ASST RACT

CARBOHYDRATE UTILIZATION AND ITS RELATION TO THE
PRODUCTION OF ANTITUMOR SUBSTANCE IN
SUILLUS pggggg

by Flora Zaibun Majid

Suillus luteus (Fr.) S.F. Gray. (Syn. ggigggg
luteus) possess antitumor activity against Crocker mouse
Sarcoma 180 (Beneke, unpublished data). A review of the
literature revealed no reports on the carbohydrate require—
ments of this fungus.

The main purpose of this investigation was to
determine some of the carbon requirements of g. lgggyg, to
determine if carbon sources would affect yields of anti-
tumor agent, and also to determine if any morphological
changes in the fungal hyphae are induced by different
Isugars in the medium.

The utilization of the nutrients was determined
by the growth response of the organisms and measured on
the basis of the average mycelial dry weights after ten
and fifteen days of incubation. Morphological characters
of the fungi were studied under the microscope. The
samples were tested for activity against the Crocker mouse
Sarcoma 180. Weights of tumors of test animals were com-
pared with those of control animals. Results were

1 I

2 Flora Zaibun Majid
expressed as percent of control tumor growth.
There was a differential utilization of the

'various carbohydrates by the two strains of Suillus luteus

 

(8—62-03 and 8-64-45). Pentoses in general supported poor
<growth of the strains. Hexoses were more favorable of
xkhich glucose was the best for both strains. Oligo-
saocharides supported fairly good growth of both strains.
Cellobiose was best for the strain 8-62-03 and maltose for
8-64-45. Polysaccharides in general supported poor growth
of both. .

Yeast extract in medium A was found to contain a
substance stimulatory to the growth of §. luteus when
strain 8-62-03 was used. Peptone was inferior to yeast
extract.

Changes in the hyphae, including protOplasm, were
effected by various sugars. There was a correlation of
good mycelial growth and dense protOplasm. Brown deposits
and clamp connections were found in the hyphae when
several sugars were substitutes as the carbon source in
the medium. ChlamydoSpores were induced only by sorbose.
8-62-03 and 8-64-45 showed similar reSponses when differ-
ent sugars were used.

Light as used under the experimental conditions
had no significant affect on the growth of 3-62-03.

There was a differential production of antitumor

substance by §. luteus when the various carbon sources

3 Flora Zaibun Majid
xaere used in the medium. Glucose and sucrose in the
rnedium resulted in fairly good antitumor activity in
8-62-03, while cellobiose and trehalose produced a sub-
stance that stimulated tumor growth. Maltose resulted in
Tbetter antitumor activity in 8-64-45 than the other
sugars tested.

The production of antitumor activity appeared to
‘be affected by the type of shaker for submerged cultures.
’This might have been owing to the duration of incubation

period.

CARBOHYDRATE UTILIZATION AND ITS RELATION TO
THE PRODUCTION OF ANTITUMOR SUBSTANCE

IN SUILLUS LUTEUS

 

by

Flora Zaibun Majid

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1965

To my family

1.1

ACKNOWL EDGFMENT S

The author wishes to express her sincere
appreciation to Dr. E. S. Beneke whose continued interest,
direction, and encouragement has made this work possible
and facilitated its completion.

Sincere thanks are also due to Drs. V. H. Mallmann,
W. B. Drew, G. Prescott, H. A. Imshaug and L. C. Ferguson
for their many helpful suggestions, constructive
criticisms, and much'needed advice.

I would like to express my deepest appreciation
to my American family, the C. W. Pohlys, for adOpting me
and encouraging me during my stay in the United States.

I am deeply grateful for the Fullbright Fellowship

‘which made my graduate studies possible.

111

TABLE OF CONTENTS
mas
INTRODUCTION ...............o..o....oo............ 1
LITERATURE REVIEW ................................ 4
I'iATERIPLS AND METHODS ............................ 16
RESULTS AND DISCUSSION ...........o...o........... 27
SUMMARY .......................................... 65

BIBLIOGRAPHY .000.000.000.00.000CUOOOOOOOOCOOOOOOO 67

IZ‘PPENDIX 0......OIOOOOOOOOOOOCOOOCOOOOOOOOOOOOOOOO 72

iv

II.

III.

IV.

VI.

The utilization of various monosaccharides
by Suillus luteus ........................
The utilization of various Oligo-
saccharides by Suillus luteus ............
The utilization of various poly-
saccharides by §uillus luteus ............
The utilization of peptone and yeast
extract by Suillus luteus strain,

B-62-O3 ..................................
Effect of light on Suillus luteus

8-62-03 ..........................
Antitumor activity of cultures of

Suillus luteus strains, 3—62-03 and

8-64-45 against Crocker mouse

Sarcoma 180

Page

28

49

62

LIST OF FIGURSS
FIGURE Page
1. The utilization of various mono-
saccharides (arabinose, xylose and

ribose) by Suillus luteus strains,

 

5-62-03 and 3-64-45 .................... 29
2. Utilization of various monosaccharides

(glucose, galactose and mannose) by

Suillus luteus strains, 3-62-03 and

5-64-45 ................................ 30
3. Utilization of various monosaccharides

(fructose, sorbose and rhamnose) by

Suillus luteus strains, 8-62-03 and

 

3-64-45 0.0.0....OOOOOOOOOOOOOOO...0.... 3-]-

4. Utilization of various oligosaccharides
(sucrose, maltose, lactose and cello-

biose) by Suillus luteus strains,

 

8-62-03 and 3-64-45 00000000000900.0000. Pg
5. Utilization of various oligosaccharides
(melibiose, trehalose and raffinose) by

Suillus luteus strains, 3-62-03 and

 

(I)
\0

3—64-45 ................................
6. Utilization of various polysaccharides

by Suillus luteus strains, 3-62—03

and 3-64-45 ............................ 43

vi

LIST OF PLAT ES

 

 

PLAT s W
l. Fruiting body of Suillus luteus strain,
3-62-03 ................................ l7
2. Fruiting body of Suillus Lgteus strain,
8-64-45 ................................ 18
3. Fifteen day old culture of Suillus luteus

 

strains, 3-62-03 and 3-64-45 ........... l9
4. Xycelium of Suillus luteus strain,

3-62-03, grown in submerged culture

with ribose as carbon source ........... 52
S. . Mycelium of Suillus luteus strain,

3-62-03, grown in submerged culture

with various sugars as carbon source.... 53
6. Mycelium of Suillus luteus strain,

3-62-03, grown in submerged culture

with various sugars as carbon

source 0......OOOOOCOOCOOOOOOOOOOOO0.... 54

vii

Introduction

Cancer is one of the most mystifying diseases,
the fatal outcome of which was recognized as early as
300 B.C. The organs in the human body, and the cells
that form them, vary widely. In some older individuals
and occasionally in the young, these cells, which
function regularly and consistently in health, divide
and multiply capriciously. The ensuing growth is what we
know as tumor (Harris, 1962). There is a high incidence
of the disease throughout the world. In Spite of great
efforts, modern science has not yet been successful in
finding a means of prevention or cure for cancer.

Much research has been done in the past and much
more is being conducted today, to find a possible
prophylactic or therapeutic agent for cancer. Attempts
have been made to find chemicals inhibitory against
cancer in fungi, eSpecially the Basidiomycetes.

Historically, the interest and possible tumor-
inhibitory prOperties of extracts from mushrooms or
other Basidiomycetes date back to an old folklore that
was discovered by Lucas when he visited a tiny mountain
village in the Bohemian Forest near the Bavarian Border
in 1934. According to this folklore (Lucas, 1960), the
lumberjacks in this area believed that eating a certain

l

2

type of mushroom-like fungus Boletus edulis prevents

 

cancer. In 1948, some dried material of g. edulis was
obtained for the preparation of crude water extracts and
tested against Crocker mouse Sarcoma 180 by the Sloan-
Kettering Institute for Cancer research. Very striking
inhibitory effects could be demonstrated by this species
of Boletus. However, the mycelium was found to be unable
to produce the tumor-inhibiting principle $3 33559. This
led to a search for other Basidiomycetes with tumor
inhibiting property.

§gillus luteus (Fr.) 5. F. Gray. (Syn. Boletus
luteus) possesses antitumor activity against Crocker mouse
Sarcoma 180 (Beneke, unpublished data). Further physio-
logical studies of this organism are required to provide
a basis for further investigations of the environmental
conditions under which §. luteus will be able to produce
a greater quantity of the antitumor agent.

