VARIATION IN EXTRACELLULAR AND ‘
INTRACELLULAR ENZYMATIC ACTIVITlES
7 EN THEGENERA PHIALOPHORA,
FONSECAEA AND CLADOSPORIUM

Dissertation for the Degree of Ph. D.
MECHSGAN STATE UM" ERSITY
SWALEE PICHYANGKURA
1973

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VARIATION I EXTRACELLULAR AND INTRACELLUIAR ENZYflATIC
ACTIVITIES IN THE GRIN PHIALOPHORL, FONSECAEA
AND CLANSPORIUM

presented by

Sunlee Pichyangkure

has been accepted towards fulfillment
of the requirements for
7r , _ 7;, 7 we 7 /",<

I I" ' ‘ degree 1n

Doctor of Philosophy

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("’3 “4 24/27 C -—{v/) 465%

Major professor

Date December 10, 1973

 

ABSTRACT
VARIATION IN EXTRACELLULAR AND INTRACELLULAR ENZYMATIC

ACTIVITIES IN THE GENERA PHIALOPHORA, FONSECAEA
AND CLADOSPORIUM

BY

Sumalee Pichyangkura

Some of the dematiaceous fungi in the genera
Phialophora, Fonsecaea and Cladosporium may be etiological
agents of chromomycosis while others may be saprobes or
plant pathogens. There is a similarity in conidial forma-
tion and morphology of these organisms which makes differ-
entiation between pathogens and saprobes difficult.
Twenty-two species of human and animal pathogens and
forty-five species of saprobes and plant pathogens were
studied to determine variations in certain extracellular
and certain intracellular enzymatic activities. This group
of organisms included five etiological agents of chromo-
mycosis: Phialophora verrucosa Medlar, 1915, Fonsecaea
pedrosoi (Brumpt) Negroni, 1936 comb. nov. Carrion, 1940
emend, F. compactum (Carrion) Carrion 1940 comb. nov.,

F. dermatitidis (Kano) Carrion, 1950, and Cladosporium
carrionii Trejos, 1954.

Paranitrophenol (PNP) derivatives were utilized as

substrates to assay for twelve extracellular enzymes in

representative strains of these three genera. The fungi

Sumalee Pichyangkura
were grown on a medium containing casamino acids, bacto-
peptone, yeast extract, glucose and 2.0% of agar at 25°C.
After 15 and 25 days of incubation, small agar plugs were
removed from beyond the edges of the colonies, and were
placed in the PNP substrates with their appropriate buffer.
Each one was assayed for extracellular enzyme activities.

There was little or no significant extracellular enzyme
activity on the 12 paranitrophenol derivatives by most of
the human and animal pathogens. All of the saprophytic
species of Phialophora had extracellular beta-D-glucosidase
and N-acetyl-beta-glucosaminidase activity while the human
pathogens had no detectable activity. Similar patterns of
these two enzyme reactions were seen with the saprophytic
and plant pathogenic species of Cladosporium and the human
pathogens in the genera CZadosporium and Fonsecaea- In
addition all saprOphytic species of Cladosporium except
0. carpophilum have extracellular alpha—D-galactosidase
activity while no activity was detected in the human patho~
genic species of CZadosporium, Fonsecaea and Phialophora.

There was no difference in extracellular peroxidase
activity between pathogens and saprobes. A11 sixty-seven
isolates studied released the enzyme.

Also, no close relationship was detected between
adenosine triphosphatase activity and the formation of the
sclerotic cells, like the tissue phase of chromomycotic
agents which were produced by using West's liquid medium

with different nitrogen sources. The ability to release

Sumalee Pichyangkura
ATPase by these fungi seemed to be dependent on the
organisms per se.

Comparison of soluble protein banding patterns between
pathogenic and saprobic fungi was made by electrophoretics
on acrylamide gel. A greater number of matching bands of
soluble proteins was shown among pathogenic intraspecies
than among interspecies of these fungi. Cladosporium
species showed a close relationship to Fonsecaea species,
while Phialophora species showed a less close relationship
to Fonsecaea and CZadosporium. Fonsecaea dermatitidis
currently classified in this genus, also exhibited a
closer relationship to Fonsecaea and to CZadosporium than
to PhiaZophora species. The electrophoretic patterns of
specific enzymes and isoenzymes of F. dermatitidis also
showed a close relationship to Fonsecaea species which
further justifies the recent reclassification from
Hormodendrum to the genus Fonsecaea.

Furthermore, it was found that there was a distinctive
difference in the number of matching bands of soluble pro-
teins between pathogenic species and saprobes. The number
of matching bands was greater among pathogens than among

saprobes.

VARIATION IN EXTRACELLULAR AND INTRACELLULAR ENZYMATIC
ACTIVITIES IN THE GENERA PHIALOPHORA, FONSECAEA

AND CLADOSPORIUM

BY

Sumalee Pichyangkura

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1973

To my Parents
Mr and Mrs Sorn Tosunthorn

and my husband Chart

ii

ACKNOWLEDGMENTS

The author wishes to express most highly her sincere
thanks and gratitude to Dr. E. S. Beneke for his valuable
advice, encouragement and constructive criticism in pre-
paring this manuscript, as well as for his guidance through-
out her course of study.

Grateful thanks are due Dr. A. L. Rogers for his
valuable advice and useful suggestion and encouragement
throughout my graduate program.

Unforgettable thanks go to Dr. W. G. Fields for his
encouragement and for serving on my graduate committee.

Sincere thanks are due to Dr. C. J. Pollard, for his
kind advice in techniques of enzyme study as well as for
his serving on my graduate committee.

Thanks are also due to Miss Bonnie J. Wiley, Drs.

L. C. Georg, E. K103, and D. J. Dezeeuw, who kindly pro-
vided strains of dematiaceous fungi for the research.

I am very grateful to Dr. A. W. Saettler and Dr. W.
Tai for their kind instruction and for allowing me to use
their laboratory instruments.

Lastly and most sincerely, I am greatly indebted to
Dr. W. B. Drew, former Chairman of Botany and Plant
Pathology, for his kindness in offering me graduate
assistantships during my study in the United States.

iii

DEDICATION

ACKNOWLEDGMENTS.
LIST OF TABLES
LIST OF FIGURES.
INTRODUCTION

TABLE OF CONTENTS

REVIEW OF LITERATURE . . . . . . . . .

History of the Diseases

Source and Geographical Distribution
of the Disease. .

Nutritional and Biochemical Studies

Serology and Immunity

MATERIALS AND METHODS.

Collection of Cultures.

Extracellular Enzyme Procedures

Survey for extracellular enzymatic
activity in solid medium .

Survey for extracellular enzymatic
activity in liquid medium.

1.
2.

3.

Liquid CPYG medium for culture

of organisms for detecting
determination of peroxidase
activity .

West's liquid medium for culture
of organisms for detecting extra-
cellular adenosine triphosphatase
activity

Gel Electrophoresis of Intracellular Soluble
Proteins, Enzymes and Isoenzymes.

Culture preparation.

Extraction of soluble proteins

Disc electrophoresis procedure

Gel staining

1

2.
3.
4

a.

b.

Staining for general soluble
proteins . . . . . .

Specific soluble protein staining

for intracellular enzyme and iso~
enzyme locations

iv

Page
ii
iii
vi

viii

10
11
15
19

19
24

24
26

26

27

29
29
3O
31
34

34

35

RESULTS. .

Extracellular Enzyme Studies. .
1. Survey for extracellular enzymatic
.activity in CPYG solid medium.
2. Survey for extracellular enzymatic
activity in CPYG liquid medium
Peroxidase activity. . . . . .
Adenosine triphosphatase activity.
Comparative Gel Electrophoresis of Intra-
cellular Soluble Protein, Enzymes and
Isoenzymes. . . . .
1. General soluble. protein staining
2. Specific protein staining for intra-
cellular enzyme and isoenzyme
locations. . . . . . .
Peroxidase . .
Alpha- and beta- D- ~glucosidases
Catechol oxidase . . . .
Acid phosphatase . .
Alkaline phosphatase
Esterase . . . .
Amylase. . . .
Comparison of Enzymatic Common Bands Between
Pairs of Pathogens and Saprobes

DISCUSSION .

Extracellular Enzymatic Studies
Peroxidase activity.
Adenosine triphosphatase activity.
Comparative Gel Electrophoresis of Intra-
cellular Soluble Proteins .
Intracellular extracts of chromomycotic
pathogens and saprobes
General soluble proteins stained
with amido black .
2. Staining of enzymes and. isoenzymes
with their specific substrates

SUMMARY.
BIBLIOGRAPHY

Page
39
39
39
62

62
62

69
69

109
110
110
110
116
119
121

Table

LIST OF TABLES

Extracellular enzyme activities of human
pathogenic species of Phialophora,
Fonsecaea and CZadosporium grown at 25°C
on solid CPYG medium for 15 days.

Extracellular enzyme activities of saprobes
and plant pathogens of Phialophora and
Cladosporium grown at 25°C on solid medium
CPYG for 15 days.

Extracellular enzyme activities of sapro-
phytic or plant pathogenic species of
Cladosporium grown on solid CPYG medium
for 25 days at 25°C . . . . . . . . .

Extracellular enzyme activities of human
pathogenic species of Phialophora, Fonsecaea
and Cladosporium grown at 25°C on solid

CPYG medium for 25 days . . . . . . . .

Extracellular enzyme activities of human
pathogenic and saprobic Phialophora species
grown at 25°C on solid CPYG medium for 25
days. . . . . . . . . . . . . . . . .
Extracellular enzyme activities of human
pathogenic and saprobic Fonsecaea and

Cladosporium species at 25°C solid CPYG
medium for 25 days. . . . . . . . . . .

The diameter measurements of colonies of
P. jeanselmei, P. fastigiata, C. cucumeri-
num (F-26) and C. oxysporum (9496) for
determination of growth rate, when grown
on solid CPYG medium at 25°C for 33 days.

Extracellular peroxidase activity of
Phialophora, Fonsecaea and CZadosporium
human and animal pathogenic species grown
at 25°C in liquid CPYG medium .

vi

Page

40

43

46

48

50

52

57

63

Table Page

9 Extracellular peroxidase activity of
Phialophora and CZadosporium saprobic and
plant pathogenic species grown at 25°C in
liquid CPYG medium. . . . . . . . . . . . . . 64

10 Extracellular adenosine.triphosphatase of
human and animal pathogenic species
Phialophora, Fonsecaea and Cladosporium
grown at 25°C in West's liquid medium with
L-proline as the nitrogen source. . . . . . . 66

ll Extracellular adenosine triphosphatase of
human pathogenic species PhiaZophora,
Fonsecaea and Cladosporium grown at 25°C
in West's liquid medium with DL-isoleucine
as the nitrogen source. . . . . . . . . . . . 68

12 Extracellular adenosine triphosphatase
activity of some Phialophora and CZado-
sporium saprobic species in West's liquid
medium with DL-isoleucine or L-proline as
nitrogen sources. . . . . . . . . . . . . . . 7O

13 Average Rf values x 10 of general soluble
protein bands of thirty strains of pathogens
and saprobes in the electrophoretic pattern
of protein extracts in liquid CPYG medium
at 25°C for 14 days . . . . . . . . . . . . . 72

14 The number of matching soluble protein bands
of thirty human pathogenic, plant patho-
genic and saprobic strains in the gel
electrophoretic gels stained with amido
black . . . . . . . . . . . . . . . . . . . . 82

15 Migration distances of peroxidase, beta-
D-glucosidase and catechol oxidase isoenzyme
bands developed and stained in acrylamide
gels for 30 pathogens and saprobes. . . . . . 86

16 Migration distances of acid phosphatase,
alkaline phosphatase, esterase, and
amylase isoenzyme bands developed with
their specific substrates within acrylamide
gels. . . . . . . . . . . . . . . . . . . . . 93

17 The number of common isoenzyme bands de-
veloped with seven specific substrates
with gel electrophoresis of thirty human
pathogenic, plant pathogenic and some
saprobic strains. . . . . . . . . . . . . . . 102

vii

Figure

LIST OF FIGURES

Types of sporulation.found.among the
agents of.chromomycosis (following L.
Ajello at aZ., 1963). . . . . . . . .

Comparison of extracellular enzyme acti-
vities and growth rate of PhiaZophora
jeanselmei (msu) human pathogen grown at
25°C on solid CPYG medium . . . .

Comparison of extracellular enzyme acti-
vities and growth rate of Phialophora
fastigiata (8008) saprobe grown at 25°C
on solid CPYG medium. . . . . . . . .

Comparison of extracellular enzyme acti-
vities and growth rate of Cladosporium
oxysporum (9496) saprobe grown at 25° C
on solid CPYG medium. . . . . .

Comparison of extracellular enzyme acti-
vities and growth rate of CZadosporium
cucumerinum (F—26) plant pathogen grown
at 25°C on solid CPYG medium.

Electrophoretic pattern in acrylamide

gels of protein extracts stained with amido
black for thirty pathogens and saprobes of
species of Phialophora, Fonsecaea and
CZadosporium. . . . . . . . . . . . .

Photograph of electrophoretic pattern of
soluble proteins on acrylamide gels that
were stained with amido black

Photographs of.electrophoretic bands of
enzymes and isoenzymes with their specific
substrates for species of Phialophora,
Fonsecaea and CZadosporium. . . . .

Electrophoretic pattern in.acrylamide gels

of peroxidase of soluble protein extracts
from thirty pathogens and saprobes. .

viii

Page

56

59

60

61

78

79

85

88

Figure Page

10 Electrophoretic pattern in gels of beta-
D-glucosidase of soluble protein extracts
from the thirty pathogens and saprobes. . . . 90

ll tElectrophoretic pattern in acrylamide gels
of catechol oxidase soluble protein extracts
from thirty pathogens and saprobes. . . . . . 91

12 Electrophoreticpattern in acrylamide gels
of acid phosphatase of soluble protein
extracts from thirty pathogens and saprobes . 96

13 Electrophoretic pattern in acrylamide gels
of alkaline phosphatase of soluble protein
extracts from thirty pathogens and saprobes . 97

14 Electrophoretic pattern in-acrylamide gels
of esterase of mycelial-soluble protein
extracts from thirty pathogens and saprobes . 98

15 Electrophoretic pattern in acrylamide gels

of amylase of mycelial soluble protein
extracts from thirty pathogens and saprobes . 101

ix

INTRODUCTION

Although much is known about the diagnosis, histo-
pathology and epidemiology of Chromomycosis, and about the
morphology and immunology of the etiologic agents, the
taxonomy of these fungi is still not generally agreed upon.
Chromomycosis is commonly caused by any one of five
dematiaceous or dark colored fungi, Phialophora verrucosa
Medlar, 1915, Ibnsecaea pedrosoi (Brumpt) Negroni, 1936
comb. nov., Carrion, 1940 emend., F. compactum (Carrion)
Carrion, 1940 comb. nov., Fonsecaea dermatitidis (Kano)
Carrion, 1950, and Cladoaporium carrionii Trejos, 1954.
These pathogens belong in the form-family Dematiaceae of
the Fungi Imperfecti (Deuteromycetes). 'They are identified
to SOme extent on the basis of three types of conidiophores
(see Figure l), which may vary from one or more types in
each culture. The Phialophora-type is usually present in
P. verrucosa; the Cladosporium-type is usually present in
Fonsecaea spp. and always Cladosporium spp., and the
Fonsecaea-type may occur in Fonsecaea spp. The genera are
closely related and are usually separated by predominance
of one or the other of three divergent types of sporulation,
but because of multiplicity and variability in details of
spore production, they have been placed in many genera,
including Phialophora, Hormodendrum, Acrotheca,

1

Cladosporium type

 

Acrotheca type
(Fonaecaea)

 

Phialophora type

 

Figure l. T es of sporulation found among the agents
of Chromomycosis ollow1ngL. Ajello et al., 1963).

3
Trichosporium, Gomphinaria, Botrytoides, Phialoconidiophora,
Hormodendroides, Rhinocladiella and Fonsecaea (Emmons at
aZ., 1970).

The highly complex interrelationships among pathogenic
dematiaceous fungi, a cause of identification problems in
the routine laboratory procedures and in the literature, is
confusing because of the constantly changing terminology
and reclassification.

The differentiation of the etiologic agents of
Chromomycosis from saprophytic members of the dematiaceous
is difficult. The pathogens are separated from closely
related saprophytic species primarily on the basis of their
gross and microscopic morphology. However, the general
appearance of the colonies of both pathogenic and sapro-
phytic members of this group is quite similar. In general
the saprophytic species grow more rapidly than the pathogens.
At any rate, the conidial formation and the similar
morphology of human pathogens to plant pathogenic and
saprophytic members of the dematiaceous group, makes their
identification even more difficult.

Biochemical studies have shown more variation among
strains of a single species than between different species,
which leads to the lack of reliable biochemical tests to
aid in identification (Bindo, 1968; Cooper, 1970; Silva,
1958, 1960). Such physiological characteristics as rate
of growth, ability to grow at temperature higher than 30°C,
tolerance to cycloheximide, failure to hydrolyze casein,

growth stimulation by vitamins, and dimorphism in the

4
tissue are some of the useful preliminary criteria for
separating these fungi from most of the others (Silva,
1960).

Serological and immunological tests have been used by
a number of investigators as possible tools for identifi-
cation and classification of these fungi. The agar gel-
diffusion method appears promising for screening of sera
(Buckley and Murray, 1966). The fluorescent antibody
technique was utilized by Al-Doory and Gordon (1963) to
study the serological relationship between members of the
dematiaceous fungi. Biguet et al. (1965) have utilized
immunodiffusion (ID) and immunoelectrophoresis (IEP) tests
to compare the antigenic relationships among P. verrucosa,
F. pedrosoi and C. carrionii. However, at present sero-
logical methods have no important practical application
although it is possible to demonstrate the existence of
antibodies (Emmons et aZ., 1963). The agar gel-diffusion
test, which may be considered the most practical of all
serological methods for this disease, still has limited
diagnostic value in advanced cases of Chromomycosis.

The purpose of this research was to study the relation-
ship of representative species of pathogenic and saprophytic
dematiaceous fungi in the genera Phialophora, Fonsecaea,
and CZadosporium on the basis of enzymatic activities. In
this study, extracellular enzymatic activities of 22 human
and animal pathogenic strains were compared with 45
saprophytic and plant pathogenic strains of dematiaceous

fungi. Characteristic electrophoretic patterns of protein

5
fractions were obtained for comparison by disc gel electro-
phoresis from each of.the pathogenic fungi, and from some
isolates of the saprophytic Cladosporium and Phialophora
species. The specificity of intracellular enzymes and isoen-
zymes, including alkaline phosphatase, acid phosphatase, ester-
ase, peroxidase, amylase, betaeglucosidase and catechol oxidase
was identified by using the appropriate substrates on the
gels. With each enzyme, the bands for each isolate were
compared with those for other isolates of the same species

and for isolates of other species.

REVIEW OF LITERATURE

History of the Diseases

 

The term "Chromoblastomycosis" first used by Terra
et al. (1922) falsely ascribed the disease to a
Blastomyces (Carrion, 1950). 'The term Chromomycosis was
introduced later by Moore and Almeida in 1935. Since the
fungus does not multiply in the tissue by budding but by
the formation of septa, the term Chromomycosis has been
accepted in recent years (Baker, 1971).

