filamentous fungi are deficient
for this vitamin. Their requirement for thiamine varies widely depending

on time of harvest, temperature, stage of development, composition of the

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21

medium, etc., but for most fungi 100 X of thiamine per liter of medium
is near to Optimum for growth and sporulation. The structural formula

Of thiamine is:

 

 

 

 

 

 

 

CH3
N c NHZ .HCl / _
CH I (|: CH 1:.- C (i CHZOHL‘ DH
3 11 ll 2 ,” \CH 3
N .—- CH HC 1"
C12H17N4OSC10HCI

Thiamine Chloride Hydrochloride

The thiamine molecule contains two rings, a substituted pyrimidine
and a substituted thiazole. Thiamine has an important role in the regu~
lation Of carbohydrate metabolism.of fungi and is undoubtedly involved
in other metabolic functions as well.

The metabolic active form of thiamine is the pyrophosphate, long
known as cocarboxylase because Of its coenzyme function in the decarboxyla-
tion of pyruvic acid. Cocarboxylase or thiamine perphosphate is the

perphosphoric ester of thiamine.

CH2 0 o
N: c ——NH2 l u u
l l + c=C—-CH2—CH2- o—P—o—P—OH
cuz—c c —-—CH2-——-N< l | I
II n /__ CH -—3 OH on
N—-—CH c1

Thiamine Perphosphate
This molecule is the coenzyme or prosthetic group of the enzyme
decarboxylase. It also participates in a variety of enzymatic reactions
on °(-kato acids, and it is also a coenzyme for transketolase (Jensen,

1954).

22

Pyruvic acid is one of the key intermediate products of carbohydrate

metabolism; it is transformed into 602 and acetaldehyde by enzyme carboxylase.

cua-co-coou \ CH3-CH0 + coz

 

pyruvic acid ;7 acetaldehyde

carboxylase (TTP)

Lilly and Barnett (1951) reported that pyruvic acid accumulates in
the culture medium of many thiamine deficient fungi when insufficient
thiamine is present in the medium.and at the same time the pH of the
medium decreases. .They found the accumulation of pyruvic acid in the
culture medium in case Of Sordaria fimicola and lenzites £52233, especially
during the early period of growth. Haag and Dalphin (1940) mentioned
that the maximum accumulation in Phycomyces blakesleeanus cultures
occurred when about l/20th of the Optimum amount Of thiamine was added.
Nyman and'Melin (1940) noted that all the examined species of Basidio-
mycetes were heterotrophic with respect to thiamine but autotrOphic or
self-sufficient with respect to biotin when grown in nutrient solution.

Robbins and Hervey (1955) reported Stereum murraii to be deficient
for thiamine. Fries (1955) found in her physiological studies of Caprinus
species that in these thiamine deficient fungi several degrees of thiamine
heterotrOphy can be observed. Hawker (1939) stated that thiamin also
influences the reproduction of many fungi. Some grow fairly well, but
remain sterile (i.e. no reproductive structures) in the absence of
thiamine. Thus the requirements for sporulation are higher than those
for growth. Fungi with moderate powers of synthesis can produce enough

for vegetative growth, but not enough for sporulation. The role of

23

thiamine in the metabolism of pyruvic acid is clear from its accelera-
tion of ethanol formation in Rhisopus cohnii, as mentioned by Sch8pfer
and Guillaud (1945). Fraser and Fugikova (1958) observed that the pre-
sence of thiamine was necessary for an appreciable growth response to
the amino acids in the case Of.Agaricus bisporus.

Shunt reactions are Often found in mold metabolism in culture media.
Wirth and Nord (1942) reported that the metabolic shunt in Fusarium lini
cultures resulted in the accumulation Of pyruvic acid in the medium.

In these strains an induced cocarboxylase deficiency results in a re-
tarded rate of pyruvate decarboxylation, as compared to the rate of

the formation Of this acid from carbohydrate. Addition of thiamine to
the cultures restores the cocarboxylase level essential for maximum
efficiency of carboxylase activity. SO the bottleneck is eliminated

and the pyruvic acid no longer accumulates in the culture medium. Grimm
and Allen (1954) reported the effect of thiamine in promoting cytochrome
synthesis in the case of Ustilago sphaerogena.

.A great deal of work.has been done with the thiamine deficient
fungi which differ in their ability to utilize or synthesize the moieties
of thiamine. The most common requirement is for the pyrimidine moiety
according to Leonian and Lilly (1940). Most of the thiamine deficient
fungi can synthesize thiazole and couple the two moieties to make the
complete molecule.

Biotin

Biotin appears to be the most important growth factor for yeast,

but numerous filamentous fungi have been reported to be deficient for

this vitamin according to Burkholder and Moyer (1943). The structure Of

t.)

24

 

 

 

biotin is: 0
I!
C
H T C--—-H
H2 C (CH2)4 COOH

 

 

I
\S/w2

Biotin C10H1603N25
Biotin is active at greater dilution than thiamine. The absolute re-
quirement is usually less than 5 5’ [liter of medium. Some species of
Marasmius commonly occurring on the litter under forest trees require
an external supply of thiamine, while Others also require biotin. Some
have a partial need of biotin so that growth in the presence of thiamine
is further increased by the addition of biotin as has been concluded
by Lindeberg (1941).

A number of enzymatic reactions have been discovered and analyzed
in which biotin appears to participate in a direct or indirect manner.
Gyorgy (1954) suggested that enzymatic action of biotin was related
to the synthesis of asparatic acid. Later he also found that the oxi-
dation of pyruvic acid was probably the result of faulty carbon dioxide
transfer in the absence of biotin. Biotin is also involved in the
deamination Of certain amino acids and in the biosynthesis Of Oleic
acid. It probably plays a role in the succinic acid dehidrogenase
and amino acid oxidase.

Evidence from studies of Memnoniella echinata by Perlman (1948)
and Of Eremothecium ashbii by McNutt (1954) implicates biotin in the

synthesis of asparatic acid, since asparate partially replaces biotin

 

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25

in nutrition. Lardy §£_§l.(1947) reported that the inability of
biotin-deficient Lactobacillus arabinosus to synthesize asparatic acid
results from the failure to condense pyruvic acid and 002 to form
oxalacetic acid which could then be transaminated to form asparatic
acid.

Another function of biotin is as a coenzyme in the fixation of
C02 in living systems as has been mentioned by Foster (1949):

biotin
C02 4‘ CHZ-CO-COOH z: :4?) HOOC'CHZ‘CO’COOH

&

 

pyruvic acid oxalacetic acid
oxaloacetic decarboxylase

A considerable portion of the oxalacetic acid so produceh is aminated
to yield asparatic acid which is utilized for protein synthesis. Another
probable function Of biotin in fungi is in the synthesis of essential
fatty acids. According to Hodson (1949) oleic acid partially replaces
biotin for a Neurospora crassa nutant. The same is true for Ophiostoma
pini_described by'Mathiesen (1950). Mandels (1955) reported that
growth of Myrothecium verrucaria on glucose agar is interrupted shortly
after germination. Biotin at 10‘3 - 10"4 4f'lliter induces continued
growth after germination. It has been shown that biotin is released
from the spores after germination and that growth then stOps due to a
deficiency within the cells. Growth is resumed when biotin accumulates
in the environment of the sporling to a sufficient concentration.
Pyridoxine or Vitamin 86

Certain fungi are able to synthesize pyridoxine while Others re-

quire it for growth, but there are not so many pyridoxine-less fungi

as there are fungi deficient for thiamine or biotin.

) I
_)
O
. J . i
) J
J J
) V
) l; l A

 

 

26

fiHZOH 7H0 in--NH2
C\\\ C\\§ C\.\§~

0 H0 H0

H \(li/ \Cl -—-CH20H \i/ C'_CH20H \C / (i— CIIZOH

CH3-—-C C C C C

II
\N/ CH3/' \N/ CHB/C\N/

Pyridoxine Pyridoxal Pyridoxamine
C8H1103N C3119031“ C8H1202N2

The structure of the compound is shown along with two other derivatives,
an aldehyde and an amine which are found naturally and are active vita-
'mins. That pyridoxine requirement is highly specific in Ophiostoma has
been determined by Robbins and Ma (1942). Fries (1943) and (1950)
investigated the role of pyridoxine in promoting the growth of certain
Ascomycetes. Pyridoxine was the only vitamin that was required by all
species investigated.

Pyridoxine requirement for Basidiomycetes has not been mentioned
in the literature. The deficiency for this growth factor is very
characteristic for many.Ascomycetes. The fungi can be partially or
totally deficient for this vitamin. Some species reported to be de-
ficient for pyridoxine but this organism was also heterotrOphic with
respect to other vitamins. The presence of one vitamin for which a
fungus is partially deficient may enable the fungus to synthesize
other vitamins with greater ease. Stokes g£_al_(l943) found a

relationship between pyridoxine and thiamine metabolism.in Neur08pora

 

sitOphila. That is evident from the fact that at any given level of
pyridoxine nutrition the mutant - deficient for pyridoxine and thiamine

shows a growth response prOportional to the amount of thiamine added,

27

although some growth is made in the absence of thiamine. The effect

is appreciably greater at low Levels of pyridoxine nutrition. Stokes
suggested that apart from its own coenzyme functions, pyridoxine parti-
cipates in the synthesis of thiamine. The same relationship between
pyridoxine and thiamine has been noted by Tatum and Bell (1946) in
Neurospora crassa. Harris (1956) explained this phenomenon: pyri-
doxine inhibits the biosynthesis of thiamine by preventing the in-
corporation Of the pyrimidine moiety; but thiamine competitively in-
hibits the endogenous destruction of pyridoxine.

The action of pyridoxine appears to be connected with either amino
acid synthesis or amino acid utilization or both. Like the other vita-
mins, pyridoxine also acts in the cell as a part of a coenzyme. The
coenzyme form of pyridoxine is pyridoxal-S-phosphate; it constitutes
prosthetic groups of metabolic enzymes: decarboxylases, transaminases
and racemases.

Pyrodoxine is also involved in the synthesis and metabolism of
tryptOphane, described by Umbreit EEHEL (1947). Silver and McElroy
(1954) reported that pyridoxine-less mutants of Neurospora crassa
accumulate nitrite from nitrate, indicating that pyridoxal-S-phosphate
may have something to do with nitrate reduction (N03—(_'_'__-)N02).

According to Snell (1945) pyridoxine, pyridoxamine and pyridoxal
are equally used by the filamentous fungi. .The two derivatives can
easily be converted in the cell to pyridoxine. Snell also discovered
that autoclaving pyridoxine with the basal medium for 20 minutes in-
creased the activity of pyridoxine forty-one times and that this change

in activityfor certain organisms was correlated with oxidation and

28
heating with certain amino acids.

Myo-Inositol
Seven optically inactive forms and a pair of active isomers Of
hexa-hydroxy-cyclo-hexane can exist, but only one Of the inactive forms

has biological activity. H

o_m

OH /7/

H
\C /
\ / OH 03 \OH
Meso (i) - inositol C

/ H OH
(hexahydroxy - cyclohexane) H I \
~ C
l l
H

OH
Deficiency for inositol is not so common in filamentous fungi as are
the requirements for other vitamins. Kggl and Fries (1937) reported
that there are more fungi partially deficient for inositol than completely
deficient. Partial deficiency was determined for inositol in
Sclerotinia camellia by Lilly and Barnett (1948) but the response to
inositol was conditioned by temperature. Deficiencies for inositol
are usually accompanied by deficiencies for thiamine and biotin.

Shirakova (1955) concluded that Diplocarpon rosae is totally deficient

 

for thiamine and partially for inositol. The same was found earlier
in certain Marasmius species by Lindeberg (1939). Melin (1946) men-
tioned partial need for inositol in certain mycorrhizal fungi.

Inositol is active only in high concentration, therefore, the
amount of the inositol requirement is much greater than that of other
vitamins (5 mg/liter). It has been suggested by Lane and Williams (1948)
that inositol is an active part of pancreatic amylase. But later on
it has been found that the function of inositol as a coenzyme in this
system is very doubtful. Fuller and Tatum (1956) reported that in

Neurospora crassa most of the inositol present is bound in the form

29

of phospholipids. The inositol-less mutant strain, which results from
an inositol deficiency, has a characteristically low level Of inositol-
phospholipid compared to the wild type. Therefore, it was suggested
that inositol is a structural component of the cell.