Since the nutritional requirements of §. luteus
have never been studied, this investigation was concerned
with the carbohydrate requirements of the fungus. Not all
fungi are able to utilize the same sugars and whether a
sugar is utilized or not depends upon both the configura-
tion of the sugar and particular abilities of the specific
fungus. While there is an immense amount of infornation
scattered throughout the literature to the effect that a

certain sugar is utilized by various Species, much of this

3
information deals with relatively few sugars. Critical
studies on the utilization of the sugars are rare (Lilly
and Barnett, 1951).

The purpose of this investigation was to determine
some of the carbon requirements of g. luteus, to determine
if carbon sources would affect yields of antitumor agent,
and also to determine if any conspicuous morphological
changes in the fungal hyphae are induced by different

sugars in the medium.

LITERATURE REVIEW

Carbohydrates as well as lipids and proteins play
a fundamental role in the life of animals and plants.
They are an important source of energy for living
organisms and a means by which chemical energy can be
stored. In addition, some carbohydrates are structural
units of the cell. Examples of carbohydrates which
participate in the energy economy of the cell are the
polysaccharides, starch and glycogen. Cellulose and
chitin are typical structural carbohydrates (Conn and
Stumpf, 1963).

Carbon occupies a critical position among the
essential elements required by the living organisms. Al-
most half of the dry weight of a fungal cell consists of
carbon. Carbon compounds are of considerable importance
in fungal nutrition, since fungi secure energy by oxidiz—
ing organic compounds (Lilly and Barnett, 1951). An
extensive literature review on the carbon requirements
for organisms in the sub-class Homobasidiomycetidae was
made by Stevens (1957), Sedlmayr (1960), and Cook (1962).

Brannon (1923) stated that nearly all fungi which
can be cultured are capable of using glucose or fructose

as a carbon source.

5
Swartz (1933) reported maltose as a very favor-

able sugar for mycelial production in Calvatia saccata,

 

Q. caelata, and Q. gigantea.

Herrick (1940) reported that Stereum gausapatum
used xylose better than arabinose, and raffinose was
better than glucose. Glycogen was a good source of carbon
for the Species.

Johnsson (1941) noted that some of the C0prinus
Species are able to use raffinose as well as glucose.

Modess (1941) showed the influence of hydrogen ion
concentration on the growth of different mycorrhizal
fungi in pure culture. Optimum pH for the growth of

Boletus bovinus, g. edulis, and 2. luteus was found to be

 

5.5.

Norman & Fuller (1942) stated that the majority of
the fungi are capable of utilizing cellulose.

Treschow (1944) observed better mycelial production
by Psalliota biSQora in galactose or in xylose than in
glucose, and oxalic acid was a satisfactory carbon source
for the Species. .

Perlman (1949) found that the use of glucose by
gglyporus anceps depended on the prOper concentration of
thiamine in the medium.

Nokrans (1950) showed that the saprOphytic species
of Tricholoma utilized galactose better than the

mycorrhiza formers. Many Species of Tricholoma grew well

with cellobiose as a carbon source. Utilization of

6
cellulose was influenced by the presence of other carbon
sources.

Gottlieb gt a; (1950) reported that some of the
wood destroying fungi were able to grow with lignin as a
carbon source.

Lilly and Barnett (1951) gave an excellent
discussion on carbohydrates as carbon source for fungi.
The authors pointed out that not all fungi are able to
use exactly the same sugars. Whether a sugar is utilized
or not depends upon both the configuration of the sugar
and the particular ability of the specific fungi.

Glucose favors the growth of more fungi than any other
sugar, but some fungi are unable to utilize glucose. The
authors further mentioned that the general order of
availability of three common disaccharides appears to be
maltose, sucrose, and lactose. CellUlose and starch are
most abundant polysaccharides which are insoluble and
must be hydrolysed or otherwise degraded to low molecular
weight compounds. The ability to utilize a certain sugar
is dependent on the presence of the necessary enzymes.

A fungus uses a compound by a series of stepwise trans-
formations which might differ from fungus to fungus.

Khudiakov and Vozniakovskaia (1951) reported that
four different strains of Boletus were able to grow on a

medium containing glucose as the sole carbon source.

7

Siu (1951) suggested that enzymes which attacked
cellulose were liberated by microorganisms only in the
presence of cellulose.

Humfeld and Sugikova (1952) found that Agaricus
campestris was unable to grow on lactose.

Albritton (1952) showed that fewer Species of
fungi utilize lactose, maltose, sucrose, and raffinose
than the monosaccharides (glucose, fructose, and galactose)
which these oligosaccharides yield on hydrolysis.

Lilly and Barnett (1953) studied the utilization
of 12 sugars (D-glucose, D-fructose, D-mannose,
D-galactose, L-sorbose, L-arabinose, D-xylose, maltose,
lactose, sucrose, cellobiose, and raffinose), by 57
Species of fungi. All the Species studied were found to
grow on glucose, and usually fructose and mannose.
Galactose was used slowly and sorbose poorly. Of the
pentoses xylose was the most favorable, while arabinose
was best for a few Species. Most species utilized cello-
biose, and some grew poorly on maltose. Sucrose was used
poorly, lactose was unsatisfactory, and there was poor or
slow growth on raffinose. Some sugars were favorable for
growth only in presence of other utilizable sugars.
Sorbose was found to be inhibitory to utilization of
maltose, sucrose, and glucose.

Fries (1955) compared the different carbon

sources for the growth of Cogrinus species. Glucose,

8
mannose, and fructose were almost equally effective for
most of the investigated species. Maltose could be
utilized by all Species, (but to different degrees),
lactose could not be utilized by any, starch supported
excellent growth of some, and inulin was not utilized.

Lilly and Barnett (1956) demonstrated that
several fungi were able to use L-arabinose and failed to
grow or grew poorly on D-arabinose.

Madelin (1956) found that cellobiose was a good
source of carbon for most of the ggpgigug Species investi-
gated.

Bach (1956) studied the physiology of an agaric,
Pholigta aurea. Among the hexoses, glucose gave best
growth, and among the disaccharides, maltose was the best
source of carbon. Starch was the most favorable poly-
saccharide. The pentose sugars, xylose and arabinose,
were unsuitable as sources of carbon.

Wilson and Lilly (1958) studied the utilization
of oligosaccharides by some Species of Eggagggygtig.

The identity of the sugars present in the media during
incubation was determined by paper chromatography. 'Phe
original sugars were present in the media at the end of
the experiment when the sugars were not used for growth.
The expected hydrolytic products of the oligosaccharides
were found in the media when the fungi grew well.

Aschan-Aberg (1960) reported that the order of

utilization of carbon sources by Collybia velutipes

9
during the process of fructification was as follows:
glucose, sucrose, and maltose.
Robbins and Hervey (1960) showed that Polyporus

schweinitzii showed a partial deficiency for at least two

 

unidentified growth promoting substances which were widely
distributed in natural products. Malt extract, yeast
extract, tomato juice, coconut milk, etc., promoted the
growth of the fungus and appeared to contain one or both
of the unidentified substances.

Swack and Phillip (1960) determined quantitatively
the production of indigotin by a wood-rotting basidiomycete,
SchyZOphyllpm commune. Of the carbohydrates studied,
D-xylose gave the highest value for mycelium production,
D-fructose and D-glucose gave the highest value for
indigotin production.

Martin (1961) showed that the mycelium of a
fertile strain of Psalliota hortensis, used for the
preparation of commercial spawn, could not grow in absence
of calcium.

Sedlmayr, Beneke, and Stevens (1961) studied the
vitamin requirement of Calvatia gigantea. The strains
investigated were totally heterotrophic with reSpect to
thiamine, and autotrOphic with reSpect to biotin, pyridoxin,
and inositol. A partial deficiency was established for an
unidentified growth factor(s). present in the Baoto-

peptone and Bacto-yeast extract.

10

Sedlmayr, Beneke, and Stevens (1961) showed that
Calvatia Species were able to utilize a variety of carbon
sources. The amount of growth varied with the carbon
source.

Pantidou (1961) described the techniques for
culturing and studying Species of Boletaceae in culture.
A few species, Gyrodon merulioides, Boletinug cavipes,

g. pictus, a. spectabilis and B. grisellus, which had not
been previously described in culture were described.

Jayko, £5 a; (1962) studied nutritional require-
ments and fermentative products of some Lactarius
species. Glucose, maltose, and dextrin were found to be
the best carbon sources.

Santoro and Casida (1962) grew pure cultures of
some mycorrhizal fungi (Amenita Caesaria, A. rubescens,
é. muscaria, ggletus luteus, and g. bicolor) in a liquid
medium with various concentrations of gibberellin.
Gibberellin inhibited the growth of all the organisms.

Cook (1962) observed that calvacin production in
some strains of Calvatia gigantea varied depending on the
carbohydrates available in the culture medium.