The mycological literature for the history of chromo-
mycosis had been published between 1920 and 1940. Many
synonyms have been used or suggested for this disease,
such as blastomycose negra (Fonseca and Area Leao, 1930),
figueira (Rudolph, 1914), verrucous dermatitis (Pedroso
and Gomes, 1920), Chromomycosis (Moore and Almeida, 1935),
dermatite verrucosa cromomicosica (Moore and Almeida,
1935), and Pedroso's, Fonseca's or Gomes' disease (Weidman
and Rosenthal, 1941). It was apparent that some cases of
this new disease could have been diagnosed in the past as
leishmaniasis, syphilis, espundia or mossy foot, which were
recorded in Brazil and other South American countries
(Al-Doory, 1972).

Chromomycosis generally is accepted as being discovered
by Pedroso in Brazil in 1911. Pedroso noticed, as reported

6

7
later by Pedroso and Gomes (1920), the presence of large
yellowish to dark brown, spherical cells appeared in the
skin sections. The disease became known as the black
blastomycosis, blastomycose negra, according to Fonseca
and Area-Leao (1930). In 1914 Rudolph published his
observation on a skin disease popularly known as figueira
in the state of Minas Gerais, Brazil. The clinical
description and mycologic findings given by Rudolph clearly
indicate that he was dealing with the same disease observed
by Pedroso in Sao Paulo.three years before.

The first Chromomycosis report in the United States
was in 1915, when two investigators in Boston, Lane and
Medlar (Lane, 1915), found a case of verrucosa dermatitis
in a young Italian man. Medlar (1915) obtained a fungal
isolation from the case. Thaxter in collaboration with the
two workers established a new genus and a new species,
Phialophora verrucosa.

In 1922, Brumpt in his Precis de Parasitologic
described the Brazilian isolates as being different from
that of the Boston case. He named the Brazilian fungus
Hormodendrum pedrosoi, based on the branching of the
conidia in chains as the characteristic for the genus,
and gave the name pedrosoi to the species in honor of the
man who discovered the case. In the same year Terra et
al. (1922) described another Brazilian case of Chromomycosis.
The isolate displayed dominating terminal conidial clusters
characteristic of the genus Acrotheca which was established

by Fuckel in 1869. They suggested the adoption of the

8
genus named Acrotheca instead of Hormodendrum, and named
their fungus Acrotheca pedrosoi according to the report
by Fonseca and Leao (1923). From 1930 on, more cases were
reported from the American continent, including a case from
Texas caused by P. verrucosa (Wilson et aZ., 1933) and a
case from North Carolina caused by Hormodendrum pedrosoi
(Martin at aZ., 1936).

Carrion and Emmons, in 1935, reported a third etio-
logic agent for Chromomycosis from Puerto Rico caused by
a fungus different from.any previous isolates. He
described it as a new species called Hormodendrum
compactum.

In 1936, Negroni made.a thorough mycologic study on
a fungus he isolated from a case in Argentina. He reported
this fungus had both "Hormodendrum type" (CZadosporium
type) and "Acrotheca type" (Fonsecaea type) sporulation,
which made it unsuitable for inclusion in either of the
two genera. He created a new genus named "Fonsecaea" to
include fungi possessing the combined method of sporula~
tion. He named.his fungal isolate Fonsecaea pedrosoi.

In the following year, Kano (1937) isolated a fungus
from the first Japanese case of Chromomycosis. He described
it as a new agent under the name Hormiscium dermatitidis.

Another new fungal agent for Chromomycosis was estab-
lished by Simson in 1946 from a case in South Africa.
Carrion compared Simson's isolate, and found it to be
similar to a previous fungus isolated three years earlier

by O'Daly (1943) from a case in Venezuela. Simson named

9
the new agent Fonsecaea pedrosoi var. cZadosporium. This
fungus was later renamed by Trejos (1954) as a new species,
Cladosporium carrionii.

Cladosporiosis was first reported in 1911 by Guido
Banti (1911) in a woman who died with symptoms of brain
tumor which had brown septate hyphae and spherical forms
of a fungus in the lesions. In 1912 Saccardo studied and
published the name of this organism as bantiana. Clado-
sporiosis had been largely ignored until 1952, when
Binford and associates found the fungus in the brain tumor
of a man. They named this organism Cladosporium trichoides.
There can be little doubt today that both the cases of
Banti and Binford were caused by the same fungus as both
illustrations are similar. Borelli (1960) proposed a new
combination to designate the species, Cladosporium bantinum.

Phialophora jenselmei , a pathological agent of
maduromycosis, has been isolated in the United States and
in Europe (Emmons, 1945). The synonyms are: Topuza
jeanselmei Langeron, 1928; and PuZZuZaria jeanseZmei,
Dodge, 1935. The fungus was first isolated from a mycetcma
by Jeanselme, Huett and Lott and named Torula jeanseZmei
by Langeron (1928). The fungus was studied and renamed
Phialophora jeanselmei by Emmons (1945).

Schwartz and Emmons (1968) reported a new pathogenic
Phialophora pichapdsiae caused by a subcutaneous cystic
granuloma in a man. This organism was originally cited
in 1934 by Melin and Nannfeldt as one of the several fungi

responsible for the discoloration of ground woodpulp. No

10
known human infection with this fungus has been reported.
The presence of large, dark-brown to yellowish,
spherical bodies, sclerotic cells, in a biopsy is the
characteristic appearance in cases of Chromomycosis. The
sclerotic cells divide by splitting or forming septa in
cells. The physiology of sclerotic cells is obscure

(Silva, 1957).

Source and Geographical Distribution of the Disease

 

The existence of Chromomycosis has been established
throughout the world. Although Fonsecaea pedrosoi is
infrequently found in soil, Carrion (1950) found the
widespread distribution of its victims has established
its ubiquity. It is the most common etiologic agent of
Chromomycosis. Actually Conant (1937) and Conant and
Martin (1937) presented the first evidence that the etio-
logic agent of Chromomycosis was found in nature in soil,
plant debris or wood fragments. Three hundred thirty-six
samples of 5011, wood and plants from several areas in the
State of Merida, Venezuela, and adjacent areas were studied
by Salfelder et al. (1968). Six strains of Fonsecaea
pedrosoi were obtained from samples. Gezuele et al. reported
in 1972 that thirty-one strains of P. verrucosa and 34 of
P. pedrosoi were isolated from 329 samples of plant debris,
soil and other materials.by utilizing inoculated animals
and by direct cultures. ,The determination of the patho-
genic strains recovered from natural sources is presumably

based on the mycological and biological similarity between

ll
them and those isolated from man. The climatic conditions
probably play a role in the location of the fungi. This
is indicated by Carrion (1950) in his survey of 90 cases
of Chromomycosis. He found 83 per cent of the isolates
from these patients were F. pedrosoi, as these patients
seemed to have contracted the disease in tropical or sub-
tropical regions, and only 17 per cent were from the
temperate zone. It appeared that 5 out of 6 existing
isolates of P. verrucosa were from patients in colder
climates. Cole and Kendrick (1973) presented a classifi-
cation of six common.wood-inhibiting species of Phialophora
associated with bleeding of softwoods and some hardwoods
in North America, which included P. verrucosa. Chromo-
mycosis is more common in the tropical and subtropical
regions. The disease is considered rare in some areas
and even absent in some countries. Al-Doory (1972) has
compiled an extensive list showing the distribution of
case reports of Chromomycosis in the world. He listed
the following countries with the greatest number of cases
in decreasing numbers: Brazil, Costa Rica, Madagascar,
Dominican Republic, Australia, Cuba, Japan, Colombia,
Venezuela, Mexico, South Africa, Paraguay, India, Honduras,

and United States.

Nutritional and Biochemical Studies
The most recent studies.on nutrition of chromomycotic
agents were those of Silva (1957, 1958, 1960). Silva
(1958) observed that alterations in basal medium (Czapek-

Dox) by increasing the concentration of inorganic nitrogen,

12

substituting ammonium chloride for sodium nitrate, or
adding yeast extract would increase the pseudo-Acrotheca
type of Sporulation of Fonsecaea pedrosoi. No substance
was found that consistently stimulated either the
Phialophora or Cladosporium type of sporulation. Silva
(1960) found organic nitrogen essential for the growth of
a strain of P. jeanselmei, both organic nitrogen and the
vitamin supplement stimulated the growth of strains of
P. verrucosa and P. obscura, but had no effect on growth
of any other strains studied. The amount of carbohydrate
was the factor that influenced the rate of growth, rather
than the amount of nitrogen.. Substitution of glucose for
sucrose did not significantly alter the sporulation ratio
of Cladosporium and pseudo-Acrotheca types (Silva, 1958).

The vitamin requirement study by Area Leao and Cury
(1950) indicated that only thiamine is required by P.
pedrosoi, P. verrucosa, F. compactum and P. jeanselmei.
A species identified by Area Leao and Cury (1950) as
Cladosporium wernecki requires no Vitamins for growth.
Gilardi (1965) in a study of vitamin and nitrogen require~
ments found that inorganic ammonium salts are required for
F. pedrosoi, F. compactum, H. dermatitidis, C. carrionii,
C. sphaeroapermum, and P. richardsiae, and organic nitrogen
salts are required for P. jeanselmei, P. verrucosa and
P. obscura. Silva (1960) observed the superiority of
bactopeptone over neopeptone in Sabouraud's agar for

cultivation of the etiological agents of Chromomycosis.

13

Montemayor (1949) reported an optimal temperature
range of 20 to 25°C for saprophytic Cladosporium spp, 30°C
for one of the agents of mycetoma, P. jeanselmei, and 37°C
for agents of Chromomycosis. Silva (1958, 1960) reported
the optimal temperature for growth of agents of Chromomycosis
ranged between 25 and 35°C. She concluded that the faster
growth rate at 25°C for saprophytic Cladosporia does not
provide a reliable differentiation.

The physiological activity of F. compactum, F.
pedrosoi, P. jeanselmei and P. verrucosa was examined by
Montemayor (1949). None of these species liquified gelatin,
Loeffler's serum or coagulated milk, in contrast to the
positive activity of two saprophytic Cladosporium isolates.
Trejos (1954) states that C. carrionii and C. trichotdes
did not exert proteolytic activity on Loeffler's serum,
contrary to the activity.of saprophytic species of this
genus. De Vries (1952) studied the enzymatic activity of
24 saprophytic species of Cladosporium. He found that
production of tributyrine hydrolyzing lipases seemed to be
a constant characteristic of these species since all
decomposed tributyrine in varying degrees. Fuentes and
Bosch (1960) studied biochemical activities for distinguish-
ing etiologic agents of chromomymycosis, brain abscess,
and maduromycosis and certain related non-pathogenic
species. None of the strains recognized as etiologic
agents of Chromomycosis had the ability to liquify gelatin
and Loeffler's serum, to coagulate milk, to digest starch,

or to utilize tributyrine and cellulose. 0n the other

14
hand saprophytic Cladosporium spp. all are able to utilize
tributyrine or cellulose. -Rosenthal (1964) reported F.
pedroaoi, F. compactum, F. dermatitidis, P. verrucosa and
H. capsuzatum utilized tyrosine and urea but have no activity
on casein and gelatin,.and hydrolysis of starch was flexible.

West (1967) attempted without success to induce fertile
perithecia in P. verrucosa A.126, isolated from a patient
with chromoblastomycosis by varying the amounts of 19
different carbon and 44 nitrogen sources.

The parasitic phase of these agents, which are spheri-
cal, brown with thick-walled cells, is of the sclerotic
type. Moore (1941) used the surface of the chorioallantoic
membrane of the fertile egg for the cultivation of P.
verrucosa. The fungal growth reversed to the parasitic
phase morphology within 5 to 10 days.

Silva (1957) tried to elicit the parasitic phase in
many artificial media that would imitate the habitat of
these fungi both in vitro and in vivo. The conversion of
vegetative mycelium to sclerotic cells was clearly seen
from the experiments, but the factors required for the
induction of the parasitic phase of Chromomycosis are
unknown. Later on West (1967) used L-proline in a synthetic
medium to induce the formation of the sclerotic cells in
P. verrucosa.

Szaniszlo et al. (1972) attempted to differentiate
the three Chromomycosis agents on the basis of chemical
composition of the hyphal walls. They demonstrated the

similarity of the unfractionated hyphal wall composition

15
for P. verrucosa, F. pedrosoi and Cladosporium carrionii,
which consisted of large amounts of glucose (17-31%) and
protein components (29-42%), small amounts of mannose
(8-14%) and glucosamine (6-8%). Alkali-soluble wall
fractions consisted predominantly of glucose and mannose,
while alkali-insoluble wall fractions consisted mainly
of glucosamine and protein components. Chitin was identi-
fied as a wall component by release of N-acetyl glucosamine
during enzymatic digestion of the wall with Streptomyces
griseus chitinase. They concluded that the hyphal forms
of human pathogenic fungi have wall compositions inter-

mediate between those of Euascomycetes and Hemiascomycetes.

Serology and Immunity

 

Stone (1930) first studied the dematiaceous fungi
serologically by the use of the complement fixation test.
Conant and Martin (1937) and Martin et al. (1936) were the
first to employ the complement fixation test for the identi-
fication and classification of the various fungi of
Chromomycosis and some other members of the dematiaceous
groups. They used fungal extracts as antigen. Conant and
Martin (1937) noted that sera of rabbits immunized with
F. pedrosoi and F. compactum have a high complement fixa-
tion titer for their homologous antigen and for each other.
Meanwhile sera from rabbits immunized with P. verrucosa
produced a positive reaction with their homologous antigen
only. Martin (1938) determined the antigenic similarity

between Cladophora americana, isolated from wood pulp, and

16

a strain of P. verrucosa isolated from a Chromomycosis
patient in Uruguay.

Martin et al. (1936) and Conant et a1. (1937) reported
that complement-fixing antibodies have been demonstrated
in sera of patients with Chromomycosis. They found that
serum from a North Carolina patient infected with F.
pedrosoi: a) fixed complement with strains of the same
fungus isolated from the.patient as well as with isolates
from South American and.Puerto Rican strains; b) cross
reacted with a strain of P. verrucosa; and c) failed to
obtain a complement fixation with other fungi, such as
Blastomyces dermatitidis, Sporotrichum schenckii, sapro-
phytic Cladosporium spp. and other pigment-producing fungi.

Wilson and Plunkett (1965) found no correlation between
a certain serological titer and the clinical course of the
disease, which would aid in diagnosis or prognosis.

Seeliger reported (1968) that P. verrucosa, F. compactum,
F. pedrosoi and C. carrionii produce cross precipitin
reaction with antisera against Pullularia puZZuZans, P.
bergeri (Torula bergeri), P. werneckii (Cladosporium
werneekii), Sporotrichum gougerotii (Cladosporium gougerorii)
and S. schenckii.

Nielson and Conant (1967) evaluated the relationship
of the yeast-like dematiaceous fungi utilizing the agglutina-
tion tests. With few exceptions, they obtained good correla~
tion between the micromorphological differences in fungi
and their antigenic properties. Isolates from Chromomycosis

showed antigenic similarities to S. gougerotii, P.

17

jeanselmei and F. dermatitidis. However, isolates from
brain abscesses produced various patterns of agglutination.

Agar gel-diffusion appears to produce the most promis-
ing results for identifying the organisms in this disease
of any tests. Recently Buckley and Murray (1966) obtained
precipitating bands in 12 of their 13 cases tested. The
13th patient was under treatment with amphotericin B and
had no reactive precipitating bands. This is probably due
to the diminishing of antibodies during the treatment.
There was more cross reactivity between F.compactum and
F. pedrosoi than between either of them and P. verrucosa.

Biguet et a2. (1965) reported on the common antigen
factors among dematiaceous pathogens from a study of water
soluble antigens extracted from mycelia and extracellular
antigens from culture filtrates. Immunodiffusion (ID) and
immunoelectrOphoresis.(IEP) tests were used to study anti~
genic relationships among P. verrucosa, F. pedrosoi and
C. carrionii. They reported more common antigens appeared
between P. verrucosa and C. carpionii than between either
species and F. pedrosoi, and concluded that antigenically,
P. verrucosa and C. carrionii are more closely related to
one another than to F. pedrosoi. In 1970, Cooper and
Schneideu used the immunodiffusion test and obtained fewer
numbers of antigens from the filtrate (F antigen) than
from mycelial growth (M-1 and M-2 antigens) of P. verruegsa,
F. pedrosoi and C. carrionii. Common antigens were found
among all three species, indicating some degree of relation~
ship. The overall results were similar to that obtained

by Biguet et a1. (1965).

l8

Al-Doory and Gordon (1963) used the fluorescent
antibody technique to differentiate C. carrionii from
C. bantianum, as well as to differentiate these two
species from all other species tested, including C.
guogerotii, C. mansonii, C. werneckii, F. dermatitidis,

F. compactum, F. pedrosoi, P. jeanselmei, P. verrucosa,
T. bergeri and nonpathogenic Cladosporium spp.

In 1965 the fluorescent antibody technique was reported
again by Gordon and AleDoory. Ninety-two strains belonging
to 39 species of fungi related or unrelated in the dematia-
ceous group were tested with conjugates of F. pedrosoi,

F. compactum and F. dermatitidis. The~,genera <CZadosp0rium
and F. compactum were.found to be closely related sero-
logically to F. dermatitidis. There was little relation-
ship shown serologically between either of these species
and P. verrucosa. Under the same condition conjugates of
two strains of P. verrucosa failed to react with any of the
three species of Fonsecaea.

The hypersensitivity test was done by Martin er al.
(1936) in pathogenesis of.chromomycosis. The test showed
only a slight delayed reaction in the site of the intra»

cutaneous injection with an autogenous heat killed antigen.

MATERIALS AND METHODS

Collection of Cultures

Twenty-two isolates of pathogenic dematiaceous fungi,
including Phialophora verrucosa, Fonsecaea pedrosoi,
Fonsecaea compactum, Hormodendrum dermatitidis, Clado-
sporium carrionii, Cladosporium trichoides and PhiaZophora
jeanselmei, and forty-six isolates of non-pathogenic fungi
and plant pathogens were obtained from three different
sources.

A. Department of Health, Education, and Welfare,
Center for Disease Control, Mycology Section, Atlanta,
Georgia:

1. A835 Cladosporium carrionii
Record from Dr. A. Trejos (#27, 1954)
Original from Emmons #8619 "Hormodendrum
from Australia"

2. A984 Cladosporium carrionii
Record from Dr. Borelli 1955
Borelli #943 Isolated from skin scraping
in Venezuela

3. A980 Cladosporium trichoides
Record from Dr. Emmons, 1955
Original from Emmons #8590 Isolated from
Barnola case

B. Department of the Army, U.S. Army Natick Labora-

tories, Natick, Massachusetts:

19

QM

QM

QM

QM

QM

QM

QM

QM

QM

260

265

8008

267

270

1487

266

268

259

20

Phialophora compactum (NIH 8605;
ATCC 10222; CBS 285.47)

Isolated by A. L. Carrion in
Puerto Rico in 1935 from a case
clinically diagnosed as chromo-
blastomycosis

Phialophora fastigiata (NIH 8705)
Isolated by E. Melin in Sweden in
1937 from wood pulp

Phialophora fastigiata

IMI 86,982;

Culture received October 1961 from
D. Eveleigh. Isolated and identi-
fied by D. Brewer in Quebec, Canada,
in 1958 from paper-mill slime

Phialophora Zagerbergii (NIH 8707)
Isolated by E. Melin in Sweden in
1937 from wood pulp

Phialophora jeanselmei

NIH 8724; ATCC 10,224;

Culture from C. W. Emmons

Isolated by D. Symmers in New York

in 1944 from hand; clinical diagnosis
mycetoma

Phialophora malorum

No isolation data. Obtained from
L. P. McColloch, Beltsville,
Maryland, 1949

Phialophora melinii (NIH 8706)
Isolated by E. Melin in Sweden in
1937 from wood pulp

Phialophora obscura (NIH 8708)
Isolated by E. Melin in Sweden in
1937 from wood pulp

Phialophora pedrosoi (NIH 8603)
Isolated by A. L. Carrion in
Puerto Rico in 1935 from a case
clinically diagnosed as chromo-
blastomycosis

10.