No general function of inositol is known in fungi. However, a
favorable effect of inositol on the growth of certain fungi which are
inhibited by the presence of excess thiamine has been observed. Sch3pfer

(1945) mentioned that in Rhizopus sinuis inositol overcame the in-

 

hibition of growth due to excess thiamine. The high Specificity of
inositol requirement was described and discussed by Lardy (1954).

Snell (1954) concluded that even though inositol is required for growth
of certain microorganisms, nothing is known of the essential metabolic
role played by it; no distinctive metabolic aberrations due to its

lack have been reported. Presumably in those organisms inositol may

be required for the formation of essential lipid components of the

cello

30

Experimental Procedure
In this study, three strains of Calvatia gigantea and one strain

of Calvatia fragilis were utilized: Calvatia gigantea 1018F; Calvatia

 

gigantea 1019B; Calvatia gigantea 766 and Calvatia fragilis 1020.

Pyrex Erlenmeyer flasks of 125 m1 capacity were used as culture
vessels. The glassware was treated with sulfuric acid-dichromate
cleansing solution, rinsed with top water and finally with distilled
water. Each flask received 25 or 40 mls of medium according to the
method used. The flasks were stappered with non absorbent cotton and
sterilized by autoclaving 15 minutes at 15 pounds steam pressure.

The completely synthetic basal medium which was used is Linde-

berg's medium:

Glucose 20.0 gram
NHg-tartarate 5.0 gram
KH2P04 1.0 gram
Mg504.7H20 0.5 gram
FeCl3(sol 1/500) 0.5 ml
Zn504(sol 1/500) 0.5 ml
MnC12(Iol 0.1 M) 0.5 ml
CaC12(sol 0.1 M) 5.0 ml
H20 distilled 955.0 ml

The pH was determined electrometrically and adjusted to 5.5 before
autoclaving.

The vitamins were added to the basal medium in the following
quantities:

Thiamine hydrochloride _7 lOOgVYliter

31

Pyridoxine hydrochloride ‘ 10053/liter
Biotin crystalline 5 {ll/liter
i-inositol 5000 (Inter

.A stock solution was made for each vitamin, and the amounts required for
each experiment were obtained from this source. This stock solution was
kept refrigerated.

A modified Czapek's-Dox's formula, which Stevens (1957) called "Medium

A", was used as the control for each experiment. It has the following

composition:
Glucose 15.0 gram
Sucrose 15.0 gram
Bactopeptone 5.0 gram
Bacto-yeast 5.0 gram
Mg $04.7H20 0.5 gram
KH2P04 1.0 gram
KCl 0.5 gram
Fe $04.7H20 0.01 gram
H20 (Distilled) 1000 m1
Final pH (not adjusted) 5.5

Henceforth in this thesis the author shall also refer to this medium
by the title "Medium‘A". A solid version of this medium was made by
adding 1.5% BacthAgar.

Two methods of inoculation and incubation were employed and compared

in this investigation.

1. gloating method (Lindeberg 1944)

The inoculum for the experiment consisted of 3.5 mm discs cut from

32

the growing edge of a l4-day old culture, grown on "Medium A” agar in
Petri dishes. The discs of inoculum were floated on the surface of

25 m1 liquid medium in 125 m1 Erlenmeyer flasks. The inoculated culture
vessels were incubated stationary at room temperature.

2. Shake method (Derrick 1952)

 

In order to obtain reproducible quantitative results among replicate
cultures, Kluyver's (1933) method was used to prepare a standard inocu-
lum. To produce the inoculum for an experiment, mycelium from stock
cultures was transferred into 40 m1 of "Medium.A" solution and grown
for 14 days on a reciprocating shaker. The shaking machine had a stroke
of 0.5 inches, and gave 100 one-inch excursions per minute.

The mycelial pellets of the 14-day old shake cultures were frag-
mented for 30 seconds in a sterile Monel metal semi-micro Waring blendor.
The material in the resulting homogenous suspension was washed with
sterile distilled water and centrifuged at 4000 rpm for 10 minutes.

This procedure was repeated twice to give a total of three washings.
The last resuspension was standardized by suspending 1 part mycelium
fragments with 30 parts of distilled water.

This first step inoculum was then used to prepare the second step
inoculum which was to be used to inoculate the experimental flasks.

This was accomplished by putting the first step inoculum into Lindeberg;
medium and allowing the mycelium to grow for 14 days on shaker. The
mycelium thus produced was then fragmented, washed, centrifuged and res
suspended (as described previously) and this constituted the second step
inoculum used for the vitamin studies. It was felt that by employing

this technique, carry over of the nutrients could be avoided.

33

The density of the inoculum was determined with a Klett-Summerson
photoelectric calorimeter using the green filter (500-570 m #9). Each
flask was inoculated with 1.0 ml of the final blended suspension which
gave a reading of 12-15% transmission under the above described condition.

The cultures were incubated at approximately 25°C on one recipro-
cating shaker, described previously, with no additional aeration other
than that caused by the continuous agitation.

The length of incubation necessary for Optimum growth for each
method was determined previously by running a series of growth curves.

The contents of each flask were filtered on tared Whatmann No. 1
filter using a Buchner funnel. The mycelial pellets were washed with
distilled water to remove any excess medium and dried at 96°C in an oven
for 24 hours.

In every experiment, four,or sometimes even more, replicate cultures
were set up for each treatment. All quantitative data are based on the
average dry weight of mycelium.produced in the test medium in quadrupli-
cate flasks. In all the experiments, the pH was checked at the con-

clusion.

34

DISCUSSION AND RESULTS

The experiments with the different vitamins show that all four
tested strains of Calvatia are totally deficient for thiamine (Table
l and 2), and that Calvatia species do not grow on a purely synthetic
medium. Upon the addition of crystalline thiamine (100 J’lliter of
medium) a good growth of mycelium was Produced. The addition of biotin,
pyridoxine or inositol singly to the basal Lindeberg medium did not add
greatly to the growth, and if it did, just a very small amount of sparse
mycelium developed. Calvatia species are heterotrOphic with respect to
thiamine, i.e., they are unable to synthesize this vitamin. Luxurious
growth depends on an external supply of this growth factor.

There are no known environmental conditions for Calvatia species
which allow the synthesis of thiamine as far as different temperatures,
different pH and composition of the medium are concerned. According
to these results, it can be assumed that the deficiency of Calvatia
species for thiamine is absolute.

The combinations of different growth factors show that Calvatia
species have only this single deficiency. (Table 3). When thiamine
and biotin both were present in the basal medium, all four strains of
Calvatia species were able to assimilate the nutrient solutions the
same way as when thiamine alone was present, as evidenced by the mycelial
yieldS. The yields were even a little less in most instances following
the addition of biotin and thiamine. Biotin alone produced very little
mycelial growth compared to the yield in the control (synthetic media).

It is well known that some vitamins have a depressing effect on

growth of certain fungi not deficient for these particular vitamins.

35

TABLE 1 - The utilization of vitamins in surface culture by Calvatia

sp. (The average mycelial dry weight of four replicates.)

Incubation time: 28 days

BIOTIN THIAMINE PYRIDOXINE INOSITOL CONTROL
§££2£2§. as. RE. as. 2!. as. 23. as. 25. as. :EE

 

1018 F 22 5.3 50 4.7 11 5.2 10 5.3 11 5.3
1019 B 18 5.3 42 4.8 15 5.5 18 5.4 11 5.5
766 7 5.5 22 5.2 3 5.5 2 5.5 3 5.5
1020 29 5.3 60 4.9 23 5.4 20 5.4 16 5.5
TABLE 2 - The utilization of vitamins in submerged culture by Calvatia

sp. (The average mycelial dry weight of four replicates.)

Incubation time: 28 days

 

 

BIOTIN THIAMINE PYRIDOXINE INOSITOL CONTROL
§££212§. as. BE. as. 2H. as. 23. as. 22. as. 23.
1018 F 7 5.0 40 4.5 4 5.0 2 5.0 6 5.0
1019 B 8 5.2 27 4.5 4 5.5 3 5.5 4 5.5
766 4 5.5 10 5.0 o 5.5 0 5.5 2 5.5

1020 7 5.5 58 5.0 6 5.5 4 5.5 3 5.5

36

TABLE 3 -- The utilization of vitamin combinations by Calvatia sp.

(The average mycelial dry weight of four replicates.)

Incubation time: 28 days

THIAMINE THIAMINE &
THIAMINE & BIOTIN PYRIDOXINE CONTROL

___Strains ease mesa ease semi

1018 F 40 4.5 37 4.5 30 4.8 6 5.0
1019 B 27 4.5 25 4.5 23 4.8 4 5.5
766 10 5.0 9 5.0 8 5.3 2 5.5
1020 58 5.0 44 5.0 39 5.0 3 5.5
TABLE 4 - Variations in the basal medium. (The average mycelial

dry weight of three replicates.)

Incubation time: 15 days (in Medium A)

Medium.A_ Medium.A Lindeberg's Lindeberg's

 

Strains Medium A «—PEPTONE ~¥EAST EXTR. +~THIAMINE* CONTROL

232.13. 1.11.8211 £932.11 £132.11 ESE.
1018 F 300 5.5 48 4.2 250 5.3 40 4.5 6 5.0
1019 B 280 6.0 55 4.5 140 5.5 27 4.5 4 5.5
766 66 5.5 37 5.3 47 5.5 10 5.0 2 5.5
1020 407 5.5 204 6.5 492 5.5 58 5.0 3 5.5

 

* inoculation time: 28 days

TABLE 3 --

Incubation time:

36

The utilization of vitamin combinations by Calvatia sp.

(The average mycelial dry weight of four replicates.)

28 days

THIAMINE THIAMINE &

 

THIAMINE & BIOTIN PYRIDOXINE CONTROL
____Strains % all as 2:1. as 2E % all
1018 F 40 4.5 37 4.5 30 4.8 6 5.0
1019 B 27 4.5 25 4.5 23 4.8 4 5.5
766 10 5.0 9 5.0 8 5.3 2 5.5
1020 58 5.0 44 5.0 39 5.0 3 5.5
TABLE 4 - Variations in the basal medium. (The average mycelial

dry weight of three replicates.)

Incubation time: 15 days (in‘Medium A)

 

Medium.A Medium A Lindeberg's Lindeberg's
Strains 'Medium A "PEPTONE ~YEAST EXTR.‘+‘THIAMINE* CONTROL
28.25. 95.23. BEEP}. £13.25 9.8.21.1
1018 F 300 5.5 48 4.2 250 5.3 40 4.5 6 5.0
1019 B 280 6.0 55 4.5 140 5.5 27 4.5 4 5.5
766 66 5.5 37 5.3 47 5.5 10 5.0 2 5.5
1020 407 5.5 204 6.5 492 5.5 58 5.0 3 5.5

 

* inoculation time: 28 days

37

Shirakova (1955) found that Diplocarpon rosae produced more dry weight

 

in the absence of biotin than when this vitamin was added. Biotin
may reduce growth or enzyme formation in the fungus. Calvatia species
must be self-sufficient with respect to biotin. They apparently are
able to synthesize this vitamin from the chemical constituents in the
basal medium. An external excess supply of biotin in the presence of
thiamine may even depress the growth of the organism.

The addition of thiamine and pyridoxine to the Lindeberg medium
produced an even more noticeable loss of mycelial weight, when this
was compired to the growth resulting when thiamine alone was present.
Pyridoxine alone resulted in very poor mycelial growth. Calvatia
strain 1020 grew somewhat better than the control; Calvatia strains
1018 F and 1019 B were about the same as the control, and Calvatia
strain 766 did not grow at all in shaker culture. It can be concluded
that Calvatia species either do not require pyridoxine during metabolism
or they are capable of synthesizing this vitamin from the constituents
of the medium. Similar cases have been reported in the literature.
Fries (1943) mentioned that in certain species of Qphiostoma, both
the rate of growth and the maximum amount of mycelium were greater in
pyridoxine-free medium than when this vitamin was added.