Molitoris (1963) studied the growth of three
strains of Beauveria tenella in relation to carbon source
and concentration, and presence of inorganic salts. The
synthesis of cell material and lipid increased with
higher concentration of carbohydrates, and a high carbo-

hydrate nitrogen ratio favored fat production.

ll
Negrutskii (1963) studied the effect of vitamin 31
and H, and gibberellin on the growth of Fomitopsis annosa.
The fungus synthesized vitamin 31 and H from nutritive
substances in the medium. Gibberellin in any concentration
hindered growth.

Robbins, £3 a1. (1963) in their study of growth
factors for gglypprus schweinitzii identified ferulic acid
as a new cofactor. The beneficial effect on the growth of
the fungus of natural materials in a basal medium was
mainly due to the presence of ferulic, oleic, linoleic,
palmitic, and stearic acids in the natural materials.

Lingappa,§£ 21. (1963) showed that polysaccharides
like dextrin or starch, were the best carbon sources for
the growth of Aggggbasidium pullulans. Light increased
growth markedly when polysaccharides were the carbon
sources. Variation in cell morphology had been described
in response to sugars and light.

Tsu-Ning (1963) found that Pleurotus ostreatus,
if grown in submerged shaker cultures, produced oxalic acid
from simple carbohydrates as efficiently as did ASpergillus
raiser-

Robbins and Hervey (1963) studied unidentified
filtrate growth substances for several fungi. Tomato
juice, or a water extract of beech wood improved growth of
several fungi.

Beneke (1963) summarized all of the work done at

that time on Calvatia gigantea in his Presidential address

12

to the Mycological Society of America. The best carbon
sources for the mycelial growth of the fungus were
glucose, cellobiose, dextrin, glycogen and maltose.
Cellobiose appeared to be the best for calvacin production.
Calvacin is an antitumor agent or substance that inhibits
13 out of 24 types of cancer in the laboratory animals.

Falanghe, g5 21. (1964) tested various soy bean
whey media as substrate for several Species of fungi in
submerged culture. Tricholoma nudum and Boletus

indecisus had the greatest rate of growth and production

 

of mycelial protein, with much shorter incubation period
compared with those of other species.

Ward (1964) showed the difference in growth of a
low temperature basidiomycete, on filter-sterilized
as opposed to autoclaved medium. Growth on the filter-
sterilized medium was only about one-fourth of that on
the autoclaved medium, and growth was also greatly
reduced when glucose or KHzPO4 was autoclaved separately.
This suggested that interaction between glucose and
KH2P04 was reSponsible for the stimulatory effect after
autoclaving. This was confirmed by an experiment in
which the medium was (a) autoclaved, (b) filter-sterilized,
or (c) filter-sterilized without glucose and KH2P04,
which were autoclaved separately and added to the sterile

medium. The results obtained clearly demonstrated that

interaction between glucose and KH2P04 when autoclaved,

13
accounted for the stimulation of growth.

Robbins and Hervey (1965) reported the growth of
Morchella crassipes was enhanced by the addition of
natural materials to a medium containing mineral salts,
dextrose, vitamins of the B complex, and purine and
pyrimidine bases. The enhancement was due to the ash in
the natural products, primarily manganese and calcium.

Wilson (1965) studied various factors affecting
the germination of Calvatia giggggga. Gibberellic acid
and furfural had no effect on germination. Light was
slightly inhibitory to germination. Adthough many carbo-
hydrates supported germination, maltose, soluble starch,
and sucrose were the best.

A general picture regarding the utilization of
various sugars by several fungi has been provided by
Cochrane (1958) and is summarized here.

Monosaccharides: Of the hexoses D-Glucose is
biologically most important and is utilized for growth by
virtually all fungi. A few forms however, have been
reported not to grow with glucose. D-fructose and
D-mannose are equivalent to glucose for growth for a large
number of fungi. D-galactose is used by most fungi but is
not usually so good a source of carbon as glucose.
L-sorbose supports normal growth of a few fungi, however
it appears not to be utilizable by most forms and it is
definitely toxic to some. D-xylose is the most utilizable

of the pentoses generally, and has been reported to be

l4
superior to glucose for some fungi. 1t however supports
very poor growth of several fungi. In most studies
L-arabinose is inferior to xylose and glucose as a carbon
source. Rhamnose, a methyl pentose, has been reported to
be used only by a few organisms. D-ribose supports
growth of some fungi. The L-isomers of the naturally
occurring aldohexoses and of xylose do not support growth
of fungi, nor do those monosaccharides which do not occur

in nature (Cochrane, 1958).

Oligosaccharidesz The following 5 disaccharides
maltose, cellobiose, trehalose, sucrose, and lactose and
one trisaccharide, raffinose, is important in the
nutrition of some fungi. Maltose is utilized virtually
by all fungi which have been tested. Cellobiose is almost
as widely utilizable as maltose. Trehalose have been
found to support growth of a large number of fungi
including even some rather fastitious fungi. Sucrose is
generally a good source of carbon for many fungi. Lactose
is used by far fewer fungi than any of the disaccharides
so far mentioned and may therefore be considered as a
poor carbon source. Melibiose is not utilized by many
fungi. Majority of the fungi utilize raffinose but non-
utilization is also common. The utilization of an oligo-
saccharide is believed to be preceded by and dependent on
its conversion to hexoses or, possibly hexose phosphates

(Cochrane, 1958).

15

Polysaccharides: Among the polysaccharides starch,
dextrin, inulin, glycogen, and cellulose are considered to
act as carbon source for many fungi. Starch is an
excellent carbon source for most fungi, even for rather
fastidious forms. Dextrins, which are chemically or
enzymetically modified starches of uncertain structure,
are generally satisfactory source of carbon for many
fungi. Inulin, a polymer of D-fructose has been found to
be a good source of carbon for many but not all fungi.
Glycogens are quite generally available to fungi of
widely different ecological groups. Those fungi unable
to use starch can be expected not to use glycogen, since
so far as is known, the same enzymes attack both poly-
saccharides. Cellulose is by far the largest natural
reservoir of biologically utilizable carbon. It is an
excellent source of carbon for many fungi, but the
Species within a given genus are not all necessarily
alike in their reSponse to cellulose. Insolubility of
cellulose however makes the nutritional study of the
compound difficult. Whether a polysaccharide is used
or not is depended on the ability of the fungus to produce
the necessary hydrolytic enzyme and also on the configur-

ation of the sugar.

I-IAT ERIALS AN D M ETHO DS

The cultures of Suillus luteus were maintained on
a Bolete medium which had the following composition:

blalt EXtraCt OOOOOOOOOOOOOOOCOOOO Sgrams

DeXtrOSe OOOOOOOOOOOOOOOOOOOOOOO. sgra-ms

Y\H2PO4 cocooooooooooooooooooon... 0.5 grams

I‘lgm .7HO OOOOOOOOOOOOOOOOOOOOO 0.5 grams

4 2
I‘lH4C1 OOOOOOOOOOOOOOOOOO000...... OOSgrams
FeC13 (1% solution) ............. 0.5 ml

Agar 0......OOOOOOOOCOOOOOOOOOOOO 15 grams

DiStilled water OOOOOOOOOOOOOCOOC 1000 ml

Stock cultures were originally obtained by remov-
ing small pieces of tissue (pseudo-parenchyma) under
aseptic conditions, from_inside the cap or stipe of an
immature basidiocarp. Two strains were employed for the
carbohydrate studies. The collection data of which are
given below:

Strain 3-62-03 collected near Lansing, Michigan.
It was brought in for identification to the Biology
Research Center, East Lansing, Michigan, in 1962.

Strain B-64-45 collected by Dr. E. S. Beneke from
the Rose Lake area, near East Lansing, Michigan, on

October 5, 1964.

16

l7

PLATE I

 

 

Fruiting body of Suillus luteus strain, 3-62-03.

18

PLATE II

 

Fruiting body of Suillus luteus dark yellow strain, 13-64-45.