11.

12.

13.

14.

15.

l6.

l7.

QM

QM

QM

QM

QM

QM

QM

QM

261

262

263

6808

264

269

489

9485

21

Phialophora pedrosoi

NIH 8610;

Culture received from C. W. Emmons
8 May 1947. Isolated by H. Hailey
in Georgia in 1939 from a case
clinically diagnosed as chromo-
blastomycosis

Phialophora pedroaoi

NIH 8615: ATCC 10,221

Culture from C. W. Emmons
Isolated by C. Binford in New
Orleans in 1943 from a case
clinically diagnosed as chromo-
blastomycosis

Phialophora richardeiae (NIH 8703)
Isolated by E. Melin in Sweden in
1937 from wood pulp

Phialophora richardsiae

WB 1630; NRRL 1630; Conant 330;
Culture received July 1955 from
K. B. Raper, University of Wisconsin.
Sent to NRRL from Harvard in June
1940 as Harvard 260. Isolated by
Conant, June 10, 1937

Phialophora verrucosa (NIH 8704)
Isolated by E. Melin in Sweden in
1937 from wood pulp

Phialophora verrucosa

NIH 8723; ATCC 10,223;

Culture from C. W. Emmons
Isolated by M. Moore in St. Louis
in 1943 from a case clinically
diagnosed as chromoblastomycosis

Cladosporium cladosporioides

(ATCC 16,022; CMI 45,534)

Isolated by E. Sigel from caulking
compound in 1948. Determined by
G. A. deVries

Cladosporium cladosporioides
Isolated by E. G. Simmons in Bogor,
Indonesia, in 1969 from decaying
leaf. Determined by M. B. Ellis

18.

19.

20.

21.

22.

23.

24.

25.

26.

QM

QM

QM

QM

QM

QM

QM

QM

QM

3167

9466

9481

9489

9495

9496

8013

7998

9257

22

Cladosporium herbarum
Isolated from a collapsible
canteen in 1945. Det. de Vries

Cladosporium oxysporum

Isolated by E. G. Simmons in
Bogor, Indonesia, in 1969 from
dead leaf of Palmae. Det. M. B.
Ellis

Cladosporium oxyeporum
Isolated by E. G. Simmons in
Bogor, Indonesia, in 1969 from
decaying petiole of Pangium
eduZe. Det. M. B. Ellis

Cladosporium oxysporum
Isolated by E. G. Simmons in
Bogor, Indonesia, in 1969 from
rotting leaf of dicot. Det.
M. B. Ellis

Cladosporium oxysporum

Isolated by E. G. Simmons in
Bogor, Indonesia, in 1969 from
decaying woody male inflorescence
of Arenga Sp. Det. M. B. Ellis

Cladosporium oxysporum

Isolated by E. G. Simmons in
Bogor, Indonesia, in 1969 from
decaying woody male inflorescence
of Arenga Sp. Det. M. B. Ellis

Cladosporium resinae (ATCC 18215)
Isolated by G. A. Atkins in
Victoria, Australia, in 1961

from Avtur fuel, P20

Cladosporium resinae f. aveZZaneum
Isolated in 1961 in Victoria,
Australia, from Avtur fuel. Det.
de Vries

Cladosporium resinae f. aveZZaneum
(Amorphotheca resinae st. perf.)
Isolated by D. G. Parbery in
Melbourne, Australia (in 1968?),
from soil. Det. Parbery

27.

28,

29.

30.

31.

32.

QM

QM

QM

QM

QM

QM

23

9258 Cladosporium resinae f. aveZZaneum
(Amorphotheca resinae st. perf.)
Isolated by D. G. Parbery in
Melbourne, Australia (in 1966-67?),
from grassland soil. Det. Parbery

55b Cladosporium sphaerospermum
Isolated from leather band, col-
lected Finschafen, New Guinea, in
1944. Det. de Vries

8050 Cladosporium ephaerospermum
Isolated from marine habitat.
Received from S. Meyers, Marine
Laboratory, Miami, Florida, in
1961. Det. E. G. Simmons and
D. I. Fennell

9494 Cladosporium aphaerospermum
Isolated by E. G. Simmons in
Bogor, Indonesia, in 1969 from
hard decaying fruit of Alsomitra
macrocarpa. Det. M. B. Ellis

9516 Cladosporium sphaerospermum
Isolated by E. G. Simmons in
Bogor, Indonesia, in 1969 from
decaying spadix of Oncosperma
horrida. Det. E. B. Ellis

9517 Cladosporium sphaerospermum
Isolated by E. G. Simmons in
Bogor, Indonesia, in 1969 from
decaying spadix of Oncosperma
horrida. Det. M. B. Ellis

Michigan State University Culture Collection:

a.

1.

Mycology Laboratory, Dr. E. S. Beneke

PhiaZophora verrucosa G. S. Bulmer,
University of Oklahoma Med. Sch.
Oklahoma City, Oklahoma

PhiaZophora verrucosa H. D. MacCurdy,
University of Windsor, Windsor, Ontario,
Canada

Phialophora jeanselmei, Michigan State
University

Fonsecaea pedrosoi Patient in Belo
Horizonte, Brazil

Fonsecaea pedrosoi, Duke University

24

Fonsecaea compactum, Duke University

Hormodendrum dermatitidis, University

of Michigan .- .-

8. Cladosporium trichoides, Michigan State
University

9. Phialophora sp. Unfit), Duke University

\lO‘

b. Plant Pathology Laboratory, Dr. Edward Klos

l. CZadOSporium carpophylum causes peach scab
disease, identification based on U.S.D.A.
Bureau Plant Industry Bulletin No. 395

2. Cladosporium carpophylum causes apricot
scab

c. Laboratory of Dr. Donald J. Dezeeuw

l. Cladosporium cucumerinum (F-26)
Isolate I from Dept. of Plant Pathology,
University of Wisconsin

2. Cladosporium cucumerinum (A-I)
Isolated from a cucumber fruit obtained
from Arenac County, Michigan, in 1967

3. Cladosporium cucumerinum (St. Clair)
Isolated from a cucumber fruit obtained
in St. Clair County, Michigan, in 1957

d. Author's collection
Twenty isolates of unidentified species
Cladosporium, collected from air and soil
around M.S.U. campus
D. University of Michigan Culture Collection:
Unidentified fungus, caused amphibian Chromomycosis
(Rana pipiens, RAP; Leopard frog) E-286 Phialcphcra
(or p0551b1y Fonsecaea)

Extracellular Enzyme Procedures

 

1. Survey for extracellular enzymatic activity in
solid medium

 

The CPYG solid medium introduced by Beneke et a2.
(1969) was used to grow the organisms for the study of
extracellular enzymes. The'CPYG solid medium is composed
of 4 grams of casein, 4 grams of bactopeptone, 4 grams of

yeast extract, 10 grams of glucose and 20 grams of agar per

25
liter with the final pH being 5.8. Twelve-day-old stock
cultures of the fungi, which had been grown on Sabouraud's
medium, were used as inocula. A plug 2 mm. in diameter was
removed from each fungal culture for inoculum and placed on
the center of a CPYG medium plate. After incubation at
25°C for 15 and 25 days, small uniform-sized plugs of the
agar were removed from beyond the growing fungal colony with
a 0.5 cm. diameter of cork borer. These plugs were then
used directly in the enzyme assays.

Twelve paranitrophenol (PNP) derivatives were chosen
as substrates for the various enzymes investigated. The
substrates and buffers were: PNP-alpha-D-glucoside 0.5 mg.
per ml., PNP-beta-D-glucoside 0.5 mg. per ml., PNP-N-acetyl-
beta-glucosaminide 0.15 mg. per ml., all in 0.005 M, pH 5.4,
sodium acetate buffer; PNP-alpha-D-galactoside, PNP-beta-
D-galactoside, PNP-butyrate, PNP-caprylate, PNP-laurate,
PNP-palmitate, PNP-stearate, all 1.0 mg. per ml. in 0.1 M,
pH 7.0, citrate-phosphate buffer; PNP-phosphate 1.0 mg.
per ml. in 0.1 M, pH 8.6, Tris HCL buffer for alkaline
phosphatase, and in 0.1 M, pH 5.0, sodium acetate buffer
for acid phosphatase.

The assay for the hydrolysis of the above substrates
was performed by using a plug of agar as the enzyme source
and adding 1.0 m1. of substrate solution into tubes. The
assay mixtures were then incubated at 37°C for two hours.
At the end of incubation period 2.0 ml. of a 0.5 M Tris

buffer, pH 9.8, was added to stop the reaction and develop

26
the yellow color of the liberated PNP. The activity of the
enzymes is expressed as O.D. (optical density) units by
spectrophotometer at 410 nanometers.
The organisms were grown in liquid CPYG medium to
study the extracellular enzymes with these substrates
listed above, but showed no satisfactory results.

2. Survey for extracellular enzymatic activity in
liquid’medium

 

a. Liquid CPYG medium for culture of organisms
for detectingTdetermination of peroxidase

activity

Suspensions of conidia and mycelial fragments
were made from twelve-day-old stock culture tubes growing
on Sabouraud's medium, with 5 ml. physiological saline
(0.85% NaCl). Then 1 ml. of the cell suspension, which

4 to 50 x 104 cells was

consisted of approximately 45 x 10
inoculated into 150 ml. Erlenmeyer flasks with 100 ml. of
the liquid CPYG medium. The flasks were inoculated and
placed on a rotary shaker at 150 rpm at 25°C. The liquid
cultures with any extracellular enzymes were filtered at

5, 10, and 15 days through a Seitz sterile filter. Then I
1 ml. of each filtrate was used in the extracellular enzyme
assays.

The method of Lfick (1963) was used to determine
peroxidase activity. Stock solutions and buffers were:
hydrogen peroxide 0.03 M (0.66 ml. of H202 or 30% w/v, in
200 ml. of double.distilled water), p-phenylenediamine 1%

w/v, and 0.067 M, pH 7, phosphate buffer.

27

The assay of peroxidase is based on the principle
that P-phenylenediamine is the hydrogen donor. It is oxi-
dized by H202 and peroxidase to a colored derivative which
is a molecule formed.from diamine and diimine. The O.D.
activity is measured by the increase in the purple color
per unit of time at 485 nanometers.

A tube with a mixture of 1 m1. of the filtrate, 3 m1.
of 6.7 x 10.2 M, phosphate buffer, pH 7, 0.3 ml. of hydrogen
peroxide and 0.3 ml. of p-phenylenediamine was incubated at
25°C on the shaker for 20 minutes. The control tube con-
tained the same mixture except for the p-phenylenediamine
was added at the end of incubation period. The control
tube was set at time zero, and then the incubated tube was
read after 20 minutes.

b. West's liquid medium for culture of organisms
for detecting extracellular adenosine tri-
phosphatase activity
The various agents of Chromomycosis and saprobes

were grown in West's Basal liquid medium (1967) for deter-

mination of adenosine triphosphatase. The medium consists

of:
Carbon source containing C in amount of 48.00 g.
Nitrogen source containing N in amount of 0.285 g.
MgSO4-7H20 0.5 g.
KHPO4 1.3 g.
FeCl3 0.005 g.
Thiamine HCL 50.0 ug.

Distilled water to adjust total volume to 1000 ml.

28

The pH was adjusted to 6.2 with either HCL or NaOH,
then autoclaved for 10 minutes at 121°C. West's basal
medium had glucose and DL-isoleucine or glucose and 1-proline
as carbon and nitrogen sources, respectively. The sterile
filtrate of thiamine HCL was added after the medium was
autoclaved.

The inoculum was prepared by adding 5 m1. of 0.85%
NaCl to a l4-day-old potato dextrose agar slant stock
culture, then gently scraping to remove spores. A 1 ml.
conidial suspension containing approximately 15 x 104 to
20 x 104 cells/m1. was inoculated into 50 m1. of West's
liquid medium in 125 ml. Erlenmeyer flask. After incu-
bation at 25°C for the period of 7, 14, 21 and 28 days
on a rotary shaker at 150 rpm, liquid cultures were filtered
through sterilized Seitz filters. The filtrates were then
used for determination of ATPase activity.

The assay method was based on the determination of
inorganic phosphate which is released from the hydrolytic
reaction of the enzyme (Ames, 1960; Pollard and Singh,
1968). The inorganic phosphate is released from the mole-
culte of adenosine triphosphate by the hydrolytic enzyme
adenosine triphosphatase, which will combine with ammonium
molybdate to form phosphomolybdate complex. Further reduc-
tion of this complex by ascorbic acid will develop a bluish
purple colored complex which could be measured colorimetri-
cally. The reagents for the determination of the enzyme

consisted of two parts: A) 10% ascorbic acid, B) 0.42%

29

ammonium molybdate, 4H20 in 1 N H2804. Mixture of one part
of A and 6 parts of B had to be freshly made just before
the assay.

Enzymatic assays were done in a clean test tube. The
mixture of 0.5 ml. of growth media filtrate, 0.5 m1. of
0.1 micromole of adenosine triphosphate substrate, 1 ml.
of citrate buffer pH 4.8 and 1.5 ml. of mixture reagent of
A and B were mixed thoroughly and incubated at 37°C for 2
hours. A control tube in which substrate was added at the
end of incubation period was utilized. The optical density
was read at 680 nanometers.

Gel Electrophoresis of Intracellular Soluble Proteins,
Enzymes and Isoenzymes

 

1. Culture preparation

 

Twenty-two human pathogenic fungi and eight saprobic
dematiaceous fungi were subjected to gel electrophoresis.
Fourteen-day-old stock cultures of the fungi grown on
potato dextrose agar were used to prepare spore and hyphal
suspensions with 5 m1. of 0.85% NaCl (physiological saline).
Four milliliters of this suspension was inoculated into
each 250 m1. Erlenmeyer flask containing 100 m1. of CPYG
liquid medium (Beneke et aZ., 1969). The flasks were then
incubated and placed on a rotary shaker at 150 rpm at 25°C

for two weeks.

3O

2. Extraction of soluble proteins

 

After two weeks all of the cultures in the flasks were
checked for contamination before extraction. The extraction
of soluble proteins was based on Gunsalus' method (1962) by
using acetone dried preparation. The mycelia collected by
filtration on Whatman No. 1 filter paper in a Buchner
funnel under vacuum were thoroughly washed three times with
sterile double distilled water. Five grams of the mycelial
mats were then added to 15 ml. of acetone at -20°C. Three
grams of acid-washed sand and the acetone suspension were
put in a Waring micro-mixer for approximately five minutes
at 15,000 rpm with the mixer container covered with acetone
dry ice. Approximately 30 m1. of acetone at -20°C was
slowly added into the container. After stirring, the
aqueous portion was allowed to settle, and was filtered
through a Whatman filter paper No. l in a Buchner funnel
under vacuum. The powder on the paper was washed with
20 m1. of acetone.at -20°C, sucked dry on the filter, and
ground to a dry powder with a spatula. The powder was
stored at -20°C in a dried bottle.

The dried acetone powder was added to 1 m1. of 0.004
M sodium bicarbonate solution and was stored overnight at
4°C. The suspension was centrifuged at 4,500 rpm for 25
minutes at 25°C. The supernatant, collected by using a
micrOpipette, was kept at 4°C for further determination of

the protein concentration.

31
Protein concentrations were estimated by Lowry's
Folin method (Lowry et aZ., 1951). Bovine serum albumin
was used as a standard. An estimated amount of 300 ug./
0.1 m1. of protein in each fungus was used for disc electro-
phoresis. The protein extracts with 0.1 m1. of 40%

sucrose were either used immediately or stored at -20°C.

3. Disc electrophoresis‘procedure

 

The theory and method of disc electrophoresis described
by Ornstein (1964) and Davis (1964), respectively, was
followed, except for the use of Tris-glycine buffer diluted
two times and a 11% small pore gel (Frank and Berry, 1972).
Polyacrylamide gel columns were made in 5 x 100 mm. cylin-
drical glass tubes. The gels were composed of two parts.
The lower part was a small pore gel where the fractiona-
tion of proteins took place. The upper part was a large
pore gel where the protein became stacked according to their
sizes and charges before being separated in the small pore
gel. Reagents used in separation of gels were: acrylamide,
N,N'-methylenebisacrylamide (BIS), 2-amino-2~(hydroxymethy1)~
1,3-propanediol (TRIZMA Base or TRIS), N,N,N',N'-tetra~
methylethylene diamine (TEMED), riboflavine hydrochloric
acid and ammonium persulfate. Stock solutions were prepared

as follows:

32

Stock A Stock B
approx1-
lN HCL 48 ml. 1N HCL mately 48 m1.
TRIS 36.6 g. TRIS 5.98 g.
TEMED 0.23 ml. TEMED 0.46 ml.
Water to 100 m1. Water to 100 ml.

(pH 8.9) (pH 6.7)

Stock C Stock D
Acrylamide 44.0 g. Acrylamide 10 g.
BIS 0.197 g. BIS 2.5 g.
Water to 100 m1. Water to 100 ml.

Stock E Stock F
Riboflavine 4 mg. Sucrose 40 g.
Water to 100 m1. Water to 100 ml.

Working solutions were prepared every time before using.

Working Solutions

 

 

 

Small pore Small pore Large pore
Solution #1 Solution #2 Solution
1 part A Ammonium persulfate 1 part B
2 parts C 0.14 ml. 2 parts D
1 part water water to 100 ml. 1 part E
pH 8.9 (8.8-9.0) 4 parts F

pH 6.7 (6.6-6.8)

 

The stock buffer solution for the reservoirs consisted
of TRIS 6.0 g., glycine 28.8 g., water to 1 liter, pH 8.3,

and a dilution of 1 part of buffer to 2 parts of water. The

33
buffer in the upper reservoirs was kept separate in another
container from the lower one until used again.

The gels were prepared by placing clean glass tubes
upright into the rubber holder rack. The lower gel solution
was made by combination of 1 part A, 2 parts C, 1 part
water and 4 parts of small-pore solution #2. A special
long needle and 30 m1. syringe was used to place the
mixture of gel up to 7 cm. into the glass tube. A‘thin
layer of water was gently added on the surface edge of
the tube to flatten the surface of the gel. Polymerizas
tion of the gel was complete after exposure under
fluorescent light.for.20 minutes. Then the water on the
surface was blotted away. The large pore gel, the upper
gel, was made of 1 part B, 2 parts D, 1 part E and 3 parts
F. A height of 1 cm. of upper gel was laid on top of the
polymerized lower gel, and two drops of water added to
flatten the surface.. The water was removed when polymeri-
zation was completed.

The gel tubes were placed in the holes of the upper
tank. Both tanks were filled with TRIS-glycine buffer
pH 8.3, and diluted 1:2 with.double distilled water. The
tubes were promptly connected by the upper and lower buffer
tanks. A few drops of 0.5 g./m1. of bromephenol blue
(crystal) were.added to the buffer in the upper tank for
tracing the progression of electrophoresis. Fifteen micro-
liter aliquot (300 ug.) of soluble protein extract combined
with 0.1 m1. of 40% sucrose was gently placed on top of the

gel column by a micropipette.