The use of inositol as a growth factor in the Lindeberg medium
resulted in very poor mycelial growth. In most experiments, the
presence of inositol apparently caused a smaller amount of mycelial
development than produced in the control. It can be presumed that
inositol may have a certain inhibitory effect on the metabolism of

Calvatia species. The mechanism of this inhibition is not known and

 

‘Jl‘lli‘

38

has not been studied yet. Because of the results discussed above, it
did not seem necessary to combine inositol with any of the other vitamins.

There are no reports in the literature concerning any organism
requiring inositol as a coenzyme function. It is doubtful whether the
compound should be considered as a vitamin. Fuller and Tatum (1956)
suggested, as was mentioned before, that inositol is involved in a
structural component of the cell.

Judging from.the results which appear in Table 4, there is an indica-
tion that in addition to the thiamine requirement, Calvatia species
have a partial deficiency for an unidentified growth factor(s) present
in peptone and yeast extract. In.medium.A, the four strains of Calvatia
had 7 - 10 times more mycelium on a dry weight basis than in Lindeberg's
medium plus thiamine.

After comparing the chemical components of the two media, one
concludes that the slightly greater amount of sucrose in medium.A
could not be responsible for the differences in yields. The presence
of an unknown growth factor in peptone and yeast extract seems to be
evident because of the favorable effect on growth following the addition
of 5 grams of Bacto-peptone and 5 grams of Bacto-yeast extract per
liter to a medium containing the adequate amount of available carbon,
nitrogen and trace elements. The hypothetical substance is apparently
water soluble and thermostable since it is resistant to autoclaving.
It may be a single compound but more likely several. This (these)
could be in addition to those growth factors already observed in a
typical analysis of Bacto-peptone and Bacto-yeast extract which appears

in the appendix.

39

The presence of unidentified growth factors in natural media is
not an unknown phenomenon as evidenced by the various reports. Yusef
(1953) found that the addition of small amounts of malt extract to
the basal medium containing vitamins and casein hydrolysate, markedly
increased the growth of different Polyporus species. He suggested
that the presence of unidentified growth-stimulating factors in malt
extract ("malt factor") appeared to play a role in the nutrition of
this fungus. Melin and Das (1954) observed that roots of pine and other
plants exert a strong growth-promoting effect on tree-mycorrhizal
Basidiomycetes. They concluded that the roots produce one or more
metabolites designed as "factor M", which are essential to the growth
of these fungi. This growth factor could not be replaced by either the
vitamins of the B complex group or the amino acids in casein hydrolysate,
or by the components of hydrolysed yeast nucleic acid. Melin (1959)
reported that a mixture of nucleic acid components, however, seemed to
lower the growth-promoting effect of the ”M factor".

Comparing the results reported in Table 4, it is noticeable that
with one exception (strain 1020), the strains of Calvatia gave the best
growth when Bacto-peptone and Bacto-yeast extract were both present in
mediuva. However, in all four cases the yield was much lower when
Bacto-yeast extract was the sole additive with peptone lacking. The
Bacto-yeast extract results reported above can be due to pH changes
of the media during incubation in the absence of the buffering effect
of peptone, or it may be an actual inhibitory effect by the yeast ex-
tract which can be overcome by the addition of peptone. Judging from

the results obtained with strain 1020, the latter supposition seems to

40

be more likely because, in spite of the buffering effect of peptone,
the organism gave a better growth in the absence of yeast extract.

MacLead (1959) discovered while determining the Optimum yeast-
extract-dextrose concentration for Hirsutella gigantea that concentrations
higher than 1.5% yeast extract appeared to have an inhibitory effect,
and at 2.5% very little growth was produced by the fungus during the
f irst 12 days of incubation. Growth began shortly thereafter and at
‘the end of 16 days there was a substantial yield. Thus it would appear
that yeast extract has an inhibitory effect in the early stages of
growth. At the same time, in view of the fact that some growth eventually
takes place, Hirsutella gigantea must adapt itself to the high yeast-
extract concentration.‘ In the case'of Calvatia species, the adaptation
of strain 1020 to yeast extract was not an important problem and was
not investigated.

Fries (1950) mentioned the superiority of yeast extract to thiamine
alone for the growth of some Basidiomycetes, but he emphasized the fact
that yeast extract contains inhibitory compounds as well as unidentified
beneficial substances. The effect of different concentrations of
thiamine has been investigated (Table 5). Increasing the thiamine
concentration from 25 5’/liter of medium to 150 6’/1iter of medium did
not show significant differences in the amount of mycelial yield in
the four tested strains of Calvatia species.

A review of the literature disclosed that there are contradictions
about the value of surface (floating) culture and submerged (shaker)
culture methods for growing fungi. Several investigators have attributed

various effects to the u;e of a disc, floating on the surface of the

41

TABLE 5 - Different thiamine concentrations on the growth of Calvatia

Species. (The average mycelial weight of four replicates.)

Incubation time: 30 days

150 100 50 25
.__LL___ __11___ __J!t__. .__LL___
§££2122. as. 22. 22. 22. 22. 22. 22. 22.
1018 F 49 4.5 48 4.5 48 4.5 45 4.5
1019 B 30 4.5 27 4.5 28 4.5 23 4.6
766 11 5.2 11 5.2 11 5.2 10 5.2

1020 74 4.9 69 4.9 70 4.9 67 5.0

42

medium. Also several authors have claimed that nutrients could be
carried over in this fashion and, therefore, the submerged culture
method has been accepted assnperior to the surface method.

Leonian and Lilly (1939) found that the food material and growth
ifactors p:esent in the agar-disc inoculum do not exert an appreciable
influence upon the growth of the colony. They also noted that the
size of the inoculum had no effect on the growth of the culture Of
several organisms.

Margolin (1942) reported that after calculating the amount of
auxithals in a disc of agar transferred into 20 mls of nutrient solution,
the required minimum for growth was found to be infinitely larger than
any amount that can be transferred in this fashion. He based this on
the assumption that the growing fungus did not destroy any of the
auxithals in the medium.

Later on, the use of fragmented mycelium in the culture of fungi
seemed to be considered as a superior method of preparing inoculum.
Savage and Vander Brook (1946) emphasized the importance of the frag-
mentation of the mycelium by a high speed blender and evaluated the
blended inoculum as Opposed to floating discs. Dorell and Page (1947)
Observed that a closer check of replicate cultures and a shorter lag
period in initiation of growth are attained if fungal mycelium is
fragmented rather than employed as a floating disc. Kitay and Snell
(1948) concluded that time of blending, washing and suspending of
fragments and amount of suspension used for inoculum are important
influencing factors.

Foster (1949) compared the physiology of surface with submerged

43

growth. He indicated that submerged growth cultures theoretically

and practically afford the closest approach to the ideal method of
studying mold metabolism, although even these experimental conditions
differ considerably from those of the natural habitat of the fungus.
.According to Foster (1949), surface culture is entirely inadequate
because it represents the overall result of the metabolic processes of a
heterogeneous mixture of physiological systems. Submerged culture,
however, provides physiologically homogeneous fungal material, since
all cells are uniformly exposed to the environmental factors, both
physical and chemflcal, during the growth period.

Derrick (1949) reported that the organisms may store or absorb
several times their requirements of vitamins when grown on a vitamin
or a.vitamin-rich medium. The amount of inoculum transferred to a
vitamin deficient medium, therefore, is critical and repeated trans-
fers are necessary to eliminate any carry Over Of vitamins. Wiken
25,2g,(1951) compared the two methods Of inoculation and incubation
and proved the superiority Of the submerged growth statistically.

The morphological alteration of several fungi in submerged shaken
cultures have been reported by Burkholder (1945) - as a morphogenetic
variation. Foster (1949), however, questioned whether this phenomenon
should be considered as morphogenesis, since the curious growth pattern
is not a fundamental link in the biological development of the organism
but rather a manifestation resulting from these peculiar physical con-
ditions.

From the results shown in Table 6, it is evident that in spite Of

the differences in the amount Of the mycelial yield given by the floating

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45

and shaker system, both methods show the requirement of the tested
strains of Calvatia for thiamine. It seems that mycelial growth in the
[aresence of different growth factors was proportionally similar in

both methods. It is noticeable that the amounts of growth are greater
in the floating method than in the shaker method. According to the
previous review of the literature, this must be due to the "carry-over"
of nutrients by the inoculum, which is unavoidable in the use of nutrient
discs. There is present a certain amount of food material and growth
factors in the nutrient discs which influences the growth Of the colony.
If this were not so, then the amount of fungal growth produced by the
shaking method could not be so much lower, since the dry weight Of
both forms Of inocula was roughly the same, i.e., 0.2 - 0.4 mg.

It has to be considered a fact that in case Of submerged growth,
the inoculum was always standardized and was a physiologically homo-
geneous material, uniformly exposed to the environmental factors during
the growth period. In the case Of surface cultures, standardization
Of the environmental circumstances could not be guaranteed. Another
disadvantage Of the latter method is also evident from the data re-
ported in Table 6. The replicates of the floating method show greater
deviation from the mean value than in the case Of the shaker method.
The greater variability produced by the floating method is expressed
in the comparatively higher standard deviation (Si) values in Table
6. The data Obtained by the floating method Of cultivation represents
only the overall result of the metabolic processes of an extremely
heterogeneous mixture Of physiological systems because the inoculum

was not provided with a uniform and reproducible physical environment.

 

 

 

46

The results shown in Table 7 indicate that there are no significant
differences in the utilization of thiamine and thiamine-pyrophosphate
by the four strains Of Calvatia tested.

Lilly and Leonian (1940) compared the action of thiamine and thiamine-
pyrophosphate in several thiamine deficient fungi. They found no difference

in the maximum weights of mycelium.formed in the presence of equivalent 3]

.'.Atl"f' '.

quantities of these two growth factors. Therefore, they concluded that

cocarboxylase can replace thiamine, possibly entering the cell without

 

dephosphorylation. Jensen (1954) reported cocarboxylase about 30 per

is! f...- .15.. _‘
"5.

cent more active than equimolecular quantities of thiamine for most
bacteria. However, he did not mention that this is also valid for the
fungi.

Lilly and Barnett (1951) suggested that pyruvate accumulates in the
culture medium of some thiamine deficient fungi in the absence Of the
required amount of thiamine. The pyruvate accumulation was accompanied
by a decrease Of pH in the medium. That was not Observed in the case
Of Calvatia species. In the absence of thiamine, the pH of the medium
did not decrease which may suggest that pyruvic acid, instead Of
accumulating in the medium, could be further metabolized by any one of
several possible pathways, except the Krebs cycle.

The greatest mycelial yield at the end of the 28 days Of incubation
occurred at the lowest pH value.

Since all living organisms are dynamic systems and each preparation
of inoculum forms a different p0pulation which varies according to its

previous environmental contacts, constant responses would not be expected.

:15.

littl.

 

TABLE 7 -

47

The utilization of thiamine and thiamine-perphosphate by

Calvatia sp.

Strains

1018 F

1019 B

766

1020

(The average mycelial dry weight of four

 

replicates.)

THIAMINE 150 lliter
14 days 28 days
EB. 25. EB. 25.
1305 5.1 37.1 405
18.5 5.2 26.9 4.7
5.3 5.3 11.9 5.1
10.5 5.1 55.0 4.7

 

TPP 150 lliter
14 days 28 days
232.“. 9.8.2.8

12.7 5.2 39.3 4.5
20.0 5.0 28.1 4.6
6.5 5.3 10.1 5.1
11.7 5.0 52.3 4.7

 

48

CARBON UTILIZATION BY CALVATIA SPECIES
Review of literature
It is well known that carbon is one of the most essential elements
in the nutrition of organisms. The carbon sources are utilized by fungi

for the synthesis of structural and functional compounds. In addition

I27

to this, the oxidation of carbon compounds provides an energy source

for their metabolism. Since nearly half of the dry weight of fungus

m 4mnmvn —.

cells consists of carbon, fungi are dependent on their carbon supply.