19

PLATE III

 

 

 

Fifteen day old culture of Suillus luteus Strains,
8-62-03 (on the right) and B-64-45 (on the left)

20

The taxonomic characteristics of g. luteus has
been described, by Smith (1965). Plates I and II show
some of the morphological characters of this mushroom-like
fungus. The pores are deeper yellow in B-64-45 (Plate II)
than in 8-62-03 (Plate I). The mycelial culture of
8-62-03 on medium A, produces a ridged, compact colony,
with tan color on the back, in two weeks. (Plate 3). The
Sarcoma 180 tumor retarding property'Of this strain is
about 72%, as compared to the control tumor in mice, from
extracts of E. luteus (Beneke, unpublished data). The
mycelial culture of 8-64-45 on medium A produces a slightly
more cottony colony with a tan to buff color on the back,
in two weeks (Plate 3). Sarcoma 180 tumor retarding
prOperty was about 6 %, as compared to the control tumor,

with extracts of §. luteus (Beneke, unpublished data).
Experimental Media Used:

A synthetic basal medium of Lindeberg‘s supple-
mented with thiamine hydrochloride (Sedlmayr, 1960),
further modified by replacing ammonium tartarate with
ammonium chloride, was used for the carbohydrate study.
A modified Czapek's-Dox‘s medium, designated by Stevens
(1957) as medium A, was used for comparison of growth,
since the fungus grew much better on medium A than on
Lindeberg's medium. The composition of these two media

used are as follows:

21

Modified Lindeberg's Medium:

 

Glucose 20 grams
NH4C1 . 5.0 grams
KH2PO4 1.0 grams
M9804.7H20 0.5 grams
FeCl3 (sol. 1/500) 0.5 m1
ZnSO4(Sol. 1/500) 0.5 ml
MnC12(sol. 0.1 M) 0.5 ml
CaC12(sol. 0.1 M) 5.0 ml
Thiamine hydro-

chloride lOO'K/liter
Distilled water 995.0 ml

Medium A:

Glucose 15.0 rams
Sucrose 15.0 grams
Bacto peptone 5.0 grams
Bacto yeast 5.0 grams
£49804.7H20 0.5 grams
KHZPO4 1.0 gram
KCl 0.5 gram
FeSO4.7H20 0.01 gram
Distilled water 1000 m1

Apparatus and Sterilization Procedures:

Pyrex Erlenmeyer flasks of 250 m1 capacity were
used as culture vessels. Some 500 m1 Florence flasks were

also used for certain eXperiments. DiSposable Petri dishes

22

(Falcon) were used for growing inocula. Waring blendors
were used to blend inocula. The density of the inocula
was determined with a Klett-Summerson photoelectric
colorimeter using the green filter (540 m“). Pipettes
were used to dispose the inocula. The cultures were grown
in liquid media on a rotary shaker. A reciprocal shaker
was also employed for certain experiments. The culture
flasks were capped with aluminum foil (Cook,l962).

The glassware was treated with hydrochloric-nitric
acid cleansing solution, rinsed with tap water and
finally with distilled water. Acid-washed pipettes were
sterilized with dry air at 1650-1800 c for three hours.
The media were sterilized by autoclaving at 121°C,
15 pounds pressure, for 15 minutes.

Sargent No. 500 filter paper and Buchner funnel
were used for filtering contents of each flask to deter-

mine the dry weight of mycelia.
ExperimentalgProcedure:

Fifty ml of modified Lindeberg's medium was placed
in a 250 ml Erlenmeyer flask and capped with aluminum foil
as suggested by Cook (1962). The pH was checked and then
each flask was autoclaved for 15 minutes at 15 pounds
pressure at 121°C.

The various carbohydrates were added to the basal
medium in quantities whidh supplied 8 grams of carbon per

liter (Sedlmayr, Beneke and Stevens, 1961). The following

23
heat stable sugars were autoclaved with basal medium for
15 minutes at 15 pounds pressure at 121°C (Lilly and
Barnett, 1958): glucose, sucrose, galactose, arabinose,
lactose, inulin, dextrin, starch, glycogen, cellulose and
cellobiose.

To avoid the breakdown of sugars during autoclaving
the heat sensitive sugars (raffinose, melibiose, maltose,
trehalose, fructose, mannose, rhamnose, ribose, sorbose,
and xylose) were sterilized by Seitz-filter and added
individually and aseptically to the autoclaved basal medium
in 250 ml flasks.

Transfers made from stock cultures were maintained
in Bolete medium. Transfer cultures were seeded at four
points into Petri dishes containing medium A, to prepare
the inoculum of an experiment. To prepare the inoculum
for an experiment five discs (diam. Ca 2.2 cm each) were
removed from a 15 day agar culture in a Petri dish and
blended with 50 ml of sterile distilled water for 30
seconds in a sterile 360 ml semimicromonel metal Waring
blendor (Sedlmayr 1960), The density of the inoculum was
determined with a Klett-Summerson photoelectric colori-
meter using the green filter (540 m“). The blended
suspension gave a reading of 15-20% transmission under the
above described condition.

Five m1 of the blended mycelium was added to 50 ml

of the modified Lindeberg's medium in a 250 m1 Erlenmeyer

24

flask. The cultures were incubated in the dark at approx-
imately 25°C on a rotary shaker giving 120 excursions per

minute. The medium had no additional aeration other than

that caused by continuous agitation.

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

Due to limited shaker space and cost of media, in
every experiment 4 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 from
quadruplicate flasks.

The amount of growth was determined by mycelial dry
weight. The contents of each flask were filtered on
Sargent No. 500 filter paper in a Buchner funnel. The
mycelial pellets were washed with distilled water to
remove any excess medium, and dried at 100°C in an oven for
18 hours. The replicates in all instances were approx-
imately equal on the dry weight basis indicating that
sufficient inoculum was used. In all the experiments pH
was checked at the conclusion.

For the study of any conSpicuous structural change
induced by the different carbohydrates, slides were pre-
pared (water mount) from ten day-old cultures grown on
rotary shaker, and observed under the microscope. General
characters of the hyphae were drawn with the aid of a

camera lucida.

25

Four sugars, which supported good growth of
B-62-03 and 8-64-45 respectively were selected to observe
their effect on production of the antitumor substances by
the fungi. Sincernedium A supported much better growth of
Suillus luteus than Lindeberg's medium, the fungi were
grown on‘nedium A. The various sugars selected were
added to the basal medium in quantities equivalent to 12
grams of carbon per liter, which correSponded with the
original carbon content of the carbohydrates in Medium A.

The contents of the flask that was tested for
antitumor agent production were blended for 60 seconds in
a sterile Waring blendor and the solid portion was
separated from the liquid by suction flask filtration. One
hundred ml of the liquid and l m1 of 1% merthioLate (1 gram
merthiolate, 14 grams of borax per 100 ml of distilled
water) were placed in a sterile rubber capped serum
bottle (Cook, 1962). According to Stevens, the
merthiolate solution does not affect the results of the
tumor assays (Cook, 1962).

The samples were tested for activity against the
Crocker mouse Sarcoma 180 as follows. Female Swiss albino
mice weighing between 18 and 22 grams were used for
Sarcoma 180 test (Cancer Chemotherapy reports, 1962). To
grow the tumor, uniformly cut pieces (large enough to fit
into a 16 guage trocar) of tumor were implanted sub-

cutaneously in the axillary region. These were allowed to

26
grow for 7 days. The mice were then sacrificed by
cervical separation and the tumors were removed. The
tumors were then cut into equal pieces of approximately
2-4 mm in size. The mice that were used in the experiment
were implanted with one of these squares of tumor on the
right axillary region where they were allowed to grow for
7 days. The treatment (injections of the extract) began
24 hours after the implant. The mice were injected in the
interperitoneal region, with 1 ml daily for 7 days. The
control mice were injected with the same volume of an
.85% saline solution at the same time. After 7 days the
mice were sacrificed and tumors removed. Weights of
tumors of test animals were compared with those of con-
trol animals. Results are expressed as percent of con-
trol tumor growth. Each testing group consisted of 6

mice.

R ESULTS AND DISCUSS ION

Carbon is one of the essential elements required
by living organisms. Almost half of the dry weight of
fungal cells consists of carbon. ProtOplasm, enzymes, the
cell wall, and reserve nutrients stored within the cells
have compounds containing carbon. Carbon compounds are
equally important in fungal nutrition. Fungi secure
energy by oxidizing organic compounds. Organic compounds
differ in composition, structure and configuration. These
are key factors which must be considered in relation to
utilization of organic compounds by fungi. (Lilly and
Barnett, 1951).

The main purpose of this work was to determine
the carbohydrates which support good growth of the test
organism (guillus luteus), to observe if any morphological
change is produced in the fungal hyphae by different
carbohydrates, and to determine if any significant
change in antitumor activity of the fungus is caused by
various carbohydrates.

The sugars that were selected in this experiment
were the ones studied by Sedlmayr (1960) in her carbo-

hydrate study of Calvatia species.