34

A Heathkit Model.ZP-32 Power supply was connected to
an electrophoresis.apparatus by an anionic system. The
negative charge protein molecules will move along the gel
toward the anode (+) which was located in the buffer of
the lower tank; the cathode was in the upper one. A current
of 3.5 milliamperes per tube and 120 volts/cm. was applied
on the system (Bloemendal, 1967). The time required as
indicated by the dye to move about 60 mm. in the separated
gel was usually about 120 minutes.

At the completion of electrophoresis, the gel tubes
were removed from.the upper reservoir and put on crushed
ice for a few minutes. The gels were removed from the
glass tube by rimming under cold water and injecting water
between the gel and.tube wall with a long needle syringe.
The wire was then withdrawn. The gels were removed with
a slight pressure against the gel. The gel columns were
stained with amido Schwarz dye in order to determine the
general soluble protein pattern. The others were lncu~
bated with the specific substrates for determination of

enzymes and isoenzymes.

 

4. Gel staining

3. Staining for general soluble proteins

 

Fractionated.solub1e protein gels were stained
0.5% (w/v) in amido Schwarz dye in 7% acetic acid for 30
minutes, and transferred into 7% acetic acid destalning

solution overnight until the sharp dark bands appeared.

35
The individual gel was kept in a tube with 2% acetic acid.
Reading of protein bands was made by using a Gilford 2400
spectrOphotometer with a gel transport attachment at 680
nanometer, a chart speed of l minute/inch, and a scan rate
of 2 cm./min. Diagrammatic interpretations were made of
the bands in addition to photographs.

b. Specific soluble protein staining for intra-
cellular enzyme and isoenzyme locations

 

 

The gels were removed from the glass tubes
immediately and were incubated with Specific enzyme sub-
strates. There were eight enzymes, including peroxidase,
acid phosphatase, alkaline phosphatase, alpha-D-glucosidase,
lbeta-D-glucosidase, esterase, catechol oxidase and alpha
amylase, which were located within the gels after
electrophoresis.

Peroxidase enzymatic activity was located by the
method used by Macko et a1. (Macko er al., 1967, modified
by Webb et aZ., 1972). Catechol was used as HZ donor.
The gels were immersed after electrophoresis in 0.02 M
solution of catechol in 0.02 M phosphate buffer, pH 5.8,
for 30 minutes at room temperature. Gels were rinsed with
distilled water.and.then transferred to a 0.3% H202 solution
for band development. Bands developed within 10 minutes
and rapidly faded. So immediate recording of their patterns
was necessary.

Acid phosphatase was located following Jensen's method

(1962). The gels were immersed directly into a substrate

36

solution which was composed of 0.6 g. lead nitrate in 500
ml. of 0.05 M acetate-buffer at pH 4.5 to which was added
sodium beta-glycerophosphate SHZO in 0.10 M concentration,
and the final pH was adjusted to 5.0. After incubation
at 37°C for 20 minutes, gels were rinsed in 2% acetic
acid and washed with distilled water. The bands were
developed to indicate where enzyme activity was located by
the use of 1% sodium sulfite solution at room temperature.
The gels were stored in 1% sodium sulfite for measurement
records.

Alkaline phOSphatase was located following Gerhardt
et a1. (Gerhardt et aZ., 1963), which has been modified by
Webb et al. (1972).. The gels were pre-incubated in a
0.05 M borate buffer for 10 minutes at room temperature,
and then immersed in a solution containing 25 mg. of alpha~
naphthyl phosphate (sodium salt), 100 mg. Fast Red TR salt
and 100 ml. 0.05 M borate buffer for 2 hours at 37°C. The
orange brown bands developed if alkaline phosphatases were
present. Their locations were then recorded.

Alpha and beta D-glucosidases were located by using
a method described by Beneke at aZ.(Beneke et al., 1969).
After electrophoresis, the gels were immersed in 30 ml. of
sodium acetate buffer pH 5.4 containing 1.5 mg./ml. PNP-
alpha-D-glucoside for 1/2 hour at 37°C and then transferred
to 0.5 M buffer at pH 9.8 to stop the reaction and to
develop the yellow-colored bands of liberated PNP at room

temperature. Beta-D-glucosidase assay was based on the same

.37
method but had PNP-betarD-glucoside as the substrate. The
distancesof bands were recorded immediately after their
movements.

Esterase was located by the method of Desborough and
Peloquin (1967). Immediately after electrophoresis, gels
were transferred to phosphate buffer for 10 minutes, before
being placed in the substrate which consisted of 50 mg.
alpha-naphthyl acetate dissolved in 2 m1. acetone and 75
mg. fast blue 2R salt, in 100 ml. of phosphate buffer at
pH 7.4, and filtered, and then incubated at 37°C for 25
minutes. The purplish blue bands were developed within
the gels. The gels were kept in phosphate buffer pH 7.4
for further measurement.

Catechol oxidase localization was based on the color
products resulting from the oxidation of catechol. The
method followed.that given by Shannon et a1. (1973). The
substrate was prepared by adding 165 mg. of catechol to
15 ml. of sodium phosphate buffer at pH 4.2 with the addi»
tion of water up to 135 ml. Gels were incubated overnight
at room temperature and.were rinsed with distilled water,
and the locations of the bands were recorded.

Amylase localization followed the technique given
by Brewbaker et aZ..(1968), which has been modified by
Webb er a1. (1972). Gels were made up by using ammonium
persulfate dissolved in a 1%_starch solution in place of
small pore solution (Ornstein, 1964; Davis, 1964). After
electrophoresis, the gels were incubated in 0.01 M phosphate

buffer at pH 5.8, for 40 minutes at room temperature and

38
then quickly immersed in Gram's iodine solution. The gels
were removed and recorded for the development of the clear
bands. The existence of unstained regions, where the blue
color indicative of the presence of starch failed to appear,

was taken as an indication of amylase activity.

RESULTS

Extracellular Enzyme Studies

 

l. Survgy for extracellular enzymatic activity 1p
CPYG soIid medium

 

The extracellular enzyme activities of twenty-two
pathogens and forty-six saprobes were investigated after
the colonies had grown.on CPYG agar for 5-, 15-, and 25-
day periods. The plugs of CPYG agar cut from near the
edge of the individual colonies of the isolates were incu~
bated at 37°C for 2 hours with the twelve paranitrophenol
(PNP) derivative substrates.

None of the twenty-two human pathogens and forty-six
saprobes and plant pathogens had detectable enzyme activi~
ties with the twelve PNP derivative substrates at 5 days.
However, some of these pathogens and most of the saprobes
produced a sufficient.quantity of extracellular enzymes,
which diffused into the agar medium for positive plug tests
at 15 days.

Table 1 shows the extracellular enzyme activities of
the twenty-two pathogenic strains of Phialophora, Fonsecaea
and Cladosporium grown on CPYG medium at 25°C for 15 days.
There is little or no detectable extracellular enzyme
activity with the twelve PNP derivatives used as substrates

for enzyme testing on most of the twenty-two human pathogenic

39

4O

 

 

 

 

O O O O O O O O O HOOEO wheaesesssme .e
O O O OO O O O O O HcOOO EssoseeOs .H
O O O OO O O O O O Homev assesseOo .e
O O O OH O O O O O HNONO osmoeema .e
O O O OH O O O O O HHONO somsseme .H
O O O O O O O O OH HxOOO assesses .H
O O O O O O O O m HOONO Hometown .H
OH O O OO O OH O O O Hamev «eschews .e
O O O O O O O O O HonOO assesses .H
O O O O O O O O O HeonO omeHmmesmO .m
O O O OH O OH OH O m HameO omeHmmessO .H
O O m m O O O O O HOONO emooseems .H
O O O OO O O O O O HOONO cassettes .m
O O O O O O O O O onuv smosssems .m
o o o o o o o o o money omoossamo .m
o o o o o o o o o AMOV omooxasoo .m

3... wow Home One mam mm H... mm P... se....

1nd Bdl Edd .4er .Bd d1 .dd dud .dT:

91 SSE SSH. 31 83 DB 9U; d. dp

1T: 9 ._ 9 .9 12A m3 9. BB 8 8

BO . ET. I1 S S. S S

S St. UK a e e e

9 93 ST.

HOOOH x HOOH\OOHQ Hemm\ee OHO .O.oO
qu>Hpo<
as
mxmw ma How asHuoE owmu vHHom do 00mm um :SOHm seesommonoNb one oeooomeom
«easemoHewxm mo moHoomm oesomonuma does: we moHuH>HHum oequo HmHsaaoumexm .H oHan

41

.moHuH>Huom oHnmuuouow oc we: mommpoumo oOHmoum pom UHHHHEHmm .ofipxusm

 

 

 

OOOOH x Hsofas? Hama\ee OHH .H.OO
sHHH>Hpu<

x

O O O O O O O O O HOON-mO .Hm assessege
O O O O O OH O O O HOHOO assessoaes .O
O O O OO O OH O O O Hemev mesooeoess .O
o o o o o o o o o nxopv wmsowsseo .b
O O O mm O O O O O HOwO oesoossso .O
O O O OH O O OH O O HOMO Horseshoe .O

mmm mmm mmm mmm mmm Mm mm MN MW .finsfio

1nd Bdl. Edd 1dd .Ed d1 dd .dva. d1.

81 SSE SDUI GI 93 99 9U: d. dp

IT. 9.. 9.8 l/A me 8. BB 9 E

93 . 9T. Trl S S. S S

S ST: HA 9 9 8 3

9 93 GT.

 

H.e.oeouv H mHan

42

strains. There was more activity toward the PNP deriva-
tive of the fatty acid caprylate than toward other substrates
especially for one strain of P. verrucosa, 2 strains of
F. pedrosoi, 2 strains of F. compactum and 1 strain each of
C. carrionii and C. trichoides. There were no detectable
enzyme activities with PNP-stearate, PNP—palmitate and
PNP-butyrate. The yellow color of paranitrOphenol could
be differentiated visibly at about 25 O.D./agar plug/hour
X 1000.

The saprobes and plant pathogens at 15 days on CPYG
agar showed more extensive extracellular enzyme activity
on the twelve substrates (Table 2). No acid phosphatase
activity was detected except in two strains of Phialophcra
saprobes, while there was activity in many Cladosporium
saprobes and most plant pathogens. No alkaline phosphatase
activity was detected in Phialophora saprobes and Clado-
sporium resinae isolates; however, activity was evident
in all of the Cladosporium oxysporum strains. The extra~
cellular activity of alpha-D-glucosidase was varied among
Cladosporium saprobes, while none of the PhiaZophora
saprobes showed activity. ,The extracellular beta~D~
glucosidase and N-acetyl-beta-g1ucosaminidase activities
were high (with several exceptions) in the saprobic iso-
lates of Phialophoraand-CZadosporium. The extracellular
activity of alpha-D-galactosidase of Phialophora saprobes
was low or not.detected when compared with the irregular

variations for Cladosporium. Table 3 shows all the saprobic

43

 

O OO OOO ONH ONO ONO ON OOH OOH HOOOOO gsessmses .O
O O OOO OOH OON OOO OH OO OO HOOOOO EOOOOOONO .O
O O OH OO OHN OOH O OO ON HHOOOO Essssmses .O
O O OOH OO OON ONO O OO OO HOOOOO esNOOOOaO .O
O O ON O OON OOH O O OHN HOOOOO Omeeoeoamsesao .O
O O O O OOH O O O O HOOOO OmeONNOOOOOOOO .O
O O O OH OOO OOO ON OO OO HNOHOO EsssOemN .O
O O OO OO OON OO O O ON HNOOQO esssssse .O
O O ON O OO OOH O O O HNOOHO essOHse .N
O O O O OO OHH O O O HOONO ONOOOOO .N
O O O O ON ONN O O O HNONO NOONmOemOOH .N
O O O OO O OO O O O HOONO ONeONHme .N
O O O OO ON OOH O O O HOONO OOONONNOOO .N
O O OH O OH OO O O O HOOOOO OOONONOOOO .N
O O O OOH ONH OO O O O HOONO OONOOOOOONO .N
O O OH O ON OO O O OH HOOOOO msNOONOOONs .N

OOO... mew. mew. OOO ONO MN. NO. ON P... se....

1nd Bdl Edd 1dd .Bd d1 .dd dud dT:

31 Soda... SDU. 91 93 DB 9U; .d. .dD.

II. 8 .. 9.9 l/A m9 9. BB 8 B

83 . 8.1 1.1. S S. S S

S STE UIA NO 9 a a

3 83 GT.

 

HOOOH x HOOO\NOHO NONOOO: OHO .O.OO

OHH>HO8<

 

mama mH How wwmu esHpoe UHHom :o comm um ozoom sswaommomeNo one
osoxmoNowem mo mammogpmm HomHm one monoammm mo moHuO>wHom maano HmasHfioomHuxm .N oHnt

44

 

 

HOOOH x HOOONOOHO NOOONEO OHO .p.oO

OHH>HO6<

O O OO OO OO OO ON OO O HOO .OO sesseemoesNO
O O OO OO OO ON OO O O HOS .OO esONOOOOeONO
O O ONH O OO ONH OO O O HOO .OO EOOOONOOOONO
O O OO O OOO OHH OO OO OO HNO .OO EOOOONOOOOOO
O O OON O OOO OON ONH OOH ON HHO .OO EONOOOOOOONO
O O OO O OO OOH O OOH OO HNHOOO EOEOOOOONOOOOO .O
O O ON O OO OO O O ON HOHOOO EOEOOOOONOOOOO .O
O O O OOH OO O O O OO HOOOOO EOSOOOOONOOONO .O
O O OO OHN OOH OO OH OO OO HOOOOO sseemsassmsesm .O
O O OO ONN ONO OOH O OO OO HOOOO sseNOOOONOOOOO .O
HOONOO
O O OH O OOH OOO OO O ON EOOOONHOOO .O OOONOOO .O
HNONOO
O O ONH ON OHO OOO OO O ON esOeONNOOO .O screams .O
HOOONO
O O ON OO OO ONO O O OO EOOOOHHOOO .O OOOOOOO .O
O O OO O ONO OON OO O ONO HOHOOO 8:38 .O
O O O O OO OON O OO OO HOOOOO EONOOOONO .O
91d edq Ede 93d DNd dq d9 dV dV
seN INo INT. seN WIN No NT. NT. No Emwcwweo
1nd Edi Edd 1dd .Ed d1 dd dva. dr:
81 SSE SDU. 91 ED 9E 9H: d. dD.
1T: 9 ._ a . E IN me E. EE E E
ED _ ET T1 S S. S S
S St. UK a e 9 a
8 93 GT.

 

H.O.OOOOO N OHOON

45

 

 

 

HOOOH x NOOONOOHO OOOONEO OHO .0.00

OuH>Huu<

O O O O OOH O O O O HHUOOOO eOOOOOOONOO .O
O O O ONH OOH O O O OO HoouHOOOO EOOOOOOOOOO .O
OO O OOO ONH ONO OOO O OOO OOOHOHOHU.NOO EOOONOEOOOO .O
OO O OO OOH ONO OOO O O OOH HON-OO EOONOOEOOOO .O
ON O OOH OHN OOO OOO O OO OOO HH-<O EOOOOOEOOOO .O
O O ON OO OOO OO O O OO HOHO .OO EOONOOOOOONO
O O O OHH OO ON O O O HOHO .OO EOOOOOOOeOHO
O O OO ON ONO OON O OO O HNHO .HO sstosmoesOO
O O OHH ON OON OOH O O O HHHO .OO EONeoamonNO
O O OOH OO OOO ONN OO ON ON HOHO .OO EONNOOOOOOHO
O O OO O OON OOH O O OO HOO .OO esNOONOOOONO
O O O ON OO OO O O O HOO .OO EONOOOOOOOOO
O O ONH OO OOO OHN OOH OO O HNO .OO SOONOOOOOONO
O O ON OO OO O OO O O HOO .OO esOsoemowsOO

31d Edq Ede 93d ONd dq de dV dV

SEN 1N9 INI SEN WIN N9 N1 NI N3 Emflcmmao

1nd Edl. Edd 1dd .Ed d1 dd dva. dT:

31 SSE SDU. 81 ED DE DU; d. dp

II. 9.. 9 .E er me E. EE E E

E3 . ET. T1 S S. S S

S St. HA 9 e a 3

9 93 GT.

 

H.O.NOOOO N OHOOH

46

Ochcpm do Hones: A V
OoHHH>HHom oEcho o>Hummoc Ozocm O-V
moOuH>Huum oacho o>HuHmom msozm OOO

 

 

 

HOO+ HNO+ HNO+ HOO+
- - + HOO- + + HNHO- HNO- HOHO- OH HOOOOO-OHOO .OO .O
OHO+
. - u + + u u . flaw- N Estxmomsoo .b
mmv+ ,.- + + + + - + + m Sarasossoxo .9
HHO+
. - + + + + mew- + + m Exssommoaooxdm .9
.. I + + + + + I + V EONOQGNNODU c.“ mdfimwm& ob
OHO+
I flHv+ + + + + AVWI + + m E3RQR~W®80 Ob
HHO+
- - + + + + - - OHV- N momwoeeommomoNo .b
OHO+
- - + + + + - - HHV- N Eseeoeme .u
9mm mm mm mam mam mm... Mm. MN. MW mm EOHOOOOO
luau d1 dd tad .ed d1 4d .dX .3& 1.
91 q; 9H 91 as 6; OH 4? dP e
Tat. e . one 1,A mNO Os. use a e Tso
E D T. I. E I 7:1 S S _ S S u 1..
S E E St. UK a 9 a a S
o s s HOD est
9 3
00mm on mxmw mm How Edwwoe omwu wHHom :o HSOHM EswsommowoNb
mo moHuomm oficowonumm woman Ho ofiuxnmoummm mo moOuO>Huum oexnco Hmasaaoomuuxm .m oHan

47
strains of Cladosporium. There was only one isolate of
C. oxysporum (9495) among all the saprobes and plant patho-

gens that showed betaeD-galactosidase activity. Caprylic

esterase activity was varied among Phialophora and Cladosporium
saprobes. ‘Only three strains of the plant pathogen C. cucumeri-
num showed lauric esterase activity out of all the saprobes

and plant pathogens tested.

The extracellular enzyme activities were increased
for some strains at 25 days of growth. Most of human and
animal pathogens still showed low or no extracellular enzyme
activities with the twelve PNP derivative substrates,
except for two isolates of Phialophora jeanselmei which
showed an increase in alpha-D-glucosidase, beta«D-g1ucosidase,
alpha-D-galactosidase.and caprylase activities. The results
of the enzyme activities of twenty-two Phialophora,
Fonsecaea and Cladosporium species are shown in Table 4.

The extracellular.enzyme activities were compared
between the human pathogenic and saprobic strains of
Phialophopa species at 25 days of growth in Table 5.

Marked beta-D-glucosidase and N-acetyl~beta-glucosaminidase
activities were evident among the saprobe isolates, while
these activities were not detected in the human pathogenic
strains of Phialophora isolates except for two isolates of
P. jeanselmei. The other enzymes were low or not detectable
in general.