It is obvious, therefore, that the study of carbon nutrition is funda-

 

mental to the understanding of the physiology of the Calvatia species.
Carbohydrates are the most common and best sources of carbon.
All of them differ physiologically with the mirror-image configuration
of the same structure. Whether a carbohydrate is utilized or not de-
pends upon both the configuration of the certain saccharide and the
particular abilities of the Specific fungus. Usually only one enantio-
morph is utilized, Or one is utilized much more than the other. In
general, L-isomers of the naturally occurring aldohexoses and xyloses
do not support growth on fungi. It is known since 1923 (Brannon)
that nearly all fungi which can be cultured are able to use glucose or
fructose. Albritton's summary of the carbohydrates utilized by fungi
for growth (1953) shows that fewer Species utilize lactose, maltose
sucrose and raffinose than utilize the monosaccharides (glucose,
fructose and galactose) which these oligosaccharides yield on hydrolysis.
Glucose is biologically the most important hexose for fungi.
This sugar is generally used as a carbon source among the carbohydrates.

Derrick (1949) attained the maximum growth for different wood-

mif,‘

 

 

 

 

 

49

destroying Basidiomycetes in media containing 16 percent glucose.
Perlman (1949), using synthetic medium, found that the utilization Of

glucose by Polyporus anceps depended upon the concentration of thiamine.

 

An increase in the amount of thiamine resulted in increased utilization
Of glucose.

Khudiakov and Vozniakovskaia (1951) reported the culture of four

different strains Of Boletus on a medium utilizing glucose as carbon

source. Oddoux (1953) was able to grow 247 species of Basidiomycetes

gm. mm; 4“-

“EE

on glucose medium. Vorderberg (1949) claimed that Coprinus lagopus

did not grow on glucose. However, this conclusion seems to be based

on results obtained with an unhappy combination of tOO low an incu-
bation temperature and an inhibitory decoction of manure added to the
substrate. The author also used fruiting body production as an
expression of the amount of growth, which is a debatable method
according to Fries (1955). Fries (1955) compared the different car-
bon sources for the growth Of Coprinus species. She found that glucose,
mannose and fructose are almost equally effective for most of the in-
vestigated fungi. Fructose is, however, not as well utilized by

Coprinus narcoticus.

 

The failure to utilize fructose as a carbon source in the higher
fungi has been reported in several cases. Shirakova (1955) found that
Diplocarpon rosae was unable to utilize fructose for growth, but it
is possible that this was due to autoclaving fructose in the medium.

The utilization Of galactose by fungi is variable. It is a poor
carbon source for most Of the Basidiomycetes. Treschow (1944) Observed

that in Psalliota bispora, galactose resulted in a mycelial growth up

 

50

tO 73 per cent Of that on glucose. The corresponding figure for

COprinus micaceus was 47 per cent. Among the Tricholoma species in-

 

 

vestigated by Norkrans (1950) the saprophytes seemed to utilize this
carbon source much better than the mycorrhiza formers. He also found

that the addition Of a small amount of glucose ("start glucose") in-

"! 3

.‘m

creased the growth on galactose.

Among the pentoses, xylose and arabinose were used most frequently

tu—M‘

in the nutrition of fungi. Xylose ("wood sugar”), as may be expected,

is usually a satisfactory source of carbon for the wood-destroying

‘EEITH‘

fungi. Treschow (1944) found that the use of xylose resulted in a 32
per cent better mycelial growth than for glucose in the case of Psalliota

bispora. Herrick (1940) reported that Stereum gausapatum used xylose

 

better than arabinose.

In general, arabinose is distinctly inferior to glucose or mannose
in fungal growth. Margolin (1942) deduced from his investigations
that xylose was utilized either more completely or more rapidly than
arabinose in most of the studied fungi. Lilly and Barnett (1956)
demonstrated that several fungi were able to utilize L-arabinose and
failed to grow or did not give satk5factory growth on D-arabinose.
Lampert (1958) investigated the different carbon sources Of Merulius
lacrimans. Xylose and arabinose yielded a good mycelial growth along
with glucose, saccharose, maltose, lactose and raffinose but the
fungus was not able to utilize rhamnose.

The utilization of monosaccharides seems to be dependent on an
enzyme system which is constitutive in some fungi and inducible in

others. The strains of a given species may fall into different groups

51

in this regard.

The oligosaccharides are used by fungi only after conversion into
reducing sugars by the approPriate extracellular enzyme. The ability
to utilize the oligosaccharides, no matter whether the process takes
place via hydrolysis to monosaccharides or via direct phosphorolysis
depends on the production Of suitable enzymes by the organism. ' F]

Maltose, as a carbon source, is almost universally utilized by A

the fungi. Most of the Basidiomycetes give a good mycelial growth

 

using maltose in the medium; however, maltose is more easily hydrolysed '
by autoclaving than are sucrose or lactose. Fries (1955) noted that u
maltose can be utilized by all species of COprinus, but to a different
degree. Swartz (1935) reported maltose as a very favorable sugar for

mycelium production in Calvatia saccata, Calvatia caelata and Calvatia

 

gigantea.

Sucrose is generally a good source of carbonifor fungal growth,
but it is utilized by fewer fungi than maltose. The response to
sucrose is correlated with the amount of invertase produced by the
particular organism. Findlay (1944) mentioned the utilization of
sucrose by some wood-rotting Basidiomycetes. Bille-Hansen (1953)
found that sucrose appeared to be completely or almost completely un-
available to the investigated three COprOphilous species of COprinus.

Lilly and Barnett (1953) observed that Collybia velutipes utilized

 

fructose, glucose, sucrose, mannose, maltose and cellobiose best in

that order; Polyporus albellus utilized mannose, xylose, glucose and

 

maltose best in that order; Polyporus versicolor utilized glucose,

mannose cellobiose, fructose and maltose best in that order.

NRC-
F“
l
I

 

“1

SM.

 

52

Cellobiose is almost as widely utilizable as maltose for fungi, but
the data is more limied than those for maltose. Norkrans (1950)

stated that Tricholoma flavobrunneum failed to utilize cellobiose, but

 

other species Of the sane genus grow well with it as the carbon source.
'Madelin (1956) indicated that cellobiose is a good source of carbon
for most of the Coprinus species. ?j
According to the literature trehalose permits good growth for
several fungi, however, this carbohydrate was not investigated in the

nutrition of the Basidiomycetes. Lactose is a poor carbon source

 

"qv . '. ASIA-

for most Of the fungi. This may be due either to the general unsuita-
bility Of galactose which is one product of hydrolysis Of this di-
saccharide or to the failure to produce the appropriate hydrolytic
enzyme. Fries (1955) reported that lactose could not be used by any
of the investigated Coprinus species. Norkrans (1950) noted, however,

that Tricholoma nudum gave a satisfactory growth when lactose was used

 

as a carbon source in the medium. Humfeld and Sugihara (1952) men-

tioned that Agaricus campestris was not able to utilize this carbo-

 

hydrate.

Raffinose is the only trisaccharide which is important in the
nutrition Of fungi, although the utilization of this carbohydrate is
inferior to the utilization of glucose or maltose. Among the Basidio-
mycetes, some of the Coprinus species were noted by Johnson (1941) to
be able to use raffinose as well as glucose. Herrick (1940) Observed

that raffinose was an even better source Of carbon for Stereum gausapatum

 

than glucose.

The use of polysaccharides in the nutrition Of fungi also depends

53

upon the ability to produce the appropriate hydrolytic enzymes. Only
those organisms are able to utilize these carbon sources which form
the necessary enzymes to hydrolyze or to degrade in some way these
water insoluble carbohydrates to low molecular weight simple sugars.
Starch is readily used by a number Of fungi, however, only those which
produce amylase are able to utilize this polysaccharide. Nflrkrans F3
(1950) indicated that because of this fact, starch is hydrolysed
primarily to maltose and all Basidiomycetes checked gave a good mycelial

growth on "soluble starch". Fries (1955) reported excellent growth

 

for COprinus micaceus, using starch as a carbon source. The fact that g

 

starch is Often a better substrate for growth than glucose has been
explained by Cochrane (1958): "This effect presumably results either
from contamination Of the starch with growth factors or from the fact
that utilization Of a slowly hydrolysable compound is accompanied by
less accumulation of acids than utilization of glucose".

Cellulose is used by the majority Of fungi according to Norman
(1942). Fungi play an important part in the breaking down Of the
cellulose that remains in the soil. Some wood-destroying fungi break
down the lignin Of the wood cells, but Others dissolve the original
cellulose cell walls. Only those Basidiomycetes which form cellulase
are able to utilize cellulose, the most common carbon source on the
surface Of the earth. Reese (1947) deduced that the wood rotting and
litter-decomposing fungi were generally, although not universally, able
to decompose cellulose. He also found acidity an influencing factor
in the cellulose utilization. Nobles (1948) stated that the fungi which

caused white rots (as for instance Polyporus abietinus, Polyporus

 

 

 

 

 

54

cinnabarinus, Armillaria mellea, etc.) were able to attack the non-

 

 

cellulosic constituents of the wood. The fungi which attacked cellulose

in preference to lignin caused the brown rots (Merulius lacrymans,

 

Lenzites trabea, Polyporus betulinus, etc.). Norkrans (1950) suggested

 

that the species within a givengenus were not necessarily alike in
their response to cellulose. In the case of Tricholoma species, the
utilization Of cellulose varied with the presence Of other carbon sources.

It has been also suggested that Tricholoma species are able to form

 

an adaptive cellulase. Siu (1951) concluded that enzymes which attacked
cellulose were liberated by microorganisms only in the presence Of
cellulose. The cellulose attacking enzymes are adaptive enzymes formed
by fungi only as a specific response to the presence of certain sub-
strate.

Lignin is also broken down by wood-destroying fungi, but attempts
to cultivate these artifically on preparations Of lignin from wood have
not been very successful. Gottlieb (1950) reported that some of the
wood-destroying fungi were able to utilize lignin, but Others, as for
instance Polyporus abietinus, used lignin in synthetic medium only
after a process Of adaptation. Such adaptation permitted the organisms
to grow on many different kinds of lignin and lignin containing re-
sidues according to Van Vliet (1954).

Inulin is used by relatively few fungi. This is due to the
failure to secrete the apprOpriate enzyme, inulase, rather than to any
inhibitory effect Of the inulin itself, as has been concluded by Hawker
(1950). Fries (1955) noted that although all the investigated species

of Caprinus were able to grow on fructose they could not utilize inulin,

 

 

 

 

 

 

55

which is a polyfructoside. Perhaps it would be possible to promote
some growth by adding "start glucose", but it is also possible that
they lack the necessary inulase.
Glycogens, the reserve polysaccharides of animals, are used by
a number Of fungi in artificial culture. Those fungi which are unable
to satisfy their carbon requiremens from starch cannot be expected to ?7
utilize glycogens, since probably the same enzymes attack both of these JV

polysaccharides. There is not much data in the literature dealing ?

 

‘with the use of glycogen by the Basidiomycetes. Only Herrick (1940) g}
b

‘mentioned glycogen as a good source Of carbon for Stereum gausapatum.

 

Certain fungi grow better on a mixture Of several carbon sources
than when supplied with the equivalent amounts Of the individual
carbohydrates comprising the mixture. It is probably that such organisms
are able to produce small quantities of several enzymes more easily
than a large quantity Of any one alone. Fries (1955) investigated the
combinations of different carbohydrates for Coprinus species. She
found that growth in the presence of mixed carbon sources was sometimes
increased or even made possible. In nature, the fungi are usually
supplied with a mixture of carbohydrates and Other carbon sources rather
than a larger amount Of a single one. Therefore, it is possible that
carbon sources that have proven unavailable in experiments can be
utilized in nature.

Proteins and their amino acids are vital constituents of the
organisms. They are attacked by many fungi chiefly as a source Of
nitrogen. In the absence Of any other source Of carbon, however, most

fungi are able to use proteins to satisfy their carbon requirement.