27

 

28

TmfleI

 

 

- mm

The Utilization of Various Monosaccharides by Suillus luteus

 

J

C-sources

PBNTOSES

l(+) arabinose

d(+)
d(-)

xylose

ribose

HEXOSES

d(+)
d(+)
d(+)
d(-)
1(-)
1(+)

glucose
galactose
mannose
fructose
sorbose

rhamnose

CON T ROL

C-free

Medium A

1 -*

B-62-03 8-64—45
10 davs 15 days 10 days 15 days

24.2 4.4 24.7 4.8 10.4 4.6 13.0 4.55
22.3 4.87 26.0 4.85 13.1 5.15 20.6 5.15
19.4 5.5 19.6 5.5 17.3 5.7 18.6 5.7

105.3 3.0 106.4 2.6 68.5 2.8 88.5 2.49

48.7 3.6 49.5 4.1 32.6 4.1 35.3 4.0
51.8 3.1 84.8 2.5 21.3 3.3 80.5 2.3
32.3 3.55 100.6 2.25 22.5 4.1 23.4 3.4
18.2 4.99 18.8 4.98 11.9 5.19 14.4 5.7

11.4 5.8 16.2 5.7 29.3 5.7 30.5 5.4

18.5 5.5 26.4 5.8 19.7 4.7 33.5 5.4

442.5 6.1 459.2 8.2 297.2 7.0 297.3 7.85

*Average mycelial dry weight of four replicates.

+Average of four replicates.

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32

Carbon Utilization. 1. Monosaccharides

Table I contains the data depicting the utilization
of various monosaccharides by the two strains of Suillus
luteus (8-62-03 and B-64-4S). The pentoses used in this
experiment were utilized less readily than most of the
hexoses by §. luteus with the exception of l-Sorbose in
case of B-62-O3, and 3-64-45, which supported poorer
hyphal growth than some of the pentoses (Table I). The
two strains used in this experiment showed some variation
in their ability to utilize different sugars as eXpressed
by their difference in dry weight when grown under the same
nutritional and environmental condition (Tables I, II &
III).

The best carbon source among the pentoses for both
strains was xylose (Table I).

Mycelial growth of 8-62-03 and 3-64-45 at the end
of 15 days appeared to be less in most of the pentoses
studied than in the control medium. This may indicate that
most of the pentoses were not readily utilized by the
organisms. Some fungal growth might have been due to
carry over of nutrients by inocula, when it was equal to
that in control medium. (xylose and arabinose in 8-62-03
and xylose in B-64-45, Table I). B-62—O3 grew'more poorly
in ribose than in C-free medium and 8-64-45 showed much
poorer growth in arabinose and ribose than in control

Inedium. There might have been more autolysis in the

33
mycelium, thus reducing the weight as evidenced by
morphological and cytological observation of the hyphae.

The hexoses employed in this study were found to
be better carbon sources than most of the pentoses.
Glucose and fructose were the best carbon sources for
8-62-03 and glucose and mannose supported the best growth
of B-64-45.

Of the hexoses D-glucose is biologically most
important and is utilized for growth by virtually all
fungi. A few forms, however, have been reported not to
grow with glucose (Cochrane, 1958). If an attempt is made
to record on a single chart all the known biochemical
fates of glucose and all the compounds derived therefrom,
the result is a complex network. A study of this network
reveals numerous possible "pathways" whereby carbon atoms,
initially contained in glucose, may ultimately appear in

00 other hexoses, pentoses, lipids, amino acids,

2:
purines, pyrimidines, etc. (White, Handler and Smith, 1964).
Table I shows that glucose was the best carbon source for
both B-62-03 and B-64-45 among the hexoses. D-fructose

and D-mannose are equivalent to glucose for growth for a
large number of fungi (Cochrane, 1958). Table I shows that
for B-62-03 fructose is almost as good as glucose and for
B-64-45 mannose is almost equivalent to glucose. Mannose

was found to support considerable fungal growth of

B-62-03. It can be noted in Table I that glucose was

34
readily used by both strains whereas the rate of fructose
utilization was rather slow for B-62-03 during the first
10 days of incubation but later it became faster. A
sLmilar situation occurred with mannose utilization by
B-64-45. .

Both 8-62-03 and 3-64-45 grew rather poorly on
galactose. Growth of 8-64-45 was almost equal to that on
the control medium at the end of 15 days. 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, a form
able to enter the main respiratory pathways.

Rhamnose has been reported to be used only by a
few organisms (Cochrane, 1959). It supported no mycelial
growth for 3-62-03 (less than carbon free medium), while
8-64-45 was slightly better than the control medium.
Since rhamonose supported no growth, autolysis may have
occurred in the hyphae of 8-62-03, while any growth of
8-64-45 might have been due to a carry over of nutrients
with the inoculum.

sorbose supports normal growth of a few fungi:
however it appears not to be utilizable by most forms and
it is definitely toxic to some (Cochrane, 1958). Growth
on sorbose at the end of 15 days appeared to be less than
the control medium in both the strains. This might

support the above view of toxicity of sorbose. The

35
following statement of Lilly and Barnett gives a general
picture of utilization of hexoses by fungi.

Lilly and Barnett (1951, p. 121) stated: "The
following generalizations about utilization of the common
hexoses may be drawn. (1) There is no single sugar which
supports the maximum amount of growth for all the fungi.
(2) Most fungi utilize glucose, although the maximum
amount of growth was not always attained on this sugar.
(3) The more closely the configuration of another sugar
approaches that of glucose, the more fungi utilize it.

It is believed that these generalizations are valid for

all fungi which utilize sugars.“

g. Oligosaccharides

 

‘Table II indicates that among the oligosaccharides
cellobiose supported the best growth of 8-62—03 and
maltose supported the best growth of 8-64-45. It was
remarkable that cellobiose supported very poor growth of
8-64-45 while maltose did not turn out to be a very good
carbon source for 3-62-03. Maltose is utilized virtually
by all fungi which have been studied. Cellobiose is al-
most as widely utilizable as maltose (Cochrane, 1958).

In analyzing the data on cellobiose and maltose utiliza-
tion (Table II) it appears that maltose utilization
increased considerably for strain 8-62-03 in 15 days of
the incubation in comparison to the 10 day culture. The

same observation appears to hold true for 8-64-45 in the

36

case of cellobiose. This might indicate that the involved
enzyme was inducible. In Spite of the fact that both
cellobiose and maltose on hydrolysis form two molecules 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 aib-glucosidic linkage
and in the latter by an.*rglucosidic linkage. Consequently,
they require different enzymes for cleavage (Sedlmayr, 1960).
The difference between the utilization of these two carbo-
hydrates by the Suillus strains was probably due to the
difference in abilities to produce the required enzymes.

Sucrose was found to be a good carbon source for
both B-62-03 and 8-64-45 (Table II). This was expected in
B-62~03 since both components of this disaccharide
(glucose and fructose) produced a satisfactory growth when
used separately (Table I). Although one of the components,
fructose, was not very favorable for the growth of
B-64-45,'utilization of sucrose by this strain probably
indicates that the organism produces the hydrolyzing
enzyme (as also probably does 8-62-03) and the glucose
liberated acts as a carbon source for the strain.
Sucrose is generally a good source of carbon for many
fungi (Cochrane, 1958).

It could be assumed from the results shown in

Table II that trehalose was utilized satisfactorily by the

The Utilization of

37

Table II

 

 

C-sources

OLIGOSACCHARIDSS

d(+)
d(+)
d(+)
d(+)
d(+)
d(+)

d(+)

sucrose
maltose
lactose
cellobiose
melibiose
trehalose

raffinose

CONTROL

C-free medium

Medium A

*Average mycelial dry weight of

B-62-03

10 d ys 15 d y
us:

2.5 98.5
4.0 66.9
5.7 39.3
2.6 111.0
4.8 21.1
2.8 91.2
5.8 29.7
5.5 26.4
6.1 459.2

+Average of four replicates.

s
951:

8.2

four replicates.

Various Oligosaccharides by Suillus luteus.

~

8—64-45

12.53.9115. 15.31.917.82

82.2 2.9 91.7, 2.5
75.5 2.8 100.1 2.6
26.2 4.75 29.8 5.2
15.7 4.4 33.1 4.3
24.4 5.6 27.5 5.5
74.7 2.8 82.1 2.55
28.6 5.6 29.9 5.75
19.7 4.7 33.5 5.4
297.2 7.0 97.3 7.95

 

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40

strains of Suillus. Trehalose has been found to support
growth of a large number of fungi including some that are
relatively fastidious. This disaccharide is formed early
in the metabolism and used up later. However, an adequate
understanding of trehalose metabolism is not yet available
(Cochrane, 1953).

Lactose supported poor growth of both strains of

Suillus luteus. Mycelial growth was found to be more than

 

that in the control medium in 8-62—03 and slightly less in
8-64-45. Lactose is used by far fewer fungi than any of
the disaccharides so far mentioned and may therefore be
considered as a poor carbon source (Codhrane, 1958). This
might be due to the lack of the necessary hydrolytic
enzyme, lactose, or due to the inability of the fungi to
utilize one of the component hexoses, galactose (Sedlmayr,
1960).