Human and animal pathogenic Fonsecaea and Cladosporzum

species had little or no enzymatic activities with the

48

 

 

OOOOH x HOOHNOOHO NOOONEO OHO .H.OO

NOH>Hpu<

O O O OO O O O O O HOOEO OOOOOOOOEOOO .N
o o o om o o o o o ADmEV exooomsoo .M
O O O OO O O O O O HOOOO EOOOOOEOO .N
O O O OO O ON O O O HNONO NO.OOOOO .N
O O O O O O O O O HHONO OOOONOOO .N
O O O O O O O O O HOONO NOOOOOON .N
O O O O O O O O O HOONO OOOOOOON .O
O O O ON O O O O O HOOEO NOOOOOOO .N
O O O O O O O O OH HOHOOO OOOONOOO .O
O O OH OO ON ON OH OH OH HcOOO NOONOOOOOO .N
O O OO OON OH OOO OO O OH HOOeO OOeNOOOOOO .N
O O O O O O O O O HOONO OOOOONOOO .N
o o o O 0H m o o o meomv omoosssoo .m
o o o o o o o o o mxouv omoosssma .m
o o o o o o o o o Aumev omoosaaos .m
O O O O O O O O O HOOO OOOOOOOOO .m

OOO. OOO. OOO OOO OOO MO. OH... mm. MO. 52....

1nd Edi. Edd 1dd ..Ed d1 dd dun. dt.

31 SSE SSU. 81 ED SE SU; d. dp

JOE 9 ._ a .E er wad E . DOE E E

93 . 9T. T..1. S S. S S

S SOP u/A a OO 9 e

9 33 GT.

 

mzmo mm How eusoE uwmu wOHom so 00mm um oBOHm sewaommowoNb one emeeomeom
neaoxmoHeexm mo mofioomm oficowoaumm ones: we moOuO>OHom oEONno Headaaoumeuxm .O oHan

49

 

 

 

OOOOH x Hooz\msHm Hemm\ee OHO .mwpw
NHH>OHU<

O O O O O O OH OH OH HOON-OO .OO,OOOOOOOOOON
O O O ONH O OO O O O HOHOO OOONOOOONO .O
O O O O O ON O O O HsOeO OOONOOOONN .O
O O O O O ON OO OH O HOONO ONOOOOOOO .O
O O O OO O O O O O HOOO NOOONNNOO .O
O O O OO O O OH O O HOOO NOOOONOOO .O

91d Edq Ede eDd DNd dq d8 dV dV

seN INe INT. seN W._N Ne NT. NT. No EOOHHOMHO

1nd Edl. Edd 1dd .Ed d1 dd dun. dt.

91 SSE SSU; 91 ED SE SUI d. db.

1.... 9.. a .8 1A me 9. EE 8 E

E3 _ ET.L 1.1+ S S. S S

S St. HA 9 e 9 e

9 93 GT.

 

H.0.0:ouO O OHOOO

50

 

 

 

 

 

O O O O OO ONO O O O OOOONOE .N
O O O O OO ONO O O O OOOOOOO .N
O O O OO OO OOO O OO O OOOOOONOOON .N
O O O O OO ONO O O O ONONNOE .N
O O O O OO OON O ON OO HOOOOO OOOOOONOOO .N
O O O OO OOO OHN O O O HOONO OOOOOOOOOON .N
O O O OO OOO ONO ON OO ON HOOOOO OOOOONOOONN .N
O O O O O O OH OH OH HOON-mO .OO OOOOOONOOON
O O OH OO ON ON OH OH OH HOOOO OOEOOOOOOO .N
O O OO OON OH OOO OO O OH HOOEO NOENOOOOOO .N
O O O O O O O O O HOONO OOOOOOOOO .N
O O O O OH O O O O HOONO OOOOOLOOO .N
O O O O O O O O O HxOOO OOOOONOOO .N
o o o o o o o o o flumev omoosaaoe .m
O O O O O O O O O HOOO OOOOOONOO .N

mmm ”MN WNW mmm WNW MN MN MN MW fifiago

1nd Edl Edd 1dd .Ed d1. dd dun. dt.

91 SSE SSU. 81 ED SE Sq d. dp

II. 9.. 9.E 1A me E. EE E E

E3 . ET. Tel. S S. S S

S ST: UK 9 9 a 3

3 93 GT.

OOOOH x NOOONOOHO NOOONEO OHO .H.OO
OHO>HHU<
mxmv ON How Esfiwoe oxmu wOHom co Domm um :SOHM mofioomm
enormomemxm oHaoemmm one oacowozumm amass mo mowufi>muom oexnno HmHDHHoumHuxm .m oHan

51
twelve substrates at 25.days, except for a few isolates
that showed enzyme activity with the PNP fatty acid
caprylate (Table 6). One isolate of C. carrionii (tex)
and two isolates of C. trichoides also showed beta-D-
glucosidase activity, while the other Cladosporium and
Fonsecaea pathogens showed very little or no activity.
Marked enzyme activities for beta-D-glucosidase, N-acetyl-
beta-glucosaminidase, and alpha-D-galactosidase were shown
in Cladosporium saprobes and plant pathogens. A marked
difference1J1N-acetyl-beta-glucosaminidase and alpha-D-
galactosidase activities can be noted between the human
and animal pathogenic strains of Fonsecaea and Cladosporium
with no detected enzyme activity and the saprobic and plant
pathogenic strains with well defined activity.

A comparison of the growth rate of Phialophora
jeanselmei (msu), a human pathogen, with extracellular
enzyme activities on twelve PNP-derivative substrates is
shown in Figure 2. The growth rate was determined by the
increasing diameter of colonies with time in Table 7.

That there was a substantial increase in the activity of
enzyme, alpha-D-glucosidase and a marked increase of both
beta-D-glucosidase and.caprylase (assayed at 5*, 15-, and
25-day colony growth)-are shown in Figure 2. A small
amount of extracellular enzymes were released from P.
jeanselmei during.the exponential phase of growth, and a
rapid increase in beta-D-glucosidase and caprylase was
noted at approximately 15 days after inoculation or right

at the beginning of the stationary phase of growth. Two

52

 

 

 

 

O O ONO ONH ONO ONO O O OON HNOHOO EOOOOOOO .O
O O OHN OOH OOO ONN O O O HOOEO EOOOOOOO .O
O O O ONH O OO O O O HOHOO OOOOOOOONO .O
O O O O O ON O O O HOOEO OOOOOOONNO .O
O O O O O ON OO OH O HOOOOOOOONONOO .O
O O O OO O O O O O HOOOOOOOONOOO .O
O O O OO O O OH O O HOOONOOONNOOO .O
O O O OO O O O O O HOOEO OOONOOOOeOOO .O
O O O OO O O O O O HOOEO EOOOOOEOO .N
O O O OO O O O O O HOOOO EOOOONEOO .N
O O O OO O ON O O O HNONO OOOOOOOO .O
O O O O O O O O O HHONO OOOONOON .O
O O O O O O O O O HOOOO NOOOOOON .O
O O O O O O O O O HOONO OOOOOOON .N
O O O ON O O O O O HOOEO OOOONOON .O
O O O O O O O O OH HOHOOO NOOOOOON .O

Omm wmm wmn Omm mam mm mm mm. My. 5288

1nd Edl. Edd 1dd .Ed d1 dd dvn. dt.

91 SSE SSU; 31 E3 SE SUI d. dp

It. a .. a .8 llA me Do. 88 Du 9

ED . EI I...» S S. S S

s est. u,A NO 9 a a

9 93 ST.

HOOOH x SOONOOH.H NOOONEO OHO .H.oO
OOH>Hpu<
mane mm How EDHUoE owmu wHHom 00mm um mofioomm ssmoommowoNu
mam economeom oHnoummm can 0Ocomonuma amen: mo moOuH>Huum oaxnco HmHsHHoomHuxm .o oHan

53

 

O O OO OOH ONO OON O O OO HOHOOO SOENONOOOOOONO .O
O O OO OOH OOO OON O O N HOOOOO EOEOONOONOOOOO .O
O O OOH OO OOO OHN O OON OOO HOOOOO EOENONOOOOOOOO .O
O O OON OOH ONO OOO O OOO OOO HOOOO OOENONOONOONOO .O
HOONOO
OO OOH ONO OOO OO O OO EOOOOHNOOO .O OOOOOON .O
HNONOO
OOH OOH ONO ONO OO O OO EOOOONNOOO .O OOONOON .O
HOOONO
O O OO ONH ON OON OO O OO EOOOOHNOOO .O OOOOOON .O
O O ONO OOH OOO OON OOO O ON HOHOOO OOONOON .O
O O OO ON OOO OON O OH ON HOOOOO EONOOOOOO .O
O O OOO ONH ONO OON OO OOH OON HOOOOO EOOONOOOO .O
O O OHN ONH OOO OOO O OO OOH HOOOOO EONONOOOO .O
O O ONH OHH OOO OOO O OOH ONH HHOOOO EOOONOOOO .O
O O OON OO OOO OOO O OO OOH HOOOOO EONOOOOOO .O
O O OO OO OOO OON O O OON HOOOOO OOOOONOOOOOONO .O
O O OOH OO OOO OO O O O HOOOO OOOONsoemoesNO .O
old ado. ede oDd ONd do. de dV dV Emwcwweo
SOON" Inna Ian. SOON” .1. N ”N8 N71 ”NI "NO
1nd Edl Edd ldd .Ed d1 dd dva. dI.
91 SSE SSU. 31 ED SE SU. d. db.
1,! e ._ o .E JOA m.e E . use 8 e
E3 . EI I.1. S S. S S
S SI. UK 8 9 3 e
a 33 9.1

 

HOOOH x OOOONOOHO NOOONEO OHO .O.OO
OHH>HHU<

 

H.O.NeouO O OHOON

54

 

O O OOO OO OOO OOO O OOH ONN HH;<O EOOONOEOOOO .O
O O OOH OO OON OOH O O O HOHO .OO EONOOOOOOONO
O O OO ON ONN ONH O O O HOHO .OO EOOOONOOOOHO
O O OHH O ONN OOH O O OO HNHO .OO EOONONOOOOOO
O O ONN OO OON OOH O O O HHHO .OO EONNOOOOOONO
O O OOH O ON OOO O OO OOH HOHO .OO EOONONmoeeNO
O O ONH O OON OOH O O OO HOO .OO EONNOOmoesNO
O O OO O OOO OOH O O O HOO .NO EONNOOOOOONO
O O OOO OO OOO OOH OO OO O HNO .OO estosmoesNO
O O OON OO OOO OON O O O HOO .OO EONNONOOOONO
O O ONN OO ONO OHO O OO O HOO .OO EONNONOOOONO
O O OOO OO OHO OOH O ON O HOO .OO EOONONOOeOHO
O O ONO OO OOO OOH O ON O HOO .HO EONNONmoeeNO
O O OO O OOO OOH O OO OO HNO .OO esNNONOOOONO
O O OHO O OOO OOO OOH OO OO HHO .HO EONNONOOeOHO
O ON OOO OOH OOO OOO ON OON OO HNHOOOOOENONOONOOONO .O

add add. Ede 93d DNd do. do dV dV EmwcmMHo

SEN INS INI SEN WIN Na NI NT. N3

1nd Edi Edd 1dd .Ed d1 dd dvfl dt.

81 SSE SSU: 91 E3 SE SU; d. dp

1T: 8 .. 8.E l/A me E. EE E E

E3 . ET. .L.1. S. S . S S

S ST: UrA 9 9 9 3

9 93 GT.

 

HOOOH x NOoHNOOHO NOOONEO OHO nn.oO
xufi>fipo<

 

H.O.N=ouO O OHOON

55

 

 

O O O OOH ONN ‘O O O ON NOUOOOO EOOOOOOOOOO .O
Apouwpmmv
O O O OOO OO O O O O EOOOOOONNOO .O
mpfimau
o o oom mm oom omo o mov com .umv Exwwgmsxuzo .9
o 0 com ON omm moo 0 mo mmH OON-mV Exxwmmsxuxo .9
91d edq Ede 33d DNd da. de dV dV EmwcmMHo
SEN INS INT. SEN n1.N Me NT NI NO
1nd edl. OZSu {ad .ed 1. Tgu dX d?
91 592 SSH 91 93 99 DH d. dP
It. a .. 9.? 17A me E. 89 E E
93 _ BI Trl. S S. S S
S St. HA 9 a a e
9 93 GT.

 

NOO>HOu<

,hOOOH x OsoaNOsHO NOOONEO OHO .muoO

 

H.O.u=oov O OHOOO

S6

 

    

 

40* 1. acid phosphatase ' :
35, 2. alkaline phosphatase I
w 3. ‘alpha-D-glucosidase j
30 4. beta-D-glucosidase
5. N-acetyl-beta-g1ucosaminidase ;
25” 6. caprylic esterase I
7. alpha-D-galactosidase ,
8. beta-D-galactosidase ;
20” 9. lauric esterase , i
No enzyme activity of no. 8 I /
E; I f
O f I
O .
H I /
15‘x I f
u I ’
8 4 . I
.c - I I
\ , -
3° ' -'
‘10”; {I l-’ 6 “s
in I I.
cu : .
m ' !
(U I/
\ .
5 ,' , 44
c3 ; / P. jeanselmei é
'O‘ I/ U
. .5
Q. “’3
c:
s.“ 3
3 2
.H .3
:5 v2
:3 2
U .2
“’ 8
Q) 01
E
N
C.‘
d)

 

 

 

Figure 2. Comparison of extracellular enzyme activi—
ties and growth rate of Phialophora jeanselmei (msu) human
pathogen grown at 25°C on solid CPYG medium.

57

Table 7. The diameter measurements of colonies of P.
.jeanselmei, P. fastigiata, C. cucumerinum
(F-26) and C. oxysporum (9496) for determina-
tion of growth rate, when grown on solid CPYG
medium at 25°C for 33 days

 

Colony diameter in cm.

 

 

P. jean- P. fasti- C. oxy- C. cucu—

seZmei giata sporum merinum

Days (msu) (8008) (9496) (F-Z6)
3 0 7 0.3 0 S 0 7
6 1 5 0.8 1.9 2.5
9 1.9 2.1 3.4 4.0
12 2.4 3 3 4.0 4.4
15 3.0 3.8 4.2 4.5
18 3.4 4 7 4.5 4.6
21 3.5 S 4 4.5 4.5
24 3.6 5.6 4.6 4.5
27 3.6 5.8 4.6 4.5
30 3.6 6 0 4.7 4.5
33 3.6 . 6.0 4.7 4.5

 

58
other enzymes, alpha-D-glucosidase and alpha-D-galactosidase,
also increased modestly after 15 to 25 days of fungal growth.

Comparisons of the growth rate and changes in enzymatic
activities with twelve PNP-derivative substrates for the
Phialophora and Cladosporium saprobes and plant pathogens
P. fastigiata (8008), C. oxysporum (9496) and C. cucumerinum
(F-26) are demonstrated in Figures 3, 4 and 5, respectively.
These fungi were representative of their groups.

Beta-D-glucosidase showed a high rate of activity for
P. fastigiata (8008) at the beginning of the colony growth,
and increased up to 25 days as seen in Figure 3. Acid and
alkaline phosphatase and‘N-acetyl-betabg1ucosaminidase
increased in amount after exponential phase between 15 and
25 days. The alpha-D-galactosidase gradually decreased in
activity after 15 days.

Figures 4 and 5 illustrate the relationships between
growth rate and enzymatic activities of Cladosporium oxysporum
(9496), a saprobe, and 6. cucumerinum (F-Zo), a plant patho-
gen. Both beta-D-glucosidase and N-acetylebeta-glucosamini-
dase increased rapidly at the beginning of colony growth and
continued through 15 days. Caprylic esterase and acid phos-
phatase activities were greater in the first 15 days in C.
cucumerinum (F-26) than in 0. oxysporum (9496). Both fungi
had no detectable alpha-D-glucosidase and beta-D-galactosidase
activities. Alkaline phosphatase and lauric esterase were
detected on the 25th day only for C. cucumerinum (F-26),

while only caprilic esterase and alpha—D-galactosidase were

 

 

59

 

 

4o” 1. acid phosphatase ' I
350 2. alkaline phosphatase I
300 3. alpha-D-glucosidaSe /
4. beta-D-glucosidase ‘ I
S. N-acetyl-beta-g1ucosaminidase /
250 6. caprilic esterase /
7. alpha-D-galactosidase /
8. beta-D—galactosidase /
2 9. lauric esterase I
04’ I
No enzyme activities of 40/
no. 3, 6, 8, and 9 j
I
0,.
15“ .’
I P. fastiml v.
I _ 1.6
0’.
ON I
8 .I
10‘ S . i, .5
>< /' .
$3 ,/ 5
£12 ./ :
H\~ / ‘ °Ht 4
>00 -
H: / 34
PH /‘ 0
up. . “
co / 9’
3.. . E
w ./ .2
Efi? .’ vu* 3
anN ./
5 E .I E‘
’ 2
<3 .
/
S I. S ./ 8 .2
/' .-"
Q .I. /-'/ .l ’
8 I! x ’0. 1/.//
/. a / ./' //
. I ./ 2/o 14 1
./ /.-"" -//’/
/' LO"”" . “/n/
'l.’--’ ::______ __H -— ’// 7
- ..-- v -/ v ‘v v
3 6 9 112 15 18 21 24 27 30
days
Figure 3. Comparison of extracellular enzyme activities

and growth rate of Phialophora fastigiata (8008) saprobe
grown at 25°C on solid CPYG medium.

60

 

 
    

 

40+1. acid phosphatase
3502. alkaline phosphatase /
3. alpha-D-glucosidase ’
30’4. beta-D-glucosidase /
5. N-acetyl-beta-glucosaminidase /
6. caprylic esterase f ,/
25’7. alpha-D-galactosidase ,.["
8. beta-D-galactosidase ,/””/
19. lauric esterase .z”’ 5
20' f’ /
No enzyme activities of- /
no. 3, 8 and 9 I ’
/ I
/ "
1s~ E? 4. s/'
O / .°
3 ° I
O I r
.2" f ./
. ‘. I '
1o Sii. / ./ 5
3 23° . ,/ C. oxysporum
O1 / a
max ,
EN 5'
N :6 W4
:tm .
a>m :
N. // .O
E a
c: .’ 3
H 6/ 0’3
v- E
CE!
0 -H
5* Q “U
0 >~
v I:
x ' " 2’2
I / I
/ \ ‘ / o
/’ /K\~ V\\ U
/ w< \
a / 7/ \\ ‘ 1
//’ ‘\

 

 

3 6 9 i2 15 is z1 24 27 30
days

Figure 4. Comparison of extracellular enzyme activi-
ties and growth rate of Cladosporium oxysporum (9496)
saprobe grown at 25°C on solid CPYG medium.

acid phosphatase
alkaline phosphatase,
alpha—D-glucosidase
beta-D-glucosidase
N-acetyl-beta-
glucosaminidase
caprylic esterase
alpha-D-galac-
tosidase
beta-D-galac-
tosidase

lauric

esterase

A
v

40

30"

20'

0 00 \IO‘ U'I-kCNNH
O

15 ,No enzyme
activities of
no 3 and 4 5

8. - ///\
//

61

/\

./'
,/
4 ./

,z’
/<____§______.

i-/ ON

 

E ./ \\/-
10" 3 6/.’ /\ .
, / .C. cucumerznum

x -’ . _
S / / \. 5'
W ..I . . U
:gég .‘/ // \\. .EL
.55? /! Z/ ‘\ S
+:n. , / u
SE. / /
.EQ / / // g
5 ”N I / / A
$5 / . 2/ 9/ g
E; ./ ///’ / ' .30
. / U

/ / ,

Q / / // .
°/ / // J.