 

"—Jlr-

 

 

56

According to Gottlieb (1946) the proteins have high calorific value

and under certain conditions can furnish energy for growth and reSpira-

tion. Best and Taylor (1945) stated that most of the straight chain

amino acids could be assimilated by the organism, and under certain

conditions as much as 58 per cent of the protein could be converted

to glucose. I}
The products of glucose metabolism: citrate, fumarate, succinate -

and malate are the organic acids, mostly utilized by fungi. Peptone

 

may serve as a source of carbon and nitrOgen for many organisms. L}
As far as Basidiomycetes are concerned, only a few investigations

have been conducted on the utilization of amino acids as carbon source.

Treschow (1944) reported, however, that Psalliota bispora gave a good

mycelial growth using oxalic acid in the medium to satisfy the carbon

requirement. It is characteristic of fungi cultivated on amino-

acid medium as the sole source of carbon to increase the alkalinety

of the medium. This is probably due to accumulation Of ammonia which

results from deamination as was mentioned by Lilly and Barnett (1951).

57

Experimental Procedure
The same four strains Of Calvatia (1019F, 10188, 766 and 1020)
as before, were employed in these studies for the utilization of
different carbon sources. The basal synthetic medium was the same
as that used previously, i.e. Lindeberg's medium except that 100<r/1iter

of thiamine was added. The pH was determined as previously described.

Forty m1 Of the modified Lindeberg medium was placed in a 125 ml

Erlenmeyer flask and plugged with non-absorbent cotton. Only Pyrex

 

glassware was used and it was washed as described previously. Each ' '
flask was autoclaved for 15 minutes at 15 pounds steam pressure. 5)

The various carbohydrates were added to the basal medium in
quantities which supplied 8 grams of carbon per liter. In most of
the experiments, the carbohydrates were added to the basal medium
before autoclaving. Maltose, trehalose and raffinose were sterilized
separately in the autoclave for different lengths Of time at different
pounds steam pressure (according to their heat-sensitivity) and added
aseptically to the basal medium.

TO avoid the breakdown of sugars during autoclaving, the following
heat-sensitive carbohydrates, fructose, L-sorbose, xylose, melibiose
and invert sugar, were sterilized by Seitz-filtration using a filter
with l‘urpore diameter.

Once again the density Of the inoculum was determined with Klett-
Summerson photoelectric calorimeter, using the green filter (spectral
range 500 - 570 moux). Each flask was inoculated with 1.0 ml of blended
suspension at 30 - 35% transmission.

The cultures were incubated only on the reciprocating shaker with

no additional aeration. All the experiments were incubated at

.A‘u7hpfl. . ‘4

 

58

approximately 25°C. The cultures were expOsed to diffuse daylight
with intervening dark periods at night. No attempt was made to
standardize condition of illumination.

Sufficient flasks were inoculated to provide two replicates for
each strain harvested after 14 and 28 days on the shaker. The mycelium
was filtered on tared Whatmann No. 1 filter papers using a Buchner 1P]
funnel, and washed twice with distilled water. It was then dried to
constant weight for 24 hours at 960 in oven. The dry weight of mycelium

was calculated. In all experiments the pH was checked at the con-

 

clusion. '

59

Results and Discussion
In this study, all carbon sources tested were present in an
amount calculated to yield an equal supply of carbon. Therefore, it
was possible to compare their utilization by the four Calvatia strains
on an equitable basis.
Monosaccharides T3
The experimental evidence shown in Table 8 indicated that pentoses

were utilized less than most of the hexoses by Calvatia species. If

H7.
4 .

any of the 5 carbon sugars were used as the sole carbon source, the

 

strains tested grew poorly. There are slight differences, however, in L}
the utilization between the strains and also between the availability
Of the pentoses.

Ribose was the best carbon source among the pentoses for strains
1018 F, 1019 B and 1020. Strain 766 did not grow in the presence of
ribose in the basal medium.

The results Of Table 8 indicated that Calvatia species were not
able to satisfy their carbon requirements with either xylose or ara-
binose.

The growth of Calvatia species was more rapid and greater dry
weights of mycelium were produced in autoclaved than in sterile
filtered xylose, although less yield would be expected on the auto-
claved xylose because this pentose is known to be broken down to
furfural in autoclaving. I

Both isomers of arabinose were employed in this nutritional in-
vestigation of Calvatia species but neither of them promoted a satis-

factory growth. According tO Lilly and Barnett (1956) from the dis-

tribution of the isomers Of arabinose in the organisms, it would be

 

 

 

   

60

F8
F}

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61

expected that L-arabinose would be utilized readily by more fungi
than D-arabinose. In the case of Calvatia species, two Of the strains,
1019 B and 766, grew better on L-arabinose, and two strains, 1018 F
and 1020, preferred D-arabinose.

The hexoses employed in this study were glucose, fructose, mannose,
galactose, rhamnose and sorbose with growth results best in this order. F7
In general, the 6 carbon sugars were utilized much better by Calvatia
Species than the pentoses, however, the ability of the strains to use

them for growth was different.

 

Glucose, as may be expected, was the best carbon source for each L)
strain among the hexoses tested, although it was not utilized as well
as dextrin or cellobiose. Glucose is a biologically very Significant
sugar. It has an important role in the terminal respiration Of or-
ganisms. Fruton and Simmonds (1959) presented a scheme for the path«
‘way Of anaerobic breakdown Of glucose to ethanol and carbon dioxide

in the microorganism. The overall reaction is:

 

Glucose +-2ADP +'2 phosphate )» 2 ethanol + 2C02 + 2ATP + 2H20.
According to the results shown in Table 8, fructose was easily

assimilated by the four strains Of Calvatia tested, but it was in-

ferior to glucose. Fructose is also metabolized by microorganisms,

after phosphorylation, the same way as glucose. But while glucose is

converted to glucose 6-phosphate by ATP in the presence Of glucokinase,

fructose is converted to fructose-l-phosphate and in the presence of

fructokinase. Nilsson (1956) observed that fructose-1,6-diphosphate

(an intermediate product Of carbon metabolism) had a very striking growth-

promoting effect on Boletus variegatus and Collybia velutipes. He also

 

found that the effect was reversed above a certain concentration Of the

 

62

diphosphate, at least for Boletus variegatus. The investigation was

 

only preliminary and he Offered no explanation of the effect.

Mannose was readily used in Calvatia fragilis (1020), although the
fungus did not grow as well as on glucose. The rate of utilization was
slow during the first 14 days of incubation, but later on it became
faster. Limited growth took place with mannose in the strains of r?
Calvatia gigantea (1018 F, 1019 B and 766) and most Of the growth was I
completed within 14 days. Mannose can be involved in the metabolism

of microorganisms, but it has to be phosphorylated previously. Slein

 

(1950) indicated that yeast hexokinase catalyzed the phosphorylation L}
of mannose by ATP to form mannose-6-phosphate, and phospho-mannose

isomerase was responsible for the enzymic conversion of this sugar

phosphate to fructose-6-phosphate.

Calvatia gigantea strains grew poorly on galactose, but Calvatia

 

fragilis (1020) had a satisfactory mycelial growthon this 6-carbon
sugar. According to Fruton and Simmonds (1959) the ability of an
organism to use galactose, depends upon its ability to convert this
hexose into a phosphorylated derivative of glucose able to enter the
main respiratory pathways.

Rhamnose, a desoxy sugar Of mannose, was not able to satisfy the

carbon requirement of Calvatia species. Even Calvatia fragilis (1020)

 

which gave an appreciable growth on mannose was not able to utilize
this carbon source.

None Of the Calvatia strains investigated was able to utilize
sorbose. Sorbose is a keto-hexose also, but differs from fructose in
the configuration Of carbon atom 5. The toxicity of sorbose was men-

tioned by Lilly and Barnett (1953). They stated that sorbose inhibition

 

Emir

 

 

 

 

63

occurred at relatively high concentrations in certain fungi and it
was strongly affected by the temperature. However, Cochrane (1958)
suggested that sorbose may interfere with a respiratory pathway of the
organism.

The differences in the utilization of the monosaccharides by the
Strains of Calvatia can be due to the structural differences between r?

the sugars and to the particular abilities of the organisms also.

 

64

Oligosaccharides
AS seen in Table 9, cellobiose was one of the best carbon sources
for Calvatia species. Strain 1018 F produced with 10 per cent more
mycelium, strain 1019 B, 25 per cent; strain 766, 50 per cent; and
strain 1020 had 150 per cent more mycelial growth in the presence of

cellobiose than with the generally accepted glucose as a carbon source.

According to Reese and Levinson (1952) most Of the fungi are able
to split cellobiose to its constituent glucose residues. Analyzing the

data on cellobiose utilization (Table 9), Calvatia fragilis (1020) i.

 

 

seemed to be superior to Calvatia gigantea (1018 F, 1019 B and 766) U
in the production of the appr0priate extracellular enzyme. Strain

1018 F Showed a relatively low mycelial growth after 14 days incubation,

but later on the rate of cellobiose utilization increased. The result

suggested that the involved enzyme was inducible. No attempt was

made, however, to study adaptive enzyme formation with respect to

carbohydrate utilization.

A satisfactory growth was Obtained using maltose as a carbon source
in the medium for Calvatia strains. The weight Of the mycelial growth
was lower than on cellobiose, with the exception of strain 1019B,
where the yield on maltose even exceeded that on cellobiose. For

Calvatia fragilis (1020) maltose seemed to be a less available car-

 

bon source than glucose, which may be due to the lack of the necessary
hydrolytic enzyme in the fungus.

Le Mense g£_§l (1947) found that maltose was utilized almost in
every fungus investigated, although quantitative differences between

strains were common.

 

 

 

65

 

 

 

 

 

 

 

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66

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67

Although cellobiose and maltose by hydrolysis form two mole-
cules of glucose, there did not seem to be any correlation between the
capabilities of the strains to utilize these two disaccharides. In
the former carbon source the two glucose molecules are connected by
a fl -g1ucosidic linkage and in the latter one by a CL-glucosidic
linkage. Consequently, they require different enzymes for cleavage.

The difference between the utilization of these two carbohydrates by

“n"fi2u
‘
r

the Calvatia strains was probably due to the difference in abilities

to produce the required enzymes.

 

.—hA—' V- .

The smaller amount of mycelial growth with maltose could be ex-
plained by the fact that maltose is more easily hydrolyzed by auto-
claving than cellobiose. A special precaution was taken, however, to
sterilize the medium containing maltose for a minimum of time.

According to the data of Table 9, sucrose, a fl -fructofuranoside,
was not a good carbon source for Calvatia species. It couli be that
the organisms produce the hydrolyzing enzyme very slowly or in a small
quantity, as both components of this disaccharide (glucose and fructose)
produced a satisfactory growth when used separately. Apparently the
CX - /3 glucose to fructose linkage was not easily broken.

Mandels (1954) indicated that sucrose is metabolized by a non-

hydrolytic system in spores of the fungus Myrothecium verrucaria.

 

Bealing (1953) Observed that invertase preparations catalyzed the
transfer of fructofuranosyl groups not only to water, but also to
various alcohols and sugars by transglucosidation.

The fact that the small yield of the four Calvatia strains on

sucrose was due to the slow production of invertase rather than to the

 

 

68

inability to utilize the components of this sugar could be proved by

the substitution of invert sugar. Invert sugar is a mixture of glucose
and fructose with the components present in l to 1 ratio. It differs
from sucrose as its constituents are already hydrolyzed. Therefore,

it would be classified as a mixture of sugars rather than a disaccharide.

All the four Calvatia strains investigated produced a very good grooth

—-fi Hut!
.__8H

on this mixed carbon source. With exception of strain 1018 F, they
gave even a better mycelial growth than on glucose in 28 day culture.

This can be explained by the effect of combined carbon sources (in-

Ifimflm—EW'xa - r“.

duction of a new enzyme). Calvatia species did not need any specific
enzymes to utilize invert sugar as they did in case of sucrose. One
constituent, glucose, acted as an "inducer", permitting a rapid growth
on the other constituent, fructose.