Mycelial growth in lS-day cultures in melibiose
appeared to be less than that on control medium in both
the strains. This might be due to the lack of the
hydrolytic enzyme melibiase or the inability of the fungi
to utilize one of the component hexoses, galactose.
since growth was slightly less than that in C-free medium
it might indicate that autolysis had occurred in the
mycelium. Melibiose is not utilized by many fungi

(Cochrane, 1958).

41

Raffinose, a trisaccharide which contains one
linkage as in sucrose and another as in melibiose was
utilized poorly by both the strains of Suillus luteus.
Since the growth on raffinose in both the strains was less
than that of carbon free medium, autolysis may have
occurred in the mycelium of these strains. The majority
of fungi utilize raffinose but non-utilization is also
common (Cochrane, 1958).

The utilization of an oligosaccharide is believed
to be preceded by and dependent on its conversion to
hexoses or possibly hexose phOSphates (Cochrane, 1958).
The above statement appears to be true from the standpoint
that the best utilized oligosaccharide for B-62-03,
cellobiose, consisting of glucose, appeared to be the best
utilized monosaccharide for the strain. The best utilized
oligosaccharide for B-64-45 was maltose consisting of
glucose which also seemed to be the best monosaccharide
for that strain. Inability of B-62-03 to utilize maltose
and 8-64-45 to utilize cellobiose satisfactorily probably
was due to the inability of the strains to produce the
necessary hydrolytic enzymes, maltase and cellobiase

respectively.
5. Polysaccharides.

Table III indicates that none of the polysaccharides
used in this experiment supported good growth of the fungi,

particularly in comparison to the monosaccharides and the

42
Table III

The Utilization of Various Polysaccharides by Suillus luteus

*— —.——.— 4.—

 

 

 

C-sources 8-62-03 8-64-45
10 days 15 days 10 days 15 davs
281 98+ mg: 2%: £81 881 881 RE:
POLYSACCEARIDES
Dextrin 23.0 4.0 46.8 3.8 14.3 4.85 39.2 4.9
Starch 36.5 3.8 39.9 3.6 47.8 3.0 48.1 2.75
Inulin 17.0 4.8 19.3 4.5 18.5 3.7 18.6 4.5
Glycogen 20.5 3.68 20.7 3.68 25.7 3.65 32.5 4.3
CONTROL
C-free medium 18.5 5.5 26.4 5.8 19.7 4.7 33.5 5.4
Medium A 442.5 6.1 459.2 8.2 297.2 7.0 297.3 7.85

*Average mycelial dry weight of four replicates.

+Average of four replicates.

43

 

 

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44

oligosaccharides. This probably indicates the inability
of the fungi to produce the necessary hydrolytic enzymes
sufficiently, since the component of the majority of the
polysaccharides used in this experiment (Dextrin, starch,
glycogen) was glucose, the most favorable monosaccharide
for both the strains. Inulin, a polyfructoside, did not
SUpport good growth of 8-62-03, although fructose was a
good carbon source for this strain (Table I).

Dextrin was the best carbon source among the poly-
saccharides for 8-62-03 and starch was the most favorable
polysaccharide for B-64-45. Starch supported the next
best growth of 8-62-03 and Dextrin of B-64-45. This was
expected since according to Cochrane (1958) Dextrins are
chemically or enzymatically modified starches of uncertain
structure, and are generally satisfactory sources of carbon
for fungi.

Starch is composed of a mixture of two different
polysaccharides, amylose and amylopectin. The glucose
units of amylose are bound to each other in¢£rl,4—g1ucosidic
linkage. Amylopectin contains chains of glucose units
like those of amylose but it also has branches of these
glucose chains linked through an 4:1,6-glucosidic linkage
(Fairley and Kilgour, 1963). Starch is an excellent
carbon source for most fungi, even for rather fastidious
forms. Dextrins are also generally a satisfactory source

of carbon for many fungi (Cochrane, 1958).

45

Glycogen is a glucose polysaccharide similar to
the amylopectin fraction of starch. It supported better
growth of B-64-45 than 8-62-03, which correSponds with the
observation that starch favored the growth of B-64-45
more than it did B-62-03. Mycelial growth in glycogen
however was slightly less than in the control medium in
8-62-03 and equivalent to that in B-64-45. Glycogen is
quite generally available to fungi of widely different
ecological groups. Those fungi unable to use starch can
not be expected to use glycogen, So far as is known, the
same enzymes attack both polysaccharides (Cochrane, 1958).

Inulin, a polyfructoside appeared to be a poor
carbon source for both 8-62-03 and B-64-45. In case of
8-62-03, it was probably due to the inability of the fungus
to produce the necessary hydrolytic enzyme inulase, since
fructose was found to be a good carbon source for this
strain. Inulin, a polymer of D-fructose, has been found
to be a good source of carbon for many but not all fungi
(Cochrane, 1958). Hawker (1950) suggested that the in-
ability of fungi to use inulin for growth was due to the
failure to secrete the enzyme, rather than to any in-
hibitory effect Of the inulin.

Cellulose is by far the largest natural reservoir
of biolOgically utilizable carbon. It is an excellent
source of carbon for many fungi, but the Species within a

given genus are not all necessarily alike in their reSponse

46
to cellulose. Insolubility of cellulose, however, makes
the nutritional study of the compound difficult.
(Cochrane, 1958). Cellulose was a poor carbon source for
both B-62-03 and B-64-45. Since no satisfactory technique
could be devised to test the cellulose utilization by the
fungi, owing to the insolubility of cellulose, the data
obtained were considered to be inaccurate. Poor mycelial
growth was obtained in all experiments. Hence the data
were not included in Table III. Cellulose is a linear
polymer of D-glucose, where the glucose molecules are
joined together through 9-1,4-glucosidic linkage. The
poor growth obtained in cellulose, especially of B-62-03,
was probably due to the lack of cellulase formation.
However, cellobiose, the intermediate product of this
polysaccharide, was one of the best carbon sources for the
organism.

In order of utilization, cellobiose was the best
carbon source for 8-62-03 followed by glucose, fructose,
sucrose, trehalose and mannose. For 8-64-45 maltose was
the best carbon source followed by sucrose, glucose,
trehalose and mannose.

The pH of the Lindeberg's medium and medium A was
adjusted to 5.5 for the nutritional studies. It was noted
that in Lindeberg's medium good mycelial growth was mostly
associated with a lowering of the pH of the medium

(Tables I and II). The pH remained about the same when

47

the sugars were not well utilized. The pH is affected
during growth by metabolic activities, raised by
absorption of anions or production of ammonia from nitrogen
compounds, lowered by formation of organic acids or
absorption of cations. These effects of growth on pH
complicate results, particularly in poorly buffered media
commonly employed (Cochrane, 1958). The hydrogen ion
concentration of a nutrient solution may change 10,000-fold
during a few days as a result of the metabolic activities
of a fungus. These changes in pH are due to changes in
the relative amounts of acids and bases formed or with-
drawn and to the ionization constants of these compounds
(Lilly and Barnett 1951).

Fungi produce acids from non-acidic nutrients
such as the carbohydrates. Various organic acids such as
pyruvic, citric and succinic are also produced. Pyruvic
acid accumulates in the nutrient solution in which many
fungi are grown, and in some instances the formation of
this acid accounts for a considerable part of the early
lowering of pH. The eventual utilization of pyruvic acid
causes the pH of the nutrient solution to rise. Other
metabolizable acids behave similarly (Lilly and Barnett,
1951). This probably is a good eXplanation of the rapid
lowering of pH in media where the carbohydrate was well
utilized (Tables I and II) and also for a rise of pH after

a lowering at first (e.g. sucrose in B-62-O3, Table II).

48

Growth inrnedium A, however, was found to be
associated with a rise of pH, which was greater in
B-62-03 showing a more rapid growth than 8-64—45 (Tables I,
II and III). Ammonia is the most common basic substance
produced by fungi. The production of ammonia results
from the deamination of amino acids and proteins (Lilly
and Barnett, 1951). In medium A ammonia supply is greater
due to peptone and yeast extract. (See appendix for their
composition.)lkzLindeberg'smedium, on the other hand,
ammonium chloride is the only N2 source. According to
Lilly and Barnett (1951) the process which produces acid
usually dominates during early fungal growth, especially
when ammonium nitrogen is used.

It was further observed that good hyphal growth
and low pH in the Lindeberg's medium was associated with
a change of color of the nutrient solution. It was bright
orange when the growth of the fungus was good and the pH
of the medium was low. When the nutrient solution
appeared to be pale colored, the growth was poor and con-
sequently there was slight change of pH of the medium.