’\

/

3 6 . 9 112 15

 

18 21 24 27

 

days

Figure 5. Comparison of extracellular enzyme activi-
ties and growth rate of Cladosporium cucumerinum (F-26)
plant pathogen grown at 25°C on solid CPYG medium.

62
presented in the assay for the 25-day culture of C.

oxysporum (9496).

2. Survey for extracellular enzymatic activity in
CPYC'liguid'medium

 

 

Peroxidase activity,

 

The results of extracellular peroxidase activity
of human pathogenic Phialophora, Fonsecaea and Clado-
sporium species are shown in Table 8. The difference in
peroxidase activity was varied, with no distinctive enzym-
atic pattern among these genera. Phialophora jeanselmei
(msu) showed a high activity in 15 days, while 0. carrionii
had high activity at all times. Great variation of
peroxidase activity was also found in individual strains
of the fungi. The pattern of enzymatic activity was some-
what similar for all strains of Phialophora and Cladosporium
at 5, 10 and 15 days, as seen in Table 9. Two strains of
Cladosporium sp., number 5 and 6, had high enzymatic
activity in 5 days with decreasing activities in 10 and 15
days. A third isolate, C. cucumerinum (St. C1air),had

the highest peroxidase activity in 5-day cultures.

..

Adenosine triphosphatase activity

 

Glucose was used as a carbon and DL-Isoleucine
and L-proline were used as nitrogen sources for West's
liquid medium.
The extracellular adenosine triphosphatase activity
in 7-day cultures showed variation from no activity to high

enzyme activity for some strains, as shown in Tables 10

63

 

 

 

Table 8. Extracellular peroxidase activity of Phialophora,
Fonsecaea.and.Cladosporium human and animal
pathogenic species grown at 25°C in liquid CPYG
medium

Enzyme activity
L9.D. 485 nm/ml./hour x 1000)
Organism 5 days 10 days 15 days

P. verrucosa (ok) 210 330 660

P. verrucosa (mac). 330 210 270

P. verrucosa (tex) 510 210 480

P. verrucosa (264) 390 240 360

P. verrucosa (269) 450 540 150

P. jeanselmei (msu) 450 S40 1350

P. jeanseZmei (bon) 540 660 450

F. pedrosoi (belo) 300 260 450

F. pedrosoi (msu) 300 570 540

F. pedrosoi (259) 360 510 780

F. pedrosoi (tex) 260 390 570

F. pedrosoi (261) 270 240 270

F. pedrosoi (262) 390 900 150

F. compactum (msu) 270 510 510

F. compactum (bon) 210 360 90

F. dermatitidis (msu) 240 360 660

C. carrionii (g8) 180 660 270

C. carrionii (g9) 1200 1680 3600

C. carrionii (tex) 300 510 270

C. trichoides (msu) 60 360 690

c. trichoides (glo) 90 330 450

Phialophora sp. (E-286) 510 420 780

 

64

Table 9. Extracellular peroxidase activity of Phialophora
and Cladosporium saprobic and plant pathogenic
species grown at 25°C in liquid CPYG medium

 

Enzyme activity
(O.D. 485 nm/m1./hour x 1000)

 

 

Organism. 5 days 10 days 15 days
Cladosporium herbarum 300 330 150
(pear)
C. herbarum (3167) 300 810 330
C. cZadosporiodes (489) 270 510 600
C. cZadosporiodes (9485) 330 480 390
C. oxysporum (9466) 210 210 480
C. oxysporum (9481) 450 390 420
C. oxysporum (9489) 600 900 570
C. oxysporum (9495) 300 420 300
C. oxysporum (9496) 210 900 360
c. resinae f. aveZZaneum 360 480 300
(7998)
C. resinae f. aveZZaneum 360 270 840
(8013)
C. resinae f. aveZZaneum 270 270 390
(9257)

C. resinae f. aveZZaneum 270 390 390
(9258) '
C. sphaerospermum (556) 420 240 420
C. sphaerospermum (8050) 300 210 360
C. sphaerospermum (9494) 660 1530 360
C. sphaerospermum (9516) 210 360 720
C. sphaerospermum (9517) 420 2400 750
C. cucumerinum Arl 720 510 720
C. cucumerinum (F-26) 450 690 480
C. cucumerinum (St. Clair) 1740 600 840
C. carpophylum (apricot) 240 450 390
C. carpophylum (peach) 270 660 270

65

 

 

 

Table 9 (Cont'd.)
Enzyme activity
(9.D. 485-nm/m1./hour x 1000)
Organism 5 days 10 days 15 days

Cladosporium sp 1 270 270' 240
Cladosporium sp 2 570 600 810
Cladosporium Sp 3 660 390 330
Cladosporium Sp 4 570 570 330
Cladosporium Sp 5 1500 780 240
Cladosporium Sp 6 1560 360 450
Cladosporium sp 7 990 270 240
Cladosporium sp 8 510 570 330
Cladosporium Sp 9 510 330 840
Cladosporium Sp 10 330 420 300
Cladosporium sp 11 990 540 300
Cladosporium sp 12 480 210 270
Cladosporium 5p 13 600 210 330
Cladosporium sp 14 480 150 210
Cladosporium Sp 15 540 570 630
P. richardsiae (263)- 270 270 240
P. richardsiae (6808) 30 180 300
P. fastigiata (265) 750 810 420
P. fastigiata (8008) 360 570 720
P. meZZinii (266) 510 360 420
P. Zagerbergii (267) 420 360 240
P. obscura (268) 450 420 270
P. malorum (1487) 630. 570 630

 

66

Table 10. Extracellular adenosine triphosphatase of human
and animal pathogenic species Phialophora,
Fonsecaea and Cladosporium grown at 25°C in
West's liquid medium with L-proline as the
nitrogen source

 

Enzyme activity
(O.D. 680 nm/ml./hour x 1000)

 

 

Organism . 7 days 14 days 21 days 28 days

P. verrucosa (0k) 150 10 50

P. verrucosa (mac) 0 0 0

P. verrucosa (tex) O 190 10 20
P. verrucosa (264) 0 0 0 0
P. verrucosa (269) 0 30 0 0
P. jeanselmei (msu) 0 80 0
P. jeanselmei (bon) 0 0 0
F. pedrosoi (belo) o 170 170 o
F. pedrosoi (msu) 0 0 0
F. pedrosoi (259) O O 0
F. pedrosoi (tex) 0 190 10 20
F. pedrosoi (261) 0 0 0 0
F. pedrosoi (262) 0 0 0 0
F. compactum (msu) 70 100 30 0
F. compactum (bon) 0 0 0 0
F. dermatitidis (msu) 0 0 0 0
C. carrionii (g8) 70 0 0 0
C. carrionii (g9) 0 0 0 0
C. carrionii (tex) 30 0 0 0
C. trichoides (msu) 30 0 0 0
C. trichoides (glO) 0 0 O
Phialophora Sp. (E4286) 0 0 0

 

67
and 11. The activity of adenosine triphosphatase was
detected in a few additional strains at 14, 21 and 28 days
of growth. Most of the twenty-two pathogens in the genera
Phialophora, Fonsecaea and Cladosporium showed no detectable
adenosine triphosphatase activity. In all cases the
adenosine triphosphatase activity was lower at 28 days than
at 21 days.

Fonsecaea pedrosoi (tex) showed high extracellular
activity at day 14 followed by decreased activity through
day 28 (see Table 10).. Two strains of P. verrucosa and two
strains of C. carrionii had marked increase in enzyme
activity by day 21 followed by a decrease on day 28 (see
Table 10). Cladosporium plant pathogens did show variation
in enzymatic activity from day 7 through day 28 with some
strains showing no detectable activity and others showing
adenosine triphosphatase activity at varied times in the
liquid cultures.

Upon changing the nitrogen source from L-proline to
DL-isoleucine it was found that there was a difference in
the enzymatic activity of the human pathogens. In Tables
10 and 11, for instance, F. pedrosoi (259) showed an
increase in the adenosine triphosphatase activity when
DL-isoleucine was utilized as a nitrogen source. No
activity was detected when L-proline was used as the
nitrogen source. Fonsecaea compactum (msu) showed greater
enzymatic activity when L-proline was utilized, and less
activity when DL-isoleucine was used. Similar results

occurred in several other species (Tables 10 and 11).

68

Table 11. Extracellular adenosine triphosphatase of human
pathogenic species Phialophora, Fonsecaea and
Cladosporium grown at 25°C in West's liquid
medium with DL-isoleucine as the nitrogen source

 

Enzyme activity
(O.D. 680 nm/ml./hour x 1000)

 

 

Organism 7 days 14 days 21 days. 28 days
P. verrucosa (0k) 0 0 40 20
P. verrucosa (mac) 0 0 40 20
P. verrucosa (tex) 0 O 0 0
P. verrucosa (264) 0 0 0 0
°. verrucosa (269) 0 0 0 0
P. jeanselmei (msu) 0 0 O 0
P. jeanselmei (bon) 0 0 0 0
F. pedrosoi (belo) 0 10 10 0
F. pedrosoi (msu) 0 0 0 0
F. pedrosoi (259) 0 40 80 20
F. pedrosoi (tex) 220 90 110 40
F. pedrosoi (261) O 0 0 0
F. pedrosoi (262) O 0 0 0
F. compactum (msu) 10 10 0 0
F. compactum (bon) 0 O 0 O
F. dermatitidis (msu) 0 0 O 0
C. carrionii (g8) 0 0 280 80
C. carrionii (g9) 0 O 30 10
C. carrionii (tex) 0 0 0 0
C. trichoides (msu) 0 20 0 10
C. trichoides (g10) 0 0 0 0
Phialophora Sp. (E-286) 0 0 0 0

 

69

There was no enzymatic activity toward ATP in the
medium of C. cucumerinum with DL-isoleucine as a nitrogen
source, but high enzymatic activity was observed with L-
proline was utilized. Similar results occurred in the other
species, including C. carpophylum and C. cZadosporioides
(see Tables 11 and 12).

In conclusion, there were no differences seen in the
adenosine triphosphatase activity in human pathogens and
in saprobes. The results were variable among species of
the fungi. Changing the nitrogen source from L-proline to
DL-isoleucine in the West's liquid medium did not show any
promising results. This will be discussed later.

Comparative Gel Electrophoresis of Intracellular
Soluble Protein, Enzymes and Isoenzymes

 

 

1. General soluble protein staining

 

The electrophoretic patterns of soluble proteins
extracted from mycelia-of fourteen-day-old cultures of
twenty-two human pathogens of PhiaZophora, Fonsecaea
and Cladosporium species and eight saprobes were compared.
The amount of the extracted proteins from the saprophytic
mycelia was found.to be lower than that of the extracted
proteins obtained from the human pathogens both from 14-
day-old cultures. The amount of protein per 1 mg. of
dried acetone powder varied from 100 to 200 pg. in the
different isolates.t A large amount of acetone powder was
utilized in order to get the same amount of soluble proteins

from these saprobes for running electrophoresis. There

70

 

 

 

 

 

Table 12. Extracellular adenosine triphosphatase activity
of some Phialophora and Cladosporium saprobic
species in West's liquid medium with DL-isoleucine
or L-proline as nitrogen sources

Enzyme activity
(O.D. 680 nm/ml./hour x 1000)
Organism 7 days 14 days 21 days 28 days
DL-isoleucine
C. resinae f. aveZZaneum 0 10 0 0
(2958)

C. cucumerinum (F-26) 0 0 0 0

C. sphaerospermum (8050) 0 0 0 0

C. cZadosporiodes (489) 0 30 60 0

C. carpophylum (peach) 0 20 0 0

Ljproline

C. resinae f. aveZZaneum 10 0 0 0

(2958)

C. cucumerinum (F-26) 0 190 100 80

C. sphaerospermum (8050) O 0 0 0

C. cladosporiodes (489) 0 0 270 100

C. carpophylum (peach) 120 10 40 0

P. obscura (268) 0 50 80 10

P. meZZinii (266) 0 0 O 0

P. molorum (1487) 0 0 0 0

P. richardsiae (6808) 50 0 150 10

P. Zagerbergii (267) 30 0 20 0

P. fastigiata (265) 0 0 0 0

P. fastigiata (8008) 0 0 O .0

 

71

was enough protein for.detecting enzymatic activity with
the specific substrates. The saprobes used for comparison
with pathogens included C. resinae (7898), C. spherosproum
(9494), P. richardsiae (6808), P. richardsiae (236), P.
fastigiata (265), P. Zagerbergii (276), C. cucumerinum
(F-26) and Phialophora species (msu).

The migration patterns, migration distance from the
origin to the front, of-each.extracted protein were measured
and used for calculation of Rf values. The calculation was
based on the migration distance of bands divided by the
distance of front and multiplied times 10. The electro-
phoretic patterns of these fungi showed characteristic
bands varying in density, thickness and spacing enabling
identification of the individual isolate. The Rf values
were measured and calculated directly from distance on
gels. The number of bands were determined by Rf values and
also read out from the densitometer records. This Rf value
method gave number of bands almost as accurate as those
derived from densitometer records, even though many bands
were close to each other. However, not all isolates were
run by electrophoresis at the same time, so the use of both
methods helped in determination of the number of bands of
soluble proteins.

The Rf values of the twenty-two pathogenic fungi and
eight saprobes are shown in Table 13. The electrophoretic
patterns of all thirty isolates were drawn by using the Rf
.values and are seen in Figure 6. Photographs of some of

the gel electrOphoresis protein bands are shown in Figure 7.

72

 

 

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75

 

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76

 

 

 

 

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77

 

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78

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Electrophoretic pattern in acrylamide gels

of protein extracts stained with amido black for thirty

Figure 6.
pathogens and saprobes of species of Phialophora, Fonsecaea

and Cladosporium.

 

79

3 strains of C. carrionii
2 strains of C. trichoides
(left to right)

 

5 strains of P. verrucosa

 

Figure 7. Photograph of electrophoretic pattern of
soluble proteins on acrylamide gels that were stained with
amido black.

p 80
A number of matching bands can be seen in the genera,
although not all species may have the matching bands.
In some cases the matching bands extend through a portion
of the species of one genus to another genus.

Average Rf values of each protein band in the electro-
phoretic patterns were compared in all possible paired
combinations to determine the number of bands which had
equal Rf value for different organisms and strains. The
bands which were within one Rf value of each other were
considered matching.. The number of matching bands are
shown in Table 14. There are a greater number of matching
bands among intraspecies than the number of bands among
interpsecies. Three strains of P- verrucosa, including
P. verrucosa (Oklahoma), P. verrucosa (mac) and P.
verrucosa (tex) show a greater number of matChing bands
than two other.strains, P. verrucosa (269) and P.
verrucosa (264).

The list of thirty isolates used in disc gel

electrOphoresis.

No. 1 PhiaZophora verrucosa (Oklahoma strain, 0k)
2 P. verrucosa (McCurdy strain, mac)
3 P. verrucosa (Texas Univ., tex)
4 P verrucosa (Bonny, Natick, bon 269)
5 P. verrucosa (Bonny, Natick, bon 264)
6 P. jeanselmei (Michigan State Univ., msu)
7 P. jeanselmei (Bonny, Natick, bon 270)
F. pedrosoi (Belo Horizonte, belo)

9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

F.

F.

81

. pedrosoi

pedrosoi
pedrosoi

pedrosoi

. pedrosoi

compactum.

compactum

Fonsecaea dermatitidis

Cladosporium carrionii

C.

C.

P.

P.

carrionii

carrionii

trichoides

trichoides
cucumerinum

resinae f. avellanium
resinae f. aveZZanium
sphaerospermum
richardsiae
richardsiae
fastigiata

Zagerburgii

Phialophora sp.

(Michigan State Univ., msu)
(Texas Univ., tex)

(Bonny, Natick, bon 259)
(Bonny, Natick, bon 261)
(Bonny, Natick, bon 262)
(Michigan State Univ., msu)
(Bonny, Natick, bon)
(Michigan State Univ., msu)
(George, CDC, g8)

(George, CDC, g9)

(Texas Univ., tex)
(Michigan State Univ., msu)
(George, CDC, glO)
(DeZeeuw, F-26)

(Bonny, Natick, 9257)
(Bonny, Natick, 7898)
(Bonny, Natick, 9494)
(Bonny, Natick, 6808)
(Bonny, Natick, 263)
(Bonny, Natick, 8008)
(Bonny, Natick, 267)

(Univ. of Mich., E-286)

In Table 14, the number of matching bands of P.

verrucosa intraspecies appear to be greater than when com-

pared with the number of the bands of one strain to the

next strain.

At any rate, the number of matching bands of

P. verrucosa and F. pedrosoi appear to be less than that of

Fonsecaea intraspecies per se.

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83

The number of common bands of F. compactum and P.
verruéosa (see Table 14) are fewer than the number of common
bands seen between F. compactum and F. pedrosoi. There is
little difference in the number of matching bands of two
strains of P. verrucosa With P. jeanselmei and of P.
jeanselmei With F. pedrosoi.

Fonsecaea dermatitidis also shows a similar number of
matching bands with every strain of P. verrucosa, P.
jeanselmei, F. pedrosoi, F. compactum, C. carrionii and
C. trichoides. .

The number of matching bands of F. compactum and
F. pedrosoi is greater than with F. compactum and c.
carrionii. However, the number of matching bands appear
to be fewer when F. compactum is compared with F. derma-
titidis, P. verrucosa or With P. jeanselmei.

Cladosporium cucumerinum (F-26), a plant pathogen,
shows a greater number of matching bands when compared
with F. pedrosoi, C. trichoides and with C. carrionii,
but the number of matching bands is less with the human
pathogenic Phialophora species (Table 14).

The matching bands.between pairs of saprobes are less
in numbers than between pairs of plant pathogenic species.
The matching bands between saprobes and human pathogenic
strains are also less in numbers.

2. §pecific protein staining for intracellular enzxmg
and isoenzyme locations

 

,The developed bands of enzymes and isoenzymes with

1
specific substrates in gel electrophoresis showed more

84
varying number of bands among saprobes than among human
pathogenic strains. Uniform pattern of enzymatic activity

and of isoenzymes were seen in human pathogenic strains.

Peroxidase

 

-Peroxidase activity was shown as a brown-colored
band in gel electrophoresis when catechol was used as an
electron donor (see Figure 8). The Rf values of peroxidase
activity are shown in-Table 15. All five strains of P.
verrucosa showed a single band of peroxidase activity at
Rf 2.5. Each of the two strains of P. jeanselmei had
double bands for peroxidase activity, one at Rf 2.2 and
the other at Rf 2.5. ~Fonsecaeapedrosoi, F. compactum,

F. dermatitidis and C. carrionii showed a similar peroxidase
activity which was composed of two different bands: one

was at Rf 1.7 and the other at Rf 2.5. Three different
bands of peroxidase activity were observed in gel electro-
phoresis of C. trichoides. Phialophora and Cladosporium
saprobes showed a variation in the enzymatic activity as
illustrated by the varied location of representative bands

in Figure 9.

Alpha-and beta-nglucosidases

 

Human and animal pathogenic strains showed no intra-
cellular alpha-D-glucosidase activity. A rather low activity
was detected in Cladosporium resinae f. aveZZaneum (7998).

'The yellow colored band was developed for beta-D-

glucosidase activity, with P. verrucosa, P. jeanselmei,

85

 

Peroxidase

("H'nwnnfl

4.

Acid phosphatase

 

Amylase

 

Figure 8. Photographs of electrophoretic bands of
enzymes and isoenzymes with their specific substrates for
species of Phialophora, Fonsecaea, and Cladosporium.