It could be assumed from the result shown in Table 9 that trehalose
'was utilized satisfactorily by the strains of Calvatia. However,

Calvatia fragilis (1020) did not respond as well to this carbon source

 

as the strains Of Calvatia gigantea. (1018 F, 1019 B and 766.)

 

According to Cochrane (1958), in all probability trehalose in
fungi has the role of a translocateable reserve carbohydrate. This
is eSpecially common in the Basidiomycetes. This disaccharide is
formed early in the metabolism and used up later, however, an adequate
picture Of trehalose metabolism is not yet available. Calvatia species
seemed to be able to produce trehalose, the special enzyme which splits
the 0(-l-l glucosidic linkage in this disaccharide. The synthesis
of trehalase has not been studied in Calvatia species. However, a
correlation between the rate of growth and the amount of the appropriate

enzyme produced is apparent.

 

69

Lactose and melibiose appeared to be poor carbon sources for the
Calvatia strains tested. All four of them produced very small mycelial
growth on these disaccharides. The failure of Calvatia species to
utilize this carbon source could be explained by the lack of the
necessary hydrolytic enzymes, lactase or melibiase; or by the inability
to utilize one Of the component hexoses, galactose.

Just one trisaccharide, raffinose, was employed in this nutritional

-2hau,fn
_———J

study of Calvatia species. Raffinose, which contains one linkage as

in sucrose and another as in melibiose was utilized poorly by the strains

 

tested. According to the earlier discussions, Calvatia species were LJ
not able to produce melibiase, but a certain amount of sucrose was
formed by the organisms. Therefore, the small growth which was pro-
duced by the fungi investigated on raffinose could be explained by the
availability of fructose.
Polysaccharides

Data Shown in Table 9 indicates that good mycelial weights were
obtained with soluble starch in three strains of Calvatia species,
although strain 1018 F was not able to utilize this carbon source in
an appreciable amount.

Starch is a "nutrient" polysaccharide and acts as a metabolite
reserve Of monosaccharides in organism. It is composed Of a mixture
of two different polysaccharides: aamylose and amyIOpecyin. According
to West and Todd (1956), the glucose units of amylose are bound to
each other in a (l-4)-glucosidic linkage: amlepectin, while con-
taining chains of glucose units like those of amylase, also has branches

Of these glucose chains linked through a (l-6)-g1ucosidic linkage.

7O

Calvatia strains 1020 and 766 grew somewhat better on starch
than they did on maltose. This may be due to the presence of some
growth promoting substances in starch. Another explanation might be
that starch is broken down by phosphorolysis directly to glucose com-
pounds and not via maltose as was suggested by Bonner (1950). The fact
that starch was not such a good carbon source for strain 1018 F and ET;
1019 B as maltose could be explained by a slow or imprOper formation
of the necessary extracellular enzyme, maltase.

The glycogen utilization in the Calvatia strains (Table 9) was

proportional to the ability of using starch as a carbon source. A1-

 

J'. -'.
A!

though the mycelial growth on glycogen was more abundant, especially
in Calvatia fragilis (1020) where it even surpassed the yield on
cellobiose.

Glycogen is a glucose polysaccharide similar to the amyIOpectin
fraction of starch, in which straight-chain arrays of glucopyranose
units (in (l-4)-glucosidic linkage) are cross linked by means Of
(1-6)-glucosidic bonds. Fructon and Simmonds (1959) reported that
inorganic phosphate was an Obligatory participant in the degradation of
glycogen and that a phosphorylated monosaccharide, glucose-l-phos-
phate was formed in the reaction.

It may be possible, in the case of Calvatia fragilis, that a

 

phosphorolysis took place to convert glycogen into glucose-l-phosphate.
This phosphorylated glucose derivative entering the main respiratory

pathway provided the energy for growth. Calvatia gigantea strains

 

1018 F and 1019 B seemed not to be able to accomplish such a phophoro-

lysis. .

 

 

I 37".13‘

 

71

According to the results in Table 9, dextrin was far the best
carbon source investigated. With the exception of strain 1018 F,
Calvatia species were able to satisfy their carbon requirements most
effectively on this polysaccharide. Dextrin is a product Of partial
hydrolysis of starch. Dextrins, formed from amylose have unbranched

chains, while those from amyIOpectin are branched.

8.8.8.3

Each Calvatia strain tested reSponded in the same way to dextrin,
as they did to starch and glycogen. Therefore, the discussion con-

cerning the ability to utilize starch and glycogen by this fungi are

 

also valid for dextrin utilization. The quantitative differences th
between starch and dextrin as carbon sources, however, cannot be

interpreted easily until the chemistry of these polysaccharides is

better known, according to Cochrane (1958).

The results in Table 9 demonstrate that inulin was poorly utilized
by Calvatia species. Just a Small growth was Observed in each strain.
Inulin is a polyfructoside and a specific enzyme, inulase is necessary
to break it down to its constituent fructose molecules. Palmer (1951)
reported that inulin represented a linear array of about 33 fructo-
furanose units joined together by means of [3-(2-l)—g1ucosidic link-
ages. He also found a small number Of D-glucose units in inulin.
Though all the investigated strains Of Calvatia grow well on fructose,
they could not utilize inulin. The insignificant small amount of
growth on this carbon source could be explained by the few units Of
glucose present in inulin.

Hawker (1950) concluded that the inability of fungi to use inulin

for growth was due to the failure to secrete the enzyme, rather than

 

72

any inhibitory effect of the inulin itself.

The results presented in Table 9 Show that Calvatia species were
not able to use cellulose as a carbon source. Evidently they lacked
the enzyme necessary to decompose this polysaccharide.

Cellulose is a linear polymer of D-glucose, where the glucose
molecules are joined together through (1'4)“.fl3 -glucosidic linkage. e8
Norkrans (1956) noted that cellulose was apparently attacked at several
points along the chain of glucose residues.

The lack of cellulase formation by Calvatia species could be proved

 

by the fact that cellobiose, the intermediate product of this poly- i8}
saccharide, was one of the best carbon sources for the organisms.
However, it should be mentioned that the technic used for testing the
cellulose utilization Of Calvatia strains was unsatisfactory. The
ability to use this polysaccharide could be inhibited by many other
factors, as for instance the water insolubility of the powdered cellu-
lose used.
Mixture Of Sugars

The results in Table 10 indicate also the effect of the combina-
tions of carbohydrates tested on Calvatia species. Each<:ombination Of
glucose resulted in a smaller amount Of mycelium than when glucose
alone was present in the medium. However, the yields were superior
to those which were Obtained using the other components separately.
This may be due to the fact that glucose allowed sufficient growth
so that the enzymes essential to galactose, lactose or rhamnose utiliza-

tion could be synthesized.

A stimulatory effect was Observed when glucose and rhamnose were

 

 

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73

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74

both present on the growth of strain 1019 B. However, rhamnose

alone was poorly utilized by the organism. It is possible that glucose

acted as an "inducer" to form an ”adaptive enzyme" to utilize rhamnose,

although no experimental evidence is available to prove this assumption.
The mixture of the two poorest carbon sources, lactose and rhamnose,

was not able to satisfy the carbon requirement Of the Calvatia strains

pr-
investigated. 5
1:
The combination of L- and D-arabinose promoted the same mycelial g
growth for each strain as the more effective of these two sugars did
‘when used separately as a carbon source. 1}
;

 

Organic Acids

The results in Table 11 show that under these experimental con-
ditions, Calvatia species were not able to use satisfactorily the organic
acids as carbon sources. All the organic acids employed in this study
supported poorly the mycelial growth in the strains investigated.

The relatively best growth was Obtained in the presence of urea.
That this may be due to the fact that this compound is a frequent con-
stituent of the higher Basidiomycetes. However the yields were so
small that urea cannot be considered a growth supporting carbon source.
Nevertheless, strain 766 grew as well on this compound as on glucose.

Acetate, oxalate, succinate and citrate, the intermediate products
of the Krebs cycle were poorly utilized by the strains Of Calvatia.

NO appreciable yields were Obtained in the presence Of aSparagine
or ammonium tartarate as a sole carbon source.

Alanine and arginine were also poor carbon sources for Calvatia

species. Lysine was the most poorly utilized of the organic acids and

 

75

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77

leucine was not able to promote any growth at all.
Glucose and some of the organic acids (Table 11) were incorporated
into the same medium to determine whether the lack of growth was due
to the toxic effect of the organic acids. Neither compound was poisonous
to the organism because when glucose was added to these compounds the
fungus produced an abundant amount of mycelium. E”
Although there are certain<2hemical differences in the organic
acids employed in this study, it is believed that some general conclu-

sions can be drawn from the results obtained.

 

In general, the organic acids are poor carbon sources for Calvatia L}
Species. That can be due to the inability of the fungi to produce the
variety of enzymes necessary to carry out the degradations and oxidations
of these compounds. Another explanation might be that the cells are
impermeable to the organic acids in such high concentrations used (be-
cause of the equivalent amount of carbon supply) and at physiological
pH levels.

The fungi produced alkaline reaction in the organic acid medium.
Increases in Ph from 5.5 to 6.8 were frequent. Higher pH values were
associated with the medium on which better mycelial growth occurred.
However, no relationship between the acidity of the organic acids and

the final pH of the medium was apparent.

78

SUMMARY

Three strains of Calvatia gigantea (1018 F, 1019 B and 766) and

 

one strain of Calvatia fragilis (1020) were studied in this nutritional

 

investigation.

All four strains of Calvatia grew well on a modified Czapek-Dox

 

medium and on Lindeberg's medium in the presence of thiamine. It r
was found that the optimum temperature range for mycelial growth of
the organisms was between 20 and 24° C and the optimum pH at 5.5.

Each of the strains investigated were totally heterotrophic with F
respect to thiamine, and autotrOphic with respect to biotin, pyridoxine L’

and inositol. A partial deficiency was established for an unidentified
growth factor(s), present in Bacto-Peptone and Bacto-Yeast extract.

No significant differences in mycelial growth of the strains of
Calvatia were obtained when equivalent amounts of thiamine and thiamine-
pyrophosphate were compared. The submerged culture method (using
fragmented hyphae) proved to be superior to the surface culture method.

Calvatia species were able to utilize a variety of carbon sources
when grown in Lindeberg's medium in the presence of thiamine. The
amount of growth varied with the carbon source and was determined by
the average mycelial dry weight of two replicates after 14 and 28 days
of incubation.

Calvatia gigantea (strain 1018 F) grew best in the following

 

decreasing order: cellobiose, maltose, glucose, fructose, invert sugar,
mannose and dextrin. A moderately good growth was obtained using
glycogen, starch, trehalose, urea, sucrose and raffinose, while the

organic acids and inulin supported very little growth.

79

Calvatia giganetea (strain 1019 B) utilized dextrin, cellobiose,

 

invert sugar, maltose, glucose, starch, glycogen, trehalose, and
fructose, listed in the order of their decreasing effectiveness.
Considerably less, but still available to the organism as carbon

sources were sucrose, urea, galactose, mannose and ribose. The organism
grew poorly on medium incorporating inulin, raffinose and the organic
acids.

Calvatia gigantea (strain 766) produced good mycelial growth on

 

dextrin, invert sugar, glycogen, starch, glucose, urea, cellobiose,
maltose and trehalose, in that order. Somewhat lower yields were ob-
tained in the presence of galactose, fructose, sucrose and the organic
acids. Raffinose and mannose were poor carbon sources for this fungus.

Calvatia fragilis (strain 1020) satisfied its carbon requirement

 

effectively on dextrin, cellobiose, glyc0gen, invert sugar, glucose
and starch, listed in the order of their decreasing effectiveness.
Galactose, mannose and fructose were less available for the organism.
No significant growth was obtained in the presence of the other carbon

sources tested.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.
11.

12.

13.

14.

15.

16.

80
BIBLIOGRAPHY

Albritton, E.C. 1952. Standard values in nutrition and metabolism.
WADC. Tech. Report 52-301. Wright-Patterson Air Force Base.