At no time did any of the strains show mycelial
growth in test media as great as that in medium A (Tables
I, II, and III), indicating that growth factors must be
present in Medium A that were not present in the modified
Lindeberg's medium (Cook, 1962). B-62-03 showed greater

mycelial growth on medium A in comparison to B-64-45

 

49
during the 15 days incubation period. The length of in-
cubation necessary for Optimum growth for each strain was
determined previously by running a series of growth curves.
Medium A supported better growth than Lindeberg's
medium, because it contained peptone and yeast extract.

(See appendix for their composition.)

Utilization of Peptone and Yeast Extract

A study was made on the effect of peptone and
yeast extract separately and together in the medium A on
§gillus luteus B-62-03. The fungus was grown in medium A
with the usual combination of 5 grams of peptone and
5 grams of yeast extract/liter. Then it was also grown on
medium A with ten grams of peptone in one case and with

ten grams of yeast extract in the other case.

 

 

 

 

 

Table IV
Utilization of Peptone and Yeast Extract by
B-62-03*
Medium 20 days
as séi
"A" (with original combination 810.0 7.0
of peptone and yeast)
"A" (with yeast) 770.2 5.3
"A" (with peptone) 256.0 4.7

*Average mycelial dry weights of four replicates
grown on reciprocal shaker.

50

The results in Table IV indicate that the yeast
extract supported better growth of B-62-03 than peptone.
flgrphological Studies of Suillus luteus Mygelium in

Submerged Culture

Pantidou (1961) described cultural characters of
several Species of Boletaceae and reported on formation of
the fruiting bodies by the genera Boletinus and Suillus
in culture. In microscopic characteristics most of the
Species of Boletaceae showed a uniformity. The differences
in hyphal characteristics such as coloring, septation, and
branching were more in the nature of degree than of kind.

The majority of the species possessed hyphae of
an ordinary type, appearing cylindrical, hyaline with
homogeneous or granular contents in the living cells or
hyaline and empty in the non-living. Several Species of
Boletaceae however, could be differentiated from each
other on the basis of their characteristic hyphal modifi-
cations.

Hyphae with papillated appearance were found and
described in Species of Suillus and Xerocomus. Such
hyphae occurred in varied frequencies in almost all
Species studied. In this case the ordinary hyphae were
modified by depositions of amorphous yellow to brown
material on the surface of the walls. This material be-
came easily detached from the cell wall, floating off when

mounted in lacto-phenol mounting medium. The deposits

51
were either small and rather regular in shape, seen only
at the edges of the hyphae, or they were large, irregular,
and covered the whole surface. In some species they were
more firmly attached to the walls and gave an ornamented
appearance to the hyphae. It is likely that this material
contributed largely to the color of both sides of the mats.
In some isolates where papillated hyphae were less
numerous or absent, the mats and their reverse sides were
lighter colored (Pantidou 1961).

For the study of any morphological change induced
by the different carbohydrates, slides were prepared
(water mount) from ten-day old cultures grown on rotary
shaker, and observed under the microscope. Changes in
the forms of the hyphae were drawn with the aid of a
camera lucida.

The morphological changes that were induced by
the use of the different sugars as carbon sources are
indicated below. Strain 8-62-03 and 8-64-45 showed
similar reSponses in the different sugars used.
monosaccharides
Wes

Arabinose:
Both in B-62-03 and B-64-45 protOplasm was scarce,
shrunken in some, with papillated hyphae. Brown deposits

were abundant in 3-64-45 but scarce in B-62-03.

52

PLATE IV

 

 

 

Mycelium of Suillus luteus strain, B-62-03, grown in
submerged culture with ribose as carbon source.

 

 

 

KO)‘

Plats V. Mycelium of strain, B-62-03, grown

in submerged culture with various sugars as
carbon source:

1, glucose: 2. xylose: 3, ribose:
4, control (- carbon).

54

 

 

Plats VI. Mycalium of Suillus luta_us strain, B-62-03, grown
in submerged culture with various sugars as carbon
source: 5, sorbose: 6, mannose: 7, raffinose.

 

\OU

'(Y

 

 

 

 

55

Xylose:

ProtOplasm was not dense and brown deposits and clamp
connections were present.

Ribose:

ProtOplasm was thin, and brown deposits were abundant
(Plate V, Fig. 3).

Hexoses:

Rhamnose:

ProtOplasn is scarce in the cells and the hyphae were
papillated.

Glucose:

ProtOplasm was very dense. In some of the older
cells it was shrunken from the wall surface which might
indicate the beginning of autolysis. There were clamp
connections present but brown deposits were not observed
(Plate v, Fig. 1).

Galactose:

ProtOplasm was not very dense in the hyphae. Large
oil drOplets and clamp connections were present. Brown
deposits were rare on the cell wall.

Mannose:

ProtOplasm was usually dense but may be rather thin
in some hyphae. The hyphae were slightly wavy (Plate VI,
Fig. 6), but brown deposits were not observed.

Fructose:

ProtOplasm was very dense in hyphae, but brown

deposits were not observed.

56
Sorbose:
ProtOplasm was thin with large oil droplets present in
the hyphae. Large conSpicuous chlamydOSpores were present,
being more abundant in the strain B-62-03 than in B-64-45

(Plate VI, Fig. 5). Brown deposits were present.

Oligosaccharides:

Sucrose:

ProtOplasm was very dense. Autolysis began in some
hyphal cells which might be due to the low pH of the
medium (Table II). Brown deposits were not observed.

Maltose:

Dense protoplasm, shrunken from the cell wall in some
hyphae. Brown deposits were not observed.

Lactose:

Thin protOplasm in hyphae and brown deposits were
more abundant in B-64-45 than in B—62-03.

Cellobiose:

ProtOplasm in the strain 8—62-03 was very dense
whereas that in B-64-45 was scarce and shrunken. Brown
deposits were present in both strains.

Melibiogg:

ProtOplasm was more dense in 3-62-03 than in B-64-45.
Brown deposits were more abundant in B-64-45.

Trehalose:

Hyphae with very dense protOplasm and clamp connec-

tions were present.

57
Brown deposits were not observed.
Raffinose:
Hyphae had shrunken or thin protOplasm in the cells
with large oil drOplets. Hyphae were wavy and contained
clamp connections (Plate VI, Fig. 7). Brown deposits

were not observed.

Polysaccharides:

Dextrin:

Hyphae had fairly dense protOplasm with large oil
drOplets. Clamp connections were observed. Brown
deposits were not found.

Starch:

Hyphae showed fairly dense protOplasm, some cells
were shrunken and vacuolated. Clamp connections were
observed, but no brown deposits were found.

Inulin:

ProtOplasm was thin and shrunken in some hyphal
cells. Brown deposits were abundant in strain B-64-45
and scarce in B-62-O3.

Glycogen:

ProtOplasm was less dense, large oil drOplets were
present in protOplasm. No brown deposits were observed.

Cellulose:

ProtOplasm was found to be fairly dense in the cells

and no brown deposits were found.

58

Contrgl_fiedium (without carbon):

ProtOplasm of the elongated hyphae was very thin with
large oil drOplets. Brown deposits were more abundant in
the strain B-64-45 than in 8-62-03 (Plate V, Fig. 4).

Medium A:

Hyphae grown in "medium A" develOped very dense
protOplasm and no brown deposits were observed.

The descriptions given above indicate that
structural changes in the hyphae and protOplasm were
directly or indirectly affected by various sugars. It
was noted that in most instances there was a correlation
between good growth and dense protOplasm. Poor growth in
most instances were associated with thin protoplasm.
Brown deposits were more abundant in the strain B-64-45
and in sugars which supported poor growth.

It was noted that brown deposits were rare when
the hyphae had very dense protOplasm. Since the hyphae
were taken from the various sugars under the controlled
conditions of these experiments it is possible that the
brown deposits may occur in more cases if changes were
made in pH, temperature, and length of time for growth of
the hyphae in the various sugars. Chlamydospores were
found to be induced by only one sugar, Sorbose. Clamp
connections were common in most instances. However, an
eXplanation can be given for the cases where no clamp

connections were found, as changes may be necessary in the

59
environmental conditions when a different carbohydrate is
utilized to induce clamp formation. Further study of the
conditions for formation of clamp connections in submerged

culture might indicate the basis for this phenomen.
Effect of Light on Growth of B-62-03

Most reports on the effect of light on the fungi
have been concerned with reproduction rather than
vegetative growth. However, light has been found to
depress or promote growth in some fungi (Lilly and
Barnett, 1951).