86

Table 15. Migration distances of peroxidase, beta-D-
glucosidase and catechol oxidase isoenzyme
bands developed and stained in acrylamide gels
for 30 pathogens and saprobes

 

Migration distances

 

 

beta-D- catechol
Organism peroxidase glucosidase. oxidase
P. verrucosa (ok) 2.5 1.8 1.3
P. verrucosa (mac) 2.5 1.8 1.3
P. verrucosa (tex) 2.5 1.8 1.9
P. verrucosa (264) 2.5 1.8 2.8
P. verrucosa (269) 2.5 1.8 2.5
P. jeanselmei (msu) 2.2 1.8 2.1
2.5
P. jeanselmei (270) 2.2 l 8 2.1 3 5
2.5
F. pedrosoi (belo) 1.7 1.8 0 8 1.9
2.5
2. .8
F. pedrosoi (msu) 1.7 1.8 .9 2.1
2.8
2.5 3.0
F. pedrosoi (259) 1.7 1.8 .9 2.5
2.8
.0
F. pedrosoi (tex) 1.7 1.8 9 2.5
2.8
.5
F. pedrosoi (262) 1.7 1.8 0.7 1.9
2.5
2 S 3.0 3.4
2.8
F. pedrosoi (261) 1.7 1.8 0.8 1.9
2.5
0
F. compactum (msu) l 1.8 2 ;.9
.5
F. compactum (bon) 1 7 l 8 0.8 1.9
2.8

87

Table 15 (Cont'd.)

 

Migration distances

 

 

beta-D- catechol
Organism peroxidase glucosidase oxidase
dermatitidis (msu) l. .8
C. carrionii (g8) 1.7 .3
2.8
2 3.4
C. carrionii (g9) 1.7 2.3 2.8 3.0
4.4
2 4.8
C. carrionii (tex) l 7 2.3 l 3 1.9
2.8
2» 4.4
C. trichoides (msu) 1 1.7 1.8 21. 2.8
3.0
5 4.
C. trichoides (g10) l 6 1.7 1 8 l 1.9
2.8
2.5 3.4 3.6
Phialophora Sp. (E-286) 1.7 2.2 1.8 2.1
P. Zagerbergii (267) 1.7 2.2 1.8 0
richardsiae (263) 1.7 2.2 1.8 2.3 0
P. richardsiae (6808) 1.7 2.2 1.8 2.3 0
P. fastigiata (265) 1.7 2.5 1.5 0
C. resinae f. aveZZaneum 1.6 2.2 2.3 0
(7998) 2.5 3.0
C. resinae f. aveZZaneum 1.7 2.2 2 3 0.8 2 l
(9257) 2.5 3.0
C. sphaerospermum(9494) 1.7 2.2 1.5 0
2.5 3.0
3.7
C. cucumerinum (F-286) 1.9 2.2 1.5 1.8 O
3.0

3.7

 

88

Electrophoretic pattern in acry

Figure 9.
of peroxidase of soluble protein.extracts from thirty

pathogens and saprobes.

lamide gels

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OOONOOUOOOOOOUOO.O

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.

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mommvssrmsoszozo.b

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axouvwwzomnaoo.b
nmmvfiwxowasoo.b
mmwvmm=owhsoo.b
Owwmnmuossew.m
muonvszuoomsoo.m
mamevsxuoomsoo.h
ONONOOQOQOOOO.O
nHONvOoOosnmm.m
OOONOOQOQOOOO.O
Axouvmomoawom.m
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moaonvwomoawom.m
ncoavmeNomroo%.m
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89
F. pedrosoi, F. compactum, H. dermatitidis, C. trichoides
and Phialophora species (E286) showing a single band of
beta-D-glucosidase at-Rf 1.8 (see Table 15). CZadosporium
carrionii also showed a single band of the enzymatic
activity at Rf 2.3. Two isolates of P. richardsiae showed
two different bands of isoenzymes, one at Rf 2.3 and one
at Rf 1.8. A single band of the enzymatic activity at Rf
1.5 was detected in Cladosporium saprobes and in P.
fastigiata. The pattern of beta-D-glucosidase and its

isoenzymes is illustrated in Figure 10.

Catechol oxidase

 

The activity.of this enzyme was shown as multiple
bands in gel electrOphoresis for most of the strains.
Human pathogens gave multiple bands of the enzymatic
activity which were noted in different locations (see
Table 15). A uniform.pattern of this enzymatic activity
was not encountered.in the pathogenic strains. Saprobes
and plant pathogens showed no enzymatic activity, except
for C. resinae f. aveZZaneum (7998) which gave double
bands of the catechol oxidase. The number of enzymatic
bands appeared to be greater in F. pedrosoi and Clado-
sporium pathogenic strains than for P. verrucosa and P.
jeanselmei. Fonsecaea dermatitidis had two bands out of

three in common with F. compactum (Figure 11).

Acid phosphatase

 

Acid phosphatase activity was seen as white bands

in acrylamide gels after the staining procedure (Figure 8).

90

Figure 10.
D-glucosidase of soluble protein extracts from the thirty

pathogens and_saprobes.

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Electrophoretic pattern in gels of beta-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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1 2 3 4 5 6 7 8

92

Twenty-two pathogenic strains showed a common straight
band at Rf 3.5 and multiple bands of isoenzymes among
species of pathogens (Table 16). A rather non-uniform
enzymatic pattern was found among saprobes. Multiple
bands were seen at different Rf values and this is illus-
trated in Figure 12. Five isoenzymes were encountered for

acid phosphatase in P. verrucosa (tex).

Alkaline phosphatase

 

An orange-red color was developed upon the addi~
tional substrate when alkaline phosphatase activity was
present in the acrylamide gels. The Rf values are recorded
in Table 16. The enzymatic patterns are illustrated in
Figure 13. There were four isoenzymes detected in the
twenty-one isolates.- Twenty-one pathogenic strains gave
two common straight lines of alkaline phosphatase activity
at Rf 1.5 and 3.3. .The intracellular alkaline phosphatase
activity seen in pathogenic species and in saprobes
appeared distinctively different from the extracellular
alkaline phosphatase.activity of human pathogens previously
reported as very low, with none detected when the organisms

were grown on solid CPYG medium.

Esterase

Blue-purple bands of esterase activity were
developed in acrylamide gel electr0phoretic column after
incubation with the esterase specific substrate. Five
bands of isoenzymes of esterase were detected as shown in

Figure 14 and Table 16. No esterase activity was found in

93

 

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94

 

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96

 

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Electrophoretic pattern in acrylamide gels

 

mxouvwomoawom.m

 

namEvmomoawom.m

 

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Figure 12.

mmowvemooxhsoa.m

 

axouvomooxssoa.m _ _ _

 

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of acidphosphataseof soluble protein extracts from thirty

pathogens and saprobes.

 

 

Oxogemooxsnma.m ooemumfla :OOHOOMOE

 

 

1 2 3 4 5 6 7 8

97

'Electrophoretic pattern in acrylamide gels

of alkaline phosphatase of soluble protein extracts from

thirty pathogens and saprobes.

Figure 13.

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6 7 oo

 

98
Electrophoretic pattern in acrylamide gels

Figure 14.
of esterase of mycelial soluble protein extracts from

thirty pathogens and saprobes.

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mamevonNomreo%.m
mvomvomooxssoo.m
mmoNUemooxssme.m
mxouvomoozsgma.m
mumevoeoonasoa.m
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7 8

B* - The dark blue band was developed when alpha-naphthyl
acetate was used for esterase specific substrate.

 

99

F. jeanselmei and F. compactum. Enzymatic bands with the
same Rf values were.abserved for P. verrucosa isolates.
Phiazophopa verrucosa isolated from wood pulp showed five
bands for esterase.while P. verrucosa isolated from human
infection had one to.five bands for esterase. The enzyma-
tic band seen at Rf 3.8 was a common band among most
pathogenic strains.- Six out of eight strains of saprobes
showed no esterase activity. The remaining two strains,
C. resinae f. aveZZaneum No. 7998 and No. 9275, had a
single band each.

The same Rf values for the enzymatic bands are seen
in F. dermatitidis, C. carrionii, C. trichoides and
Phialophora sp. (E?286L which was isolated from-a frog.
There was an unidentified dark blue band recognized in

C. trichoides and in F. dermatitidis (see Figure 14).

Amylase

Colorless bands of amylase were noted in the
blue gel electrophoretic column after staining the column
with Gram's iodide as.a-control. The large starch mole-
cule was digested with amylase and the products gave no
color with Gram's.iodide staining (Figure 8, lower picture).

A few of the human and animal pathogens showed amylase
activity. These included P. verrucosa (tex), C. carrionii
(tex), Phialophora sp. (286), and two isolates of P.
jeanselmei. All.eight isolates of saprobes showed this
enzymatic activity.. The maximum number of isoenzymes

detected among theSe thirty isolates was four bands. The

100
Rf values and diagrams of the enzymatic bands are shown
in Table 16 and in Figure 15, respectively. Determination
of amylase activity.in the mycelial extracts of these fungi
was carried out by using liquid CPYG medium in the previous
experiment but failed to show any promising results.

Comparison of Enzymatic Common Bands Between
TPairs of Pathogens and Saprobes

 

 

Comparison of enzymatic bands or enzymatic activity
of all tested enzymes, including acid phosphatase, alkaline
phosphatase, betaeD2g1ucosidase, peroxidase, catechol
oxidase, esterase and amylase, of the thirty isolates are
shown in Table 17. Sequential combination of this table
would give a complete chart of pair comparative gel electro—
phoresis of the seven enzymes. Several pathogens, includ-
ing F. pedrosoi (261) and C. trichoides (g10) had an
especially high number of common bands for all strains.
A greater number.of.enzymatic matching bands were found
among human pathogenic strains. A lesser number of the
matching bands was seen among saprobes, animal pathogens
and among plant pathogens. The results of the enzyme
patterns here show somecorrelation to the previous results
of soluble protein patterns seen in Table 14. The number
of enzymatic matching bands were greater in intraspecies
organisms than for-interspecies organisms, including: P.
verrucosa, P. jeanselmei, F. pedrosoi and F. compactum.
Cladosporium carrionii, C. trichoides and F. dermatitidis

showed closer relationship to F. pedrosoi when the matching

101

Electrophoretic pattern in acry

Figure 15;

lamide gels

of amylase of mycelial soluble protein extracts from thirty

pathogens and saprobes.

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OOOOOUeOeOOOOOeO.m
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OH O O O O O N N O O N N O N N N O N N O

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OH O O O O 5 O O O O O O O

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NH O O O O O O O O O O

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103

bands of the enzymes-were compared on the bases of intra-
species and interspecies.

Table 17 shows.that six pairs of matching enzymatic
bands occurred between P. jeanselmei and each strain of
F. pedrosoi. The matching bands of all seven enzymes of
F. pedrosoi and P. verrucosa were similar to that of P.
verrucosa and Cladosporium pathogenic strains.

Phialophora sp..(286) showed a great number of match-
ing bands with P. jeanselmei, C. cucumerinum (F-26), C.
resinae (9257) and with P. richardsiae No. 6808 and No.
236. The plant pathogen of cucumber, C. cucumerinum (F-26),
also showed a great number of matching bands with those of
human and animal pathogens. The intraspecies of pathogenic
strains showed an equal number of matching bands with the

plant pathogen as indicated in Table 17.

DISCUSSION

Extracellular Enzymatic Studies

The dematiaceous pathogenic fungi, P. verrucosa, F.
pedrosoi, F. compactum, F. dermatitidis, C. carrionii, C.
trichoides and P. jeanselmei, showed low extracellular
enzymatic activity in most strains with twelve paranitro-
phenol derivatives, including the alpha-D-glucoside, beta-
D-glucoside, alpha-D2galactoside, beta-D-galactoside,
N-acetyl-beta-glucosaminide, caprylate, stearate, laurate,
palmitate and butyrate. Low acid phosphatase and alkaline
phosphatase were also observed when the organisms were
grown fknr 15 and 25 days at 25°C on solid CPYG medium,
which was considered to be a good synthetic medium for
production of extracellular enzymes of BZastomycee derma-
titidis (Beneke et aZ.,.1969). The extracellular enzymatic
activities of forty-five dematiaceous fungi that are
saprobes and plant pathogens, including Phialophora species
and Cladosporium species, were high in most cases at 15
days of mycelial growth in the medium and usually increased
through 25 days on the same medium.

The lack of detectable or low extracellular enzyme
activities for the human and animal pathogenic dematiaceous
fungi in contrast to the higher extracellular enzyme activities
for the saprobes and plant pathogens is similar in pattern

104

105

to other reports in the literature. Montemayer (1949) found
from the studies of biochemical activities of the fungi
that none of these, including F. compactum, F. pedrosoi,
P. jeanselmei and P.-verrucosa, could liquify gelatin,
Loeffler's serum or coagulated milk, in contrast to the
positive activity for-two.saprophytic Cladosporium isolates.
In 1952 De Vries also reported that C. carrionii and C.
trichoides did not exert proteolytic activity on Loeffler's
serum while on the other hand.extensive activity occurred
with the saprophytic species of this genus. In addition,
he found that twentyefour saprophytic species of Clado—
sporium produced tributyrine hydrolyzing lipases in varying
degree whereas the.pathogenic strains showed no activity.

The extracellular.fatty acid esterase activities
varied in the organisms investigated. Activity toward
PNP caprylate was found in most of the pathogenic and
saprophytic species-while only an occasional strain showed
activity toward PNP lauric acid. None of the strains tested
showed any extracellular activity toward PNP derivatives
of butyrate, palmitate-or stearate. It is possible that
these enzymes may.be bound to the cell in some of the
strains of CZadosporium, Fonsecaea and Phialophora, and
do not escape into the medium unless cell disintegration
occurs or those which are liberated to a significant extent
into the medium are from intact cells under varied physio-
logical conditions of the organism. There may be good
reasons to believe that several cell bound enzymes are

situated outside the relatively impermeable barrier provided

106
by the cytoplasmic membrane, so only certain enzymes may
diffuse through pores of the cell wall as proposed by
Pollock (1962), who-called these kinds of enzymes "par-
tially cell-bound enzymes."

Mitchell andeoyle (1959) state that it is not sur-
prising that a small-amount of the potentially extracellular
enzymes should be detected on the outer layers of the cell.
The problem of the.enzymatic liberation would be devolved
upon the cell wall,.thus.partially or completely preventing
enzyme released from the cell. Thus, as Mitchell and Moyle
speculated, it would-be plausible to say that the extent
of enzymes produced would presumably depend upon the
properties both of the cell wall and of the enzyme per se.

Szaniszlo et a1. (1972) analyzed the chemical compo-
sitions of hyphal.walls of three chromomycotic agents,
including P. verrucosa, F. pedrosoi and C. carrionii, and
found that the hyphal walls were composed of glucose,
mannose, glucosamine, and protein components which presented
the similar amount of.the chemical compositions in these
three genera. No records of analyses of the chemical
compositions of the.hyphal wall of Cladosporium and
Phialophora saprobes exist, to my knowledge. The mycelial
mats of plant pathogens and saprobes were more difficult
to homogenize than those of human and animal pathogens.

It seems reasonable to conjecture the chemical compositions
of the cell walls-of plant pathogens and saprobes may be
different from those of the human pathogens. This may be

a factor in the variation that could account for the marked

107

differences in certain-extracellular activities between
the human pathogens,and-the plant pathogens and the saprobes.

Extracellular-enzymatic activity of intraspecies of
plant pathogens.and-saprobes, Phialophora and Cladosporium
species, showed a variation in relationship to one another
at 25 days of growth in culture. Phialophora saprobe
strains showed high activities for beta-D-glucosidase and
N-acetyl-Bdglucosaminidase, while no activity was detected
for the pathogenic strains. Cladosporium saprobe also
showed a high activity for beta-D-glucosidase, and N-acetyl-
D-glucosaminidase, whereas these activities were not
detected for the human pathogens. Both genera showed
similar extracellular enzyme patterns, except alpha-D-
galactosidase activity was high. This could possibly be
used for differentiation of Phialophora and Cladosporium
saprobes.

Since N-acetyl-beta—glucosaminidase was detected in
a rather high activity in Cladosporium and Phialophora
saprobes and none in human pathogens, it might be specu-
lated that the growth rate of the saprobes would be faster
than that of the pathogens. The speculation is based on
the fact that NeacetyI-B‘glucosaminidase will hydrolyze
PNP-N-acetyl-B‘glucosaminide and a free group of N-
acetyl-B-glucosaminidewill be released and directly
absorbed for chitin synthesis. It is reasonable to con-
clude that the rate of cell wall formation of the saprobes
is faster than that of the pathogens, and reflect in the

faster rate of growth of the saprobes. The observations

108

on the rate of growth seem to correlate with the observa-
tions in the work of Silva (1960), who found that rate of
growth of Cladosporium saprobes was faster than that of the
pathogens.

From the results of the twelve enzymes studied, it
was noted that there were differences of extracellular
enzymatic activity among Cladosporium interspecies. For
instance, five isolates of C. oxysporum showed extracellu-
lar alkaline phosphatase while four strains of C. resinae
and two strains of C. cZadosporioides had no enzymatic
activity. The combination of morphologic appearance of
these fungi and the differences in the enzymatic activity
may be good criteria for taxonomic classification of C.
oxysporum.

The relationship of increase in extracellular enzyma-
tic activity and growth rate in culture of all human and
animal pathogens, and.the plant pathogens and saprobes
except Fonsecaea pathogens were compared (Figures 2, 3,

4 and 5).

Most of the increased enzymatic activity, including
beta-D-glucosidase,.N-acetylabeta-glucosaminidase, caprylase,
acid phosphatase,.alkaline phosphatase and alpha-D-
galactosidase of.some of these fungi appeared to increase
during the logarithmic phase of growth at 15 days. This
evidence would indicate that extracellular enzymes were
liberated from the series of individual cells during the
normal growth and metabolism of the cells and not released

after cell autolysis (Pollock, 1962).

109

It is interesting-to note that P. jeanselmei, a human
pathogen, showed surprisingly low enzymatic activity through
the first 15 days of growth and rapidly increased enzymatic
activity right after the logarithmic phase of growth. The
increased enzymatic activities might have been due to
released enzymes from autolytic cells, which seems to be
less convincing because it is unlikely to assume that there
was an autolysis of the fungus right at the beginning of
the stationary phase. There may be a correlation between
the rapid rate of enzyme activity at about the lS-day growth
period and the change that occurs in the yeast type to
mycelial type colony after the first 10 days of colony

growth.

Peroxidase activity

 

There was no significant difference of peroxidase
activity seen among dematiaceous pathogenic and saprophytic

isolates.

Adenosine triphosphatase activity

 

According to a report by West (1967) the sclerotic

cells of P. verrucosa.(A:126) were formed when L-proline
was utilized as a nitrogen source. Basically about 60-70%
of total nitrogen of.the fungal wall is incorporated into
protein and a small amount into nucleic acids and chitin
according to Moore and Landecker (1972).

By using L—proline as a nitrogen source, it was found
that all five strains of P. verrucosa did form the sclerotic

cells. There was no significant relationship between

 

110
formation of the sclerotic cells and the release of adeno-
sine triphosphatase as three of the five strains of P.
verrucosa had some enzyme activity.

Fonsecaea and.CZadosporium species failed to form
sclerotic cells in West's liquid medium. However, sclero-
tic cells were formed.when species of both genera were
cultured on six synthetic-media by Silva (1967).