Anchelm.M., A. Hervey and W.J. Robbins. 1952. Antibiotic sub-
stances from Basidiomycetes. X. Fomes juniperuus, Schrenk. Proc.
Nat. Acad. 5C1. 38: 6550

Aschan, K. 1954. The production of fruit bodies in Collybia w~8
velutipes. 1. Influence of different culture conditions. Physiol.
Plantarum. 7: 571.

Atkinson, N. 1954. Psalliotin, the antibiotic of Psalliota
xanthoderma. Nature 174: 598.

Barnett, H.L. and V.G. Lilly. 1948. The interrelated effects of ' '
vitamins, temperature and pH upon vegetative growth of Sclerotinia J
camelliae. Am. Jour. Bot. 35: 297

 

V?!

Bealing, F.J. 1953. Mould "Glucosaccharase": a fructosidase.
Biochem J. (London) 55: 93.

Best, C.H. and N.B. Taylor. 1945. The physiological basis of
medical practices. William.and Wilkins, 2nd Ed.

Bille-Hansen, E. 19538. Fructification of three copr0philous
species of Caprinus, using glucose, sucrose and maltose as carbon
source. Bot. Tidsskr. 50: 81

Bille-Hansen, E. 1953 b. Fructification of copr0philous Caprinus
on synthetic medium. Physiol. Plantarum. 6: 523.

Bonner, J. 1950. Plant Biochemistry. Academic Press. New York.
Bose, S.R. 1947. Antibiotic in a Polyporus. Nature 158: 292.

Brannon, J.M. 1923. Influence of glucose and fructose on growth
of fungi. Bot. Gaz. 76: 257

Burkholder, P.R. and D. Moyer. 1943. Vitamin deficiencies of
fifty yeasts and molds. Bull. Torrey Bot. Club 70: 372.

Burkholder, P.R. and E.W. Sinnott. 1945. Morphogenesis of fungus
colonies in submerged shaken cultures. Am. Jour. Bot. 32: 424.

Cochrane, V.W. 1958. Physiology of Fungi. Wiley and Sons, New York
1928.

Coker, W.C. and J.N. Couch. /The gasteromycetes of the Eastern United

States and Canada. Univ. of North Carolina Press, Chapel Hill.

 

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

81

Cunningham, G.H. 1944. The Gasteromycetes of Australia and New
Zealand. 236 pp. Dunedin.

Dennis, R.W.G. 1953. Some West Indian Gasteromycetes. Kew.
Bulletin: 307

Derrick, M.J. 1949. Fundamental studies of the nutrition and
physiology of wood destroying Basidiomycetes. Ph.D. thesis,
Syracuse University.

Doery, H.M., J.F. Gardner, H.S. Burton and P. Abraham. 1951. Fr
Antibiotics from a Basidiomycete, Coprinus Quadrifidus. Antibiotics
and Chemotherapy 1: 409. P

fir \-
A...

Darrell, W.W. and R.M. Page. 1947. The use of fragmented mycelium ~
in the culture of fungi. J. Bact. 53: 360. f

Etheridge, D.E. 1955. Comparative studies of North American and
European cultures of the root rot fungus, Fomes annosus (Fr.)
Cooke. Can. J. Bot. 33: 416.

 

.14 ,— -
H...

 

Findlay, W.P.K. 1941. Effect of addition of sugar on rate of
decay of wood. Ann. Applied Biol. 28: 19.

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

I!
Fries, R.E. 1919. Ubfir einige Gasteromyceten aus Bolivia und
Argentinian. Arkiv. fur Bot. 8:1

Fries, N. 1943. Vitamin B1, vitamin B6 and biotin auf das Wachstum
einiger Ascomyceten. Symb. Bot. Upsalienses 7:5

Fries, N. 1949. Culture studies in the genus Mycena. Svensk Bot.
Tidsskr. 43: 316

Fries, N. 1950. Ophiostoma multiannulatum as a test object for
the determination of pyridoxine and various nucleotide constituents.
Arkiv Bot. 1:271.

 

Fries, N. 1950. Growth factor requirements of some higher fungi.
Svensk Bot. Tidsskr. 44: 370

Fries, L. 1953. Factors promoting growth of C0prinus fimetarius
(L.) under high temperature conditions. Physiol. Plantarum 6:551

Fries, L. 1955. Studies in the physi010gy of Ceprinus. I. Growth
substance, nitrogen and carbon requirements. Svensk Bot. Tidsskr.
49: 475

Fries, L. 1956. Studies in the physiology of Coprinus, II. In-
fluence of pH, metal factors and temperature. Svensk. Bot. Tidsskr. 50:1

I-u’

 

 

82

33. Froser, L.M. and B.S. Fugikova. 1958. The growth promoting effect
of several amino acids on the common cultivated mushroom Agaricus
bisporus. Mycologia 50: 538.

34. Fruton, J.S. and S. Simmonds. 1959. General Biochemistry. Wiley,
New York, 2nd Ed.

35. Fuller, R.C. and E.L. Tatum. 1956. Inositol-phospholipid in
Neurospora and its relationship to morphology. Am. Jour. Bot.
43: 361

 

36. Garner, J.H.B. 1956. Gasteromycetes from Panama and Costa Rica.
Mycologia 48: 757.

37. Gottlieb, D. 1946. The utilization of amino acids as a source of
carbon by fungi. Arch. Biochem. 9: 341.

38. Gottlieb, 8., W.C. Day and M.J. Pelczar, Jr. 1950. The biological
degradation of lignin II. The adaptation of white-rot fungi to
growth on lignin media. Phytopath. 40: 926.

39. Grimm, P.W. and P.J. Allen. 1954. Promotion by zinc of the forma-
tion of cytochromes in Ustilago sphaerogena. Plant PhysiOIOgy 29: 369.

SI
40. Gyorgy, P. 1954. The Vitamins. In Sebrell and Harris (eds)
(527 - 593). Academic Press Inc., New York

41. Haag, E. and C. Dalphin.. Une réaction biochimique de l'aneurine
d' une grande sensibilite. Compt. rend. soc. phys. hist. mat.
Geneva 57: 76. 1940

42. Harris, D.L. 1956. Interaction of thiamine and pyridoxine in
Neurospora. 11. Competition between pyridoxine and the pyrimidine
precursors of thiamine. Arch. Biochem. BiOphys. 60: 35.

43. Hawker, L.E. 1939. 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: 455

44. Hawker, L.E. 1950. Physiology of Fungi. Univ. of London Press,
London.

45. Herrick, J.A. 1940. The carbon and nitrogen metabolism of Stereum
ggusapatum Fries. Ohio Jour. Sci. 40: 123

 

46. Hervey, A.H. 1947. A survey of 500 Basidiomycetes for antibacterial
activity. Bull. Torrey Bot. Club 74: 476.

47. Hodson, A.Z. 1949. Oleic acid interference in the Neurospora
crassa assay for biotin. J. Biol. Chem. 179:49.

 

 

 

 

83

‘48. Hollos, L. 1904. Die Gasteromyceten Ungarns. Leipzig.

49. Humfeld, H. and T.F. Sugikova. 1952. The nutrient requirements

of Agaricus campestris grown in submerged culture. Mycologia
44: 605

50. Jansen, B.C.P. 1954. The Vitamins. In Sebrell and Harris (eds)
(426 - 448), Academic Press Inc., New York

51. Johnsson, G.T. and A.C. Jones. 1941. Data on the cultural
characteristics of species of Coprinus. Mycologia 33: 424.

52. Kavanagh, F., A. Hervey and W.J. Robbins. 1949. Antibiotic sub-
stances from Basidiomycetes. IC. Marasmius conigenus. Proc. Nat.
Acad. Sci. 35: 343.

 

53. Khudiakov, J.F. and U.M. Vozniakovskaia. 1951. Pure cultures of
mycorrhizal mushrooms. Mikrobiologia (USSR) 20: 13

54. Kitay, E. and E.E. Snell. 1948. Effect of size of inoculum on
the apparent vitamin requirements of lactic acid bacteria. Soc.
Exptl. Biol. and Med. 68: 648

55. Kluyver, A.J. and L.H.C. Perquin. 1933. Zur Methodik der Schimmel -
Stoffwechseluntersuchung. Biochem.Z. 266: 68

56. Koch, W. 1958. Untersuchungen aber'Mycelwachstum.und Fruchtkgr-
perbildung bei einigen Basidiomyceten (Polystictus versicolor,
Polyporus annosus, Pleurotus ostreatus and Psalliota bispora.)
Arciv £11? Mikrobiologie 30: 409

II
57. Kggl, F. and N. Fries. 1937. Uber den Einfluss von Biotin,
Aneurin and meso-Inosit auf das Wachstum.verschiedenen Pilzarten.
Zeit. physiol. Chem. 249: 93

58. Lamprecht, L. 1957. Zur Physiologie des echten Hausschwamms.
Dieuverarbeitung anorganischer Stick stoffverbindungen in
Abhangigkeit von Konzentration, pH-Wert und Isoelektrischem Punkt.
Archiv fur Midrobiologie. 27: 182.

59. Lamprecht, L. 1958. Beitrgge zur Physiologie des echten
Hausschwamms. Uber die Verfiertung organisch gebundenen Stickstoffs
und Kohlenstoffs. Archiv fur Mikrobiologie. 30: 190

60. Lane, R.L. and R.J. Williams. 1948. Inositol, an active con-
stituent of pancreatic (alpha) amylase. Arch. Biochem. 19: 329

61. Lardy, H.A., R.L. Potter and C.A. Elvehjem. 1947. Involvement
of biotin in the biosynthesis of oxalacetic and -ketog1utaric
acids. J. Biol. Chem. 169: 451

 

'r

 

 

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

Lardy, RCA. 1954.
351) Academic Press Inc., New

Le Mense, E.H., J. Corman, J.M3 Van Lanen and A.F. Langlykke.

Production of mold amylases in
54: 1490

Leonian, L.H. and V.G. Lilly.
fungi II.
path. 29: 592

Leonian, L.H. and V.G. Lilly.
some filamentous fungi. Plant

Lilly, V.G. and H.L. Barnett,
McGraw Hill Book Co. Inc., New

Lilly, V.G. and H.L.
fungi. W. Va. Univ.

Barnett o
Agr. EXPO

Lilly, V.G. and H.L.
arabinose by fungi.

Barnett.
Am. Jour.

fl
Lindeberg, G. 1939. Uber den
auf das Wachstum verschiedener
Tidsskr. 33: 341.

H
Lindeberg, G. 1941. Uber die
androsaceus (L.) Fr. Arch. fur

Lindeberg, G. 1944. fiber die
Bodenhymenomyceten.
Symb. Bot. Upsalienses. 8(2) :

Lindeberg, G. 1946.

The Vitamins.

Effect of inoculum on the growth of the colony.

84

In Sebrell and Harris (323 -
York

1947.

submerged culture. J. Bacteriol.

1939. Studies in the nutrition of

Phyto-
1940. Auxithals synthesized by
Physiol. 15: 515

1951.
York

Physiology of the Fungi.
1953. The utilization of sugars by
Sta. Bull. 362 T: 5

1956. The utilization of D and L-
Bot. 43: 709

Einfluss der Wasserstoffionenkonzentrations
Marasmius Arten. Svensk Bot.

Wirkung von Biotin auf Marasmius
Mikrobiologie 12:58

Physiologie ligninabbauender

Studien on Swedischen Marasmius Arten.

1

The effect of biotin and thiamine on the

growth of Collybia dryophila Fr. Svensk Bot. Tidsskr. 40: 63

 

Lucas, E.H., R.U. Byerrum, D.A.
1958 - 1959.
in vivo and in vitro by species of the genus Calvatia.

and Co Ch. StQCko
biotics annual

MacLead, D.M. 1959.

1. Growth reSponse in an enriched liquid medium.

Bot. 37: 695

Madelin, MoFo 1956.

Fr., especially as affecting fruiting.

Mandels, GoRo 1954.

Clarke, H. Ch. Reilly, J.A. Stevens
Production of oncostatic principles
Anti-

Nutritional studies on the genus Hirsutella

Can. Jour. of

Studies on the nutrition of Coprinus lagopus

Ann. Bot. (N.S.) 20: 303

Metabolism of sucrose and related oligo-

saccharides by spores of the fungus‘Myrothecium verrucaria.