To study the effects of light, a number of plates
(B-62-03 inoculated on medium A) were kept in a constant
temperature room (22-24OC) and exposed to the fluorescent
lights in the laboratory for the part of the light period
mentioned in Table V. These plates were then removed to
the constant temperature dark room for the remaining
period. Growth was measured after the total 15 days of

incubation was completed.

Table V

Effect of Fluorescent Light on B-62-03
W

 

 

Total days Diameter of Colony
Light Dark
0 15 2.26 cm.
-3 12 2.40 cm.
7 8 2.33 cm.
10 5 2.25 cm.

15 O 2.41 Cm.

60
Table V shows that light does not have any

significant effect on the growth of B-62-03. It

appeared to be slightly stimulatory in some instances (3
days and 15 days). Results obtained from plates exposed
to light for 7 and 10 days were almost equivalent to that
kept in the dark all the time. This may indicate that
slight variation in growth might have been due to the
variation in size of inocula, and individual variability,

which is not significant.

Production of antitumor substance

Stevens (1957) showed variations in tumor-
retarding activity of cultures of Collybia radicata,
when different carbon sources were utilized. Cook (1962)
reported that there was a differential production of
Calvacin (an inhibitor of Crocker mouse Sarcoma 180) by
Calvatia gigantea, when the various carbon sources were
used. Cultures grown in cellobiose generally produced
the most Calvacin. There was no correlation between the
amount of growth and the amount of Calvacin produced when
the selected carbon sources were used.

In this study an attempt was made to determine if
variation in antitumor activity was evident in Suillus
luteus when other carbon sources were substituted in the
basal medium.

Table VI indicates the results of the Crocker

mouse Sarcoma 180 tumor tests using six mice in each test.

61
The designation T/C in Table VI was determined by taking
the average tumor weight of the experimental mice, injected
with the extract of Suillus luteus culture grown in
different carbon sources, and dividing this figure by the
average tumor weight of the control mice, which were
injected with normal Saline(.85% Saline).

This method of determining tumor retardation is
followed by the Cancer Chemotherapy National Service
Center.

Table VI indicates that glucose (in rotary shaker)
and sucrose (in reciprocal shaker) as a carbon source
result in the greatest antitumor activity in strain
B-62-O3. Whereas antitumor activity was much reduced
when this strain was grown on a reciprocal shaker with
glucose as a carbon source. When grown on a rotary shaker
with sucrose as a carbon source it stimulated, rather than
retarded the growth of the tumor.

Lucas, gt al., (1953-59) reported that compounds
in the filtrate of Calvatia Species which stimulated tumor
growth appeared in some instances where the T/C was
reported to be 100 or more. This was found to be due to
the production of these substances in younger cultures
prior to the deveIOpment of Calvacin. Calvacin once
produced i3 vitro, could be lost with further mycelial

growth or that more time may be needed for Calvacin pro-

duction (Stevens, 1957).

62
Table VI

Antitumor Activity of Cultures of Suillus luteus Strains
B-62-03 and B-64-45 against Crocker Mouse Sarcoma lBO

 

 

 

 

Carbon Source Days of Shaker Strain Strain
Incubation B—62-03 T/C B—64—45 T/C
Glucose lS rotary 66 81
20 reciprocal 92 124
Maltose 15 rotary - 84
20 reciprocal - 6O
Cellobiose 15 rotary 102 -
20 reciprocal 93 -
Trehalose 15 rotary 145 95
20 reciprocal 83 120
Sucrose 15 rotary 128 78
20 reciprocal 69 ll2

T/C = Experimental tumor weight divided by the control
tumor weight.

63

The strain 3-62-03 when grown on a rotary shaker
with cellobiose as a carbon source showed some stimulation
of tumor growth, whereas it showed very slight retardation
of tumor growth when grown on a reciprocal shaker with the
sane carbon source.

When trehalose was used as a carbon source the
strain B-62-O3 showed some antitumor activity when grown
on a reciprocal shaker. But it stimulated tumor growth
when grown on a rotary shaker with the same carbon source.

Maltose was the best sugar for the antitumor
activity of the strain 3-64-45 (when grown on a reciprocal
shaker). The strain still showed antitumor activity when
grown on a rotary shaker with maltose as a carbon source,
but the activity was reduced.

Glucose supported some antitumor activity of
B-64-45, when grown on a rotary shaker. The fungus how-
ever stimulated tumor growth when grown on a reciprocal
shaker with glucose as a carbon source.

When trehalose was utilized as a carbon source,
D-64-45 showed slight antitumor activity when grown on a
rotary shaker, but it stimulated tumor growth when grown
on a reciprocal shaker.

some retardation of tumor growth was obtained
when B—64-45 was grown on a rotary shaker with sucrose
as a carbon source. But the fungus stimulated tumor
growth when grown on a reciprocal shaker with the same

carbon source.

64

It was evident that the two strains 3-62-03 and
B-64-45 showed some variation in their antitumor activity
in different sugars and also in different shakers which
might have been due to the duration of incubation period.

None of the sugars used in this experiment how-
ever, seemed to be as effective as having both sugars
present in medium A, as Suillus luteus when cultured in
medium A, retarded tumor growth up to 72A of the controls
(Beneke unpublished data). Greater antitumor activity
inxnedium A probably is due to the presence of two
different sugars (glucose and sucrose) in the medium and

also due to difference in experimental condition.

SUI'E'IARY

Two strains of Spillus luteus (8-62-03 and
B-64-45) were used to determine utilization of carbon
sources and the effect of some of the carbon sources on
the production of antitumor activity. Morphological
changes induced by different carbon sources were studied
under the microsc0pe.

There was a differential utilization of the
various carbohydrates by the two strains of Suillus luteus.
Pentoses in general supported poor growth of the strains.
Hexoses were more favorable, of which glucose was the
best for both strains.

.Oligosaccharides supported fairly good mycelial
growth of both. Cellobiose was the best oligosaccharide
for the strain 8-62-03 and maltose was best for 8—64-45.

Polysaccharides in general supported poor growth
of both strains.

Yeast extract in medium A contained substances
stimulatory to the growth of the strain 3-62-03.

Structural changes in the hyphae and protOplasm
were affected by various sugars. There was a correlation

of good mycelial growth with dense protOplasm. Brown

65

66

deposits and clamp connection were found to develOp when
certain sugars were placed in the medium. Chlamydospores
were induced only by sorbose. B-62-03 and B-64-45
showed similar reSponse structurally in different sugars
used.

Light as used under the conditions of this
eXperiment had no effect on the growth of B-62-03.

Antitumor activity of g. luteus was affected by
various carbon sources used under these eXperimental con-
ditions. Of the sugars tested glucose and sucrose
resulted in the greatest production of antitumor activity
of 3-62-03 while cellobiose and trehalose produced a
substance(s) which stimulated tumor growth. Maltose
supported best antitumor activity of the strain 5-64-45,
while trehalose induced the production of a stimulatory
substance for tumor growth in the fungus. Type of shaker
used affected the antitumor activity of the fungus. This

might be however owing to the duration of incubation period.

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71

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APPENDIX

Typigal analysis of Bacto-Yeast Extract. (Furnished by

the Difco Laboratories Incorporated, Detroit 1, Michigan).

Ash 10.10
Total N 9.18
Chloride 0.19
Total Sulphur 1.39
Lead 16.00
Arsenic 0.11
Manganese 7.60
zinc 83.00
Conper 19.00

"J «“0 " 3.?“

I «41‘. vul-

Phosphorus 0.89
Iron 0.028
5102 0.052
Potassium 0.042
Sodium 0.32
Magnesium ’ 0.03
Calcium 0.0406
Arginine 0.78

72

73
Asparatic acid
Glutamic acid
PER CENT
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
TryptOphane
Tyrosine
Valine
I-‘lICROGRAi-is PER GRAN
Pyridoxine
Biotin
Thiamine
nicotinic acid
Riboflavine

Folic acid

3.4
0.83
0.60

3.4

74
Typical analysis of Bacto-Peptone (Furnished by Difco

Laboratories Incorporated, Detroit 1, Michigan).

Total NitrOgen 16.16%
Primary Proteose N 0.0 %
Secondary Proteosa N 0.68%
Peptone N 15.38%
:mmonia 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 0.05%
Magnesium 0.056%
Manganese Nil
Iron 0.0033%
Ash 3.53%

Lead 15.00 ppm

Arsenic

Zinc

Copper

Si02

Arginine
Asparatic acid
Glutamic acid
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Valine

:tcuocsmws I 3.9 GRm

 

Pyridoxine

Biotin

Riboflavine

75

5.90ES
11.00%
23.00%
0.965

2.006

     

  

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