Plant Physiol. 29: 18

 

a
o
o
o
o o
I
n
O
. O I I
u
o o
o n
I
o
o .
Q
.0
ad. . liulil1§
, I.

 

77.

78.

79.

80.

81.

82.

83.

84.

85.

86.

87.

88.

89.

90.

91,

85

Mandels, G.R. 1955. Biotin and interrupted growth of Myrothecium
verrucaria. Am. Jour. Bot. 42: 921

 

 

Margolin, A.S. 1942. The effect of various carbohydrates upon
the growth of some fungi. (Thesis) W. Va. Univ.

Mathiesen, A. 1950. The nitrogen nutrition and vitamin require-
ment of Ophiostoma pini. Physiol. Plantarum 3:93

 

McNutt, W.S. Jr. 1954. The direct contribution of adenine to the
biogenesis of riboflavin by Eremothecium ashbyii. J. Biol. Chem.
210: 511

 

Melin, E. 1925. Untersuchungen uber die Bedeutungen der Baummy-
korrhiza. Eine okologisch physiologische Studie. Gustav Fischer.
Jena.

Melin, E. and B. Nyman. 1940. Weitere Untersuchungen fiber die
Wirkungnvon.Aneurin und Biotin auf das wachstum.von Wurzelpilzen.
Arch. fur Mikrobiologie. 11:313.

Melin, E. 1946. Der Einfluss von Waldstre extracten auf das
Wachstum von Boden'ilzen mit besonderer Berucksichtigung der
Wurzelpilze von Baumen. Symb. Bot. Upsalienses. 8: 3.

Melin, E. and V.S.R. Das. 1954. Influence of root-metabolism
on the growth of tree mycorrhizal fungi. Physiol. Plant. 7: 851

Melin, E. 1959. Studies on the physiology of tree mycorrhizal
Basidiomycetes. 1. Growth response to nucleic acid constituents.
Svensk. Bot. Tidsskr. 53: 135.

Mikola, P. 1948. On the physiology and ecology of Cenococcum
graniforma. Comm. Inst. Forest fennicae. 36: 3.

‘Modess, 0. 1941. Zur kenntnis der Mykorrhizabildner von Kiefer
and Fichte. Symb. Bot. Upsalienses. 1: 1

Nilsson, H. 1956. The growth-stimulating effect of fructose-1,
6-diphosphate on Boletus variegatus and Collybia velutipes.
Physiol. Plantarum. 9: 74

 

Nobles, M.K. 1948. Studies in forest pathology. VI. Identifi-
cation of cultures of wood-rotting fungi. Can. Jour. Research
Sec. C., 26: 281

Norkrans, B. 1950. Studies in growth and cellulolytic enzymes
of Tricholoma. Symb. Bot. Upsalienses. 11:5

Norkrans, B. and B.G. Ranby. 1956. Studies of the enzymatic de-
gradation of cellulose. Physiol. Plantarum. 9: 198

 

 

“r—

 

 

l
. s
/ j ,
’ o
v

92.

93.

94.

95.

96.

97.

98.

99.

100.

101.

102.

103.

104.

105.

86

Norman, A.C. and W.H. Fuller. 1942. Cellulose decomposition by
microorganisms. [Advances in Enzymol. 2: 239

Oddoux, L. 1953. Essai de culture de 508 especes 1' homobasidio-
mycetes. Inernat. Conf. on Sci. Aspects of mushroom growing.
Proc. 20 MUSho 8C1. 2: 28

Palmer,.A.A. 1951. A specific determination and detection of
glucose as a probably constituent radical of certain fructosans
by means of Notatin. Biochem J. 48: 389.

Perlman, D. 1948. On the nutrition of Memnoniella echinata and
Stachybotrys atra. Am. Jour. Bot. 35: 36

Perlman, D. 1949. Studies on the growth and metabolism of Poly-
porus anceps in submerged culture. Am. Jour. Bot. 36: 180

Plunkett, B.E. 1953. Nutritional and other aspects of fruit-body
production in pure cultures of Collybia velutipes (Curt.) Fr.
Ann. Bot. (N.S.) 17: 193.

 

Reese, E.T. 1947. On the effect of aeration and nutrition on
cellulose decomposition by certain bacteria. J. Bacteriol. 53:
389.

Reese, E.T. and H.S. Levinson. 1952. A comparative study ofthe
breakdown of cellulose by microorganisms. Physiol. Plantarum 5:
345.

Ritchie, D. 1948. The development of Lycoperdon oblongisporum.
Am. Jouro Bat. 35: 21.5

Robbins, W.J. and R. ma. 1942. Specificity of pyridoxine for
Ceratostomella ulmi. Bull. Torrey Bot. Club 69: 184

Robbins, W.J. and A. Hervey. Growth substance requirement of
Stereum.murraii. ‘Mycologia 47: 155. 1955.

 

Savage, G.M. and Vander Brook. 1946. The fragmentation of the
mycelium of Penicillium notatum and Penicillium chrysogenum

by a high speed blender and the evaluation of blended seed.

J. Bacteriol. 52: 385.

Siu, R.C.H. 1951. Microbial decomposition of Cellulose. Rein-
hold Pub1., New York

I
Schgpfer, W.H. 1934a. Les vitamines cristallisees B comme
hormones de croissance chez un microorganisme. (Phycomyces).
Arch. fur Microbiologie. 5: 511

106.

107.

108.

109.

110.

111.

112.

'113.

114.

115.

116.

117.

118.

119.

120.

87

H
Schgpfer, W.H. 1934 b. Uber die Wirkung von reinen kristallisierten
Vitaminen auf Phycomyces. Ber. deut. bot. Gas. 52: 308

 

Schgpfer, W.H. and M. Guillaud. 1945. Recherches experimentales
sur les facteurs de croissance et le pouvoir de synthese de'Rhizpous
cohnii Berl. et de Toni (Rhizopus suinus Neilson.) Zeit. fur
Vitaminforsch. 16: 181.

 

Scheler-Correns E. 1957. Uber die Fruchtkgrperbildung von
Coprinus lagopus bei verschiedenen Stickstoffquellen. Archiv fur
Mikrobiologie. 26: 52

 

Shirakawa, H.S. 1955. The nutrition of Diplocarpon rosae. Am.
Jour. Bot. 42: 379

Silver, W. and W.D. McElroy. 1954. Enzyme studies on nitrate and
nitrate mutants of NeurOSpora. Arch. Biochem. Biophys. 51: 379

Slein, M.W. 1950. Phosphomannose isomerase. J. Biol. Chem. 186: 753

Small, B.E., B.M. Guirard and R.J. Williams. Occurrence in natural
products of a physiologically active metabolite of pyridoxine.
Jour. Biol. Chem. 143: 519. 1942.

Snell, B.E. and A.N. Rannefelt. 1945. The vitamin B6 group. III.
The vitamin activity of pyridoxal and pyridoxamine for various
organisms. Jour. Biol. Chem. 157: 475.

Snell, B.E. 1954. The Vitamins. Sebrell and Harris (366 - 367)
Academic Press Inc., New York

Stevens, J.A. 1957. Studies of environmental factors influencing
in vitro growth of Basidiomycetes and their elaboration of biolOgically
active substances. (Ph.D. thesis) Michigan State University

Stokes, J.L., J.W. Foster and G.R. Woodward. 1943. Synthesis of
pyridoxine by a "pyridoxineless" X-ray mutant of Neurospora
sitophila. Arch. Biochem. 2:235.

 

Swartz, D. 1933. Some developmental characters of Species of
Lchperdaceae. Am. Jour. Bot. 20: 440

Tatum, B.E. and T.T. Bell. 1946. Neurospora. III. Biosynthesis
Of thiamine. Am. Jouro Bet. 33: 150

Treschow, C. 1944. Nutrition of the cultivated mushroom. Dansk.
Bot. Arkiv. 11:1.

Umbriet, W.W. 1954. The Vitamins. Sebrell and Harris (234 - 242).
Academic Press Inc., New York

121.

122.

123.

124.

125.

126.

127.

128.

88

Van Vliet, W.F. 1954. The enzymic oxidation of lignin. Biochem.
et BiOphys. 15: 211

Vorderberg, K. 1948. Nutritional requirements of Coprinus lagopus.
Z. Naturforsch. 3b: 272

 

West, E.S. and W.R. Todd. 1956. Textbook of Biochemistry. The
MacMillan Co. (2nd Ed.), New York

Niken, T., H.G. Keller, C.L. Schelling and A. Stockli. 1951.
Uber die Verwendung von Myzelsuspensionen als Impfmaterial in
Wchstumversuchen mit Pilzen. Experientia. 7: 237

Wilkins, W.H. 1945. Investigation into the production of bacterio-
static substances by fungi. Cultural work on Basidiomycetes.
Trans. Br. Mycol. Soc. 28: 110

Wirth, J.C. and F.F. Nord. 1942. Essential steps in the enzymatic
breakdown of hexoses and pentoses. Interaction between dehydro-
genation and fermentation. Arch. Biochem. 1: 143

Wolpert, F.S. 1924. 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. Miss. Bot. Gard.
11: 430

Yusef, H.M. 1953. The requirements of some Hymenomycetes for
essential metabolites. Bull. Torrey Bot. Club 80: 43

Typical analysis of Bacto-Ycast Extract -

89

(Furnished by the Difco

Laboratories Incorporated, Detroit 1, Michigan)

PER CENT

Ash

Total N
Chloride
Total Sulphur
PPM.

Lead

Arsenic
Manganase
Zinc

Copper

PER CENT
Phosphorus
Iron

SiOz
Potassium
Sodium
Magnesium
Calcium
Arginine
Asparatic acid
Glutamic acid
Glycine

Histidine

10.10
9.18
0.19

1.39

16.00
0.11
7.80

88.00

19.00

0.89
0.028
0.052
0.042
0.32
0.03
0.0406
0.78
5.1
6.5
2.4

0.94

 

75 3.1.: 1.. .3...

 

Typical analysis of Bacto-Yeast Extract

PER CENT
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Tryptophane
Tyrosine
Valine

MICROGRAMS PER GRAN

 

Pyridoxine
Biotin
Thiamine
NicOtinic acid
Riboflavine

Folic acid

cort inued

2.9
3.6
4.0
0.79
2.2
3.4
0.88
0.60

3.4

20.00
1.40
3.20

279.00

19.00

0.30

90

Typical analysis of Bacto-Peptone -

Incorporated, Detroit 1, Michigan)

91

(Furnished by Difco Laboratories

 

Total Nitrogen 16.16%
Primary Proteose N 0.06%
Secondary Proteose N 0.68%
Peptone N 15.38% F
Ammonia N 0.04%
Free amino N (Van Slyke) 3.20%
Amide N 0.49%
Mono-amino N 9.42%
Di-amino N 4.07%
Tryptophane 0.29%
Tyrosine 0.98%
Cystine (Sullivan) 0.22%
Organic Sulphur 0.33%
Inorganic Sulphur 0.29%
Phosphorus 0.22%
Chlorine 0.27%
Sodium 1.08%
Potassium 0.22%
Calcium 0.058%
Magnesium 0.056%
Manganese Nil
Iron 0.0033%
Ash 3.53%

Lead 15.00 ppm

Typical analysis of Beets-Peptone

 

Arsenic

Zinc

Capper

Si02

Arginine
Asparatic acid
Glutamic acid
Glycine
Histidine
Isoleucine
Leucine

Lysine
Methionine
Phenylalanine
Threonine
Valine
Pyridoxine
Biotin
Thiamine
Nicotinic acid

Riboflavine

continued

92

18.00 ppm

‘17.00 ppm

0.042%

8.00%

5.90%

11.00%
23.00%

0.96%

2.00%

3.50%

4.30%

0.83%

2.30%

1.60%

3.20%

2.50 gamma/gm.
0.32 gamma/gm.
0.50 gamma/gm.
35.00 gamma/gm.

4.00 gamma/gm.

 

CHI AN 15 AVE ‘UNNERSI‘H LIBRARIE

 

31293 02365 0678