Again, West (1967) found there was no significant
difference in adenosine triphosphatase activity among the
fungi when DL-isoleucine was utilized as a nitrogen source
in experiments designed to get more mycelial mat weight
without formation.of.the sclerotic cells.

In conclusion,.it would be reasonable to propose that
there was no significant difference in adenosine triphos-
phatase activity between human pathogens and saprobes when
L-proline or DL-isoleucine was used as nitrogen source.

On the other hand, the ability to produce extracellular
ATPase in the fungi not only depends on the nutrient
utilized but also is dependent on the releasing capability
of the organism.

Comparative Gel Electrophoresis of Intracellular
Soluble Proteins

 

Intracellular extracts of chromomycotic
pathogens and saprobes

 

 

1. General soluble proteins stained with amido black
The results.showed that low concentrations of soluble

protein were extracted from mycelium of saprobes harvested

 

“4

111

at the same period of time on identical medium when compared
with those from pathogens. It seems possible to expect a
difference in the type of proteins obtained, which appear

to depend on the physiological activity of the cell at the
time of harvesting; since the soluble proteins reflect

the physiological state of the cell rather than morpho-
logical strucutre, it is reasonable to anticipate that the E1
saprobes not only have a variation in composition of the ‘4‘

cell wall but also have a physiological difference from

 

pathogenic species. '§.

It is possible to separate a mixture of globular
proteins in solution on the basis of their different rates
of migration in the electric field at a given pH. The
migratory distance depended on charges and sizes of the
protein molecule. The matching bands between a pair of
gel electrophoresis could be considered as similar kinds
of proteins in the different isolates.

Gel electrophoresis permits high resolution analysis
of extremely small samples of complex mixtures of proteins.
It is also used for detecting mutant forms of hemoglobin
and other proteins. .Lehninger (1970) stated that differ-
ence of a single charged group per molecule is sufficient
to distinguish the mutant from the normal form of protein.
Gel electrophoresis was an accurate method to use for the
study of interspecies.relationship in the protein bands of
the dematiaceous fungi.

Schechter (1972) devoted a great deal of effort to

making interesting studies on the difference in soluble

112
proteins of Candida.species by using gel electrophoresis,
although his diagrammatic interpretation seems to create
more complications than those of Shannon (1972), who
utilized starch gel electrophoresis for studying of intra-
cellular enzymes of Polyporus. The later diagrammatic
interpretation was, therefore, adopted here for the human
and animal pathogenic.species of PhiaZophora, Fonsecaea
and Cladosporium with the saprobic and plant pathogenic
species of Phialophora and Cladosporium.

The number of matching bands by gel electrophoresis
was low between pairs.of five strains of P. verrucosa and
six strains of F. pedrosoi (Table 14). The findings indi-
cate a distinctive difference of interspecies relationship
between F. pedrosoi and P. verrucosa.

The results of comparison of matching bands of soluble
proteins by gel electrophoresis showed a greater relation-
ship between F. pedrosoi and F. compactum than that of F.
compactum and P. verrucosa. The number of matching bands
was low between pairs.of five strains of F. pedrosoi and
six strains of F. pedrosoi. The findings indicate a
distinctive difference of interspecies relationship between
F. pedrosoi and P. verrucosa. This finding was comparable
to the serological studies of Conant and Martin (1937).
They found that the sera of rabbits immunized with Fonsecaea
(Hormodendrum) pedrosoi and F. compactum not only had a
high titer of complement fixing antibodies for their
respective antigens but also had a high titer for each

other, and reacted to appreciable degree only with the

113

homologous fungus. However, Martin et a2. (1936) demon-
strated a crOSSrantigenic relationship between strains of
P. verrucosa and Fonsecaea (Hormodendrum) pedrosoi. Promis-
ing results on relationships were demonstrated by Buckley
and Murray (1966), who utilized the agar gel-diffusion
test to determine that there was more cross-reactivity
between F. pedrosoi and F. compactum and very little with
P. verrucosa. Their.findings by the agar gel diffusion
method support the results obtained by gel electrophoresis
that there are similar kinds of protein molecules in the
matching bands between P. verrucosa and F. pedrosoi which
can serve as an antigenic protein for inducing antibodies.
If the matching bands of soluble proteins contained
antigen-determinant groups which can induce antibodies,
they could show antigenic relationship between them.

In another study Gordon and Al-Doory (1965) utilized
fluorescent antibody technique to compare relationships
of the dematiaceous fungi. They noted that there was very
little serological relationship between P. verrucosa and
F. compactum or Cladosporium sp. The conjugates of two
strains of P. verrucosa did not react with any species of
Fonsecaea. They also found that F. dermatitidis conjugate
did not react with any strain of P. jeanselmei, when using
the fluorescent antibody technique. However, by using disc
gel electrophoresis, there were three matching bands in one
strain and two matching bands in another strain of P.

jeanselmei with F. dermatitidis. One might conclude that

114

either the molecular size of these similar soluble pro-
teins are not big enough to serve as antigens, or they
lack capable antigenicity.

By using immunodiffusion (ID) and immunoelectrOphor-
esis (IEP), COOper et a1. (1970) and Biguet et az. (1965)
found that there were.more common antigens between P.
verrucosa and C. carrionii than between either these two
species and F. pedrosoi. They concluded that F. pedrosoi
should be retained in-the genus Fonsecaea. They found a
low number of different lines of identification between
pairs of C. carrionii - P. verrucosa and C. carrionii -
F. pedrosoi. On the other hand, C. carrionii showed a
closer relationship to P. verrucosa and F. pedrosoi than
to P. verrucosa and F. pedrosoi., These results were
comparable to the results obtained by acrylamide gel
electrophoresis. Moreover, there was a greater number
of matching bands for Cladosporium human pathogens,
Cladosporium saprobes and Cladosporium plant pathogen
with Fonsecaea pathogenic.species. These results might
suggest a closer relationship between Cladosporium patho‘
gens, Czadosporium saprobes and Fonsecaea sp. There are
a considerable number of matching bands between Cladosporium
saprobes and two species of Fonsecaea, including F.
pedrosoi and F. compactum as well as F. dermatitidis.
It would be appropriate to say that there was a close
relationship between Cladosporium saprobes and the two
species of Fonsecaea and F. dermatitidis. These results

appear to be comparable to those reported by Gordon and

 

 

115
Al-Doory (1965), who applied fluorescent antibody technique
to the study and comparison of three Fonsecaea sp., includ-
ing F. pedrosoi, F. compactum and F. dermatitidis and
Cladosporium saprobes. They found that all three Fonsecaea
sp. showed a considerable reaction with the saprophytic
Cladosporium sp. and F. dermatitidis was found to be most
closely reactive with saprobe Cladosporium.

Again, a lower number of matching bands was seen when
Fonsecaea pathogens were compared with Phialophora saprobes
and pathogens. The findings indicate a closer relationship
of Fonsecaea in). with Cladosporium sp. than to Fonsecaea
and Phialophora species.

It appears that comparable results are obtained from
both techniques, namely, fluorescent antibody test and
disc electrophoresis. Moreover, it appears that more
detailed findings can be obtained from the latter technique
than the former one.

Vaughan et a1. (1965) compared protein bands of
several Brassica species obtained by gel immunoelectro-
phoresis against those obtained by the relatively simple
acrylamide gel electrophoresis. They concluded that the
latter method was-at least equal to the former in resolution
of protein bands.- In addition, Lester et a1. (1963) found
unique protein bands were in electrophoresis patterns from
protein of Baptisia while no such difference could be
detected by the serological techniques. It would be worth-
while to compare the results obtained from disc electro-

phoresis with the aforementioned methods.

116

In a comparison-of.the various methods utilized for
the study of the relationships of the dematiaceous fungi,
there is similar evidence from agar gel-diffusion, immuno-
diffusion (ID), immunoelectrophoresis (IEP), fluorescent
antibody technique and by disc gel electrophoresis to
indicate that F. pedrosoi and F. verrucosa are less closely
related than F. compactum and F. pedroeoi.

2. Staining of enzymes and isoenzymes with their
specific substrates

 

The diagrammatic interpretation of Shannon et a1.
(1973) was also adopted for comparison of enzymatic and
isoenzymatic bands by adding the total number of similar
bands together for pairs of organisms.

There was a distinctive difference of enzymatic bands
nOted between pathogenic isolates and saprobes. A high
.number of matching bands of enzymes and isoenzymes were
shown among pathogens. .This may reflect a closer rela-
tionship of physiological activity of these human patho-
genic fungi.

Furthermore, it.was found that there are a greater
number of common bands in pathogenic intraspecies than in
pathogenic interspecies. These results are comparable to
those of the general soluble protein pattern, which offers
some basis of support for the taxonomic classification of
these species of fungi.

In order to find the relationship between Cladosporium
sp., Phialophora sp..and Fonsecaea sp., the number of

matching bands of specific enzyme and isoenzyme of these

117
species were then compared (Table 17), and it was found
that there was similarly.close relationship between the
human and animal pathogenic.CZadosporium sp. and Fonsecaea
sp. and between Cladosporium.sp. and PhiaZophora sp.
However, there were less common matching bands between
the plant pathogenic and saprobic Cladosporium sp. and
PhiaZophora sp. .The results are somewhat different from
those obtained by.general soluble protein staining, which
shows close relationship between Cladosporium sp. and
Fonsecaea sp. but less close between Cladosporium sp. and
Phialophora sp. .These findings with a higher number of
matching bands for.interspecies of Fonsecaea and a lower
number between Cladosporium species seems to correspond
with the work by Cooper (1970), who tried to show that
Fonsecaea was a distinctive genus and was included neither
in Phialophora nor in Cladosporium.

The results obtained from this study also show a
close relationship between F. dermatitidis and F. pedrosoi
when their matching enzymatic bands are compared. This
would support the current classification as Fonsecaea
dermatitidie.

However, Frank and Berry (1972) have indicated that
disc acrylamide.gel.electrophoresis is applicable to
species identification, although it seemed to be compli-
cated within.and even between genera. The patterns were
never so close by this.method as to contradict the tradi-

tional taxonomy;.when based on morphology according to

118
Martin and Alexopoulos.(1969), this is a statement that is
also applicable to the dematiaceous fungi.

In conclusion,.the determination of soluble proteins,
enzymes and isoenzymes.by-disc electrophoresis is considered
a sensitive technique-for the study of the relationship
of dematiaceous fungi. The difference of banding patterns
of soluble proteins could be obtained from the same organism
if they were grown under the different condition and
harvested at different times according to Shannon (1973).
Therefore, it is essential that standardized parameters
be used to provide valid results with disc acrylamide
gel electrOphoresis...

The results from the.use of disc acrylamide gel
electrophoretic banding patterns of general soluble pro-
tein, enzymes and isoenzymes have contributed to support
of the current concept of speciation of the dematiaceous
fungi. The differentiation of pathogens from saprobes
by determination of-the-presence or absence of beta-D-
glucosidase and N—acetyl:beta-glucosaminidase may be a
valuable method to include in the clinical laboratory
procedures. More strains of the pathogens and saprophytes
need to be examined-for.these.extracellular enzyme activi-
ties to see if the difference continues to exist between

the saprophytic species and the pathogenic species.

SUMMARY

1. All the saprophytic species of Phialophora showed
extracellular N-acetylrbeta-glucosaminidase activity. No
activity was detected in the human pathogenic species.

2. All of the.saprophytic species of Cladosporium
had extracellular N:acety1-beta-glucosaminidase activity.
No activity was detected in the human pathogenic species
of Cladosporium and-Fonsecaea.

3. All saprophytic species of Phialophora had extra-
cellular beta-D-glucosidase activity, while the pathogenic
species had none when assayed at 15 days.

4. All saprophytic species of Cladosporium except
C. carpophilum have extracellular alpha-D-galactosidase,
while no activity was detected in the pathogenic species
of Cladosporium, Fonsecaea, and Phialophora except for
one strain of P. jeanselmei.

5. All twenty2two pathogenic species and forty-five
saprobes were capable of releasing extracellular peroxidase,
when grown in.liquid CPYG medium at 25°C, at S-, 10‘ and
15-day intervals.

6. A few of the human pathogens and saprobe isolates
showed extracellular adenosine triphosphatase activity.
Some increase in ATPase activity in strains occurred by

changing the nitrogen source from DL-isoleucine to L-proline.

119

120

7. No relationship could be found between adenosine
triphosphatase and sclerotic cells in cultural studies as
the sclerotic cells found in the tissue phase did not
develop.

8. Comparative disc gel electrophoretic patterns of
soluble proteins showed little significant relationship
between human pathogens and saprobes.

9. There was significantly greater number of matching
bands of soluble proteins of pathogenic intraspecies than
for the interspecies.-

lO. Comparative gel electrophoretic patterns of
soluble proteins showed closer relationship between
Fonsecaea sp. and Cladosporium sp. than between Fonsecaea
sp. and Phialophora sp.

ll. Fonsecaea compactum showed the closest relation-
ship with F. pedrosoi on the basis of soluble protein
and enzyme gel electrophoretic patterns.

12. Enzyme and isoenzyme gel electrophoretic patterns
showed correlative relationship with the general soluble
protein patterns.

13. The evidences from the correlative relationships
of matching bands indicated that Fonsecaea dermatitidis is
closer to other species of Fonsecaea than to the former

genus Hormodendrum (Cladosporium).

BIBL IOGRAPHY

BIBLIOGRAPHY

Ajello, L., L. K. George, W. Kaplan and L. Kaufman. 1963.
Laboratory Manual for Medical Mycology. Public
Health Service Publication 994. U. 8. Government
Printing Office, Washington,.D.C.

A1:Doory,.Y..and. . A. Gordon. 1963. Application of
fluorescent antibody procedures to the study of
pathogenic dematiaceous fungi. I. Differentia~

tion of Cladosporium carrionii and Cladosporium
bantianum. J. Bacteriol. 86: 332-338.

Al—Doory, Y. 1972. Chromomycosis. Mountain Press Pub-
lishing Company, Missoula, Montana.

Ames, B. N. 1960. ‘Assay of inorganic phosphate, total
phosphate and phosphatase. Method in Enzymology,
Vol. VIII. Academic Press, Inc., New York, p.
115-118.

Area Leao, A. E. and A. Cury. 1950. Deficiencias vitamini-
cas de cogumelos patogenicos. .Mycopath. Mycol.
Appl. 5: 79-87.

Biguet, J. and S. Andrieu. 1963. Revue criteque des
travaux mycologiques de 1940-1961 (inclus) concern-
ant les territoires africains de culture Francaise.
Mycopath. Mycol. Appl. 19: 315-341.

Biguet, J., P. TranVanky, S. Andrieu, and J. Fruit. 1965.
Analse immunoelectrophoretique des antigenes
fongiques et systematique des champignons reper-
cussions pratiques sur 1e diagnostic des mycoses.
Mycopath. Mycol. Appl. 26: 241-256.

Bindo, F. X. and K. A. Griffin. 1968. Physiological and
biological identification of dematiaceous fungi
isolated from clinical material. Bacteriol. Proc.,
p. 88.

Binford, C. H., R. K. Thompson, and M. E. Gorham. 1953.

Mycotic brain abscess due to Cladosporium trichoides,
a new species. Amer. J. Clin. Path. 22: 535-542.

121

122

Bloemendal, H. 1967. .ElectrOphoresis, theory, method
and application. Vol. II. Academic Press, New
York, p. 379-416.

Brewbaker, J. L., M. O. Upadhya, Y. Makinen and T.
MacDoald. 1968.. Isoenzyme polymorphism in flower-
ing plants. 11. Gel electrophoretic methods and
applications. Physiol. Plant 21: 930-940.

Buckley, R. H. and I. G. Murray. 1966. Precipitating
antibodies in Chromomycosis. Sabouraudia 5: 78-80.

Carrion, A. L. 1942. Chromoblastomycosis. Mycologia 34:
424-441.

Carrion, A. L.. 1950. Chromoblastomycosis. Ann. N.Y.
Acad. Sci. 50: 1255-1282.

Carrion, A. L. and C. W. Emmons. 1935. A spore form
common to three etiologic agents of Chromomycosis.
Puerto Rico J. Pub. Health 8 Trop. Med. 11 1:
114-115.

Carrion, A. L. and M. Silva-Hutner. 1971. Taxonomic
criteria for the fungi of chromoblastomycosis with
reference to Fonsecaea pedrosoi. Intern. J.
Dermat. 10: 35-43.

Cole, T. G. and B. Kendrick. 1973. Taxonomic studies of
PhiaZophora. Mycologia 65: 661-687.

Conant, N. F. 1937. The occurrence of a human pathogenic
fungus as a saprophyte in nature. Mycologia 29:
597-598.

Conant, N. F. and D. S. Martin. 1937. The morphologic
and serologic relationships of the various fungi
causing dermatitis verrucosa (chromoblastomysis).
Amer. J. Trop. Med. 17: 553-569.

Conant, N. F., D. T. Smith, R. D. Baker, and J. L. Gallaway.
1971.. Manual of Clinical Mycology. 3rd Ed. W. B.
Saunders Co., Philadelphia, p. 503-526.

Cooper, B. H., and.J. D. Scheidau. 1970. A serological
~comparison of Phialophora verrucosa, Fonsecaea
pedrosoi and Cladosporium carrionii using immuno-
diffusion and immunoelectrophoresis. Sabouraudia
8: 217-226.

Davis, B. J. 1964. Disc electrophoresis. II. Method
and application to human serum protein in animals.
N.Y. Acad. Sci. 121 Art. 2, p. 404-427.

123

Desborough, S. and S. J. Peloquin. 1967. Esterase iso-
enzymes from Solanum tubers. Phytochemistry 6:
989-994.

De Vries, G. A. 1952. Contribution to the knowledge of
the genus Cladosporium Link ex fr., vetgeverij
Drukkerij, Holandia.

Emmons, c. w. 1945. Phialophora jeanselmei, comb.n.
from mycetoma of the hand. Arch. Path. 39: 364-
368.

Emmons, C. W., C. H.-Binford, and J. P. Utz. 1970.
Medical Mycology. 2nd Ed. Lea G Febiger, Phila-
delphia, p. 177-292, 350-364.

Fonseca, O. D., and A. E. Area-Leao. 1930. Chromoblasto-
mycosis. Rev. Med. Cir. Brazil 38: 197.

Frank, R. G. and J. A. Berry. 1972. Taxonomic applica-
tion of isoenzyme patterns produced with disc
electrophoresis of some Myxomycetes in the order
Physarales. Mycologia 64: 830-840.

Fuentes, C. A. and 2. E. Bosch., 1960. Biochemical dif-
ferentiation of the etiologic agents of chromoblasto-

mycosis from non-pathogenic Cladosporium species.
J. Invest. Dermat. 34: 419-421.

Gerhardt, W., J. Clausen and H. Anderson. 1963. Electro-
phoretic pattern of extractable proteins and
enzymes in embryonic and adult brains. Acta
Neurologia Scandinavica 39: 31-40.

Gezuele, E., J. E. Mockinnon and J. A. Conti-Diaz. .1972.
The frequent.isolation of Phialophora verrucosa

and Phialophora pedrosoi from natural sources.
Sabouraudia 10:.266-273.

Gilardi, G. L.. 1965. Nutrition of systemic.and subcu-
taneous pathogenic fungi. Bacteriol. Rev. 29:
406-424.

Gordon, M. A. and Y. Al-Doory. 1965. Application-of
fluorescent.antibody procedures to study of patho-
genic dematiaceous fungi, II. Serological rela-
tionships of.the genus Fonsecaea. J. Bact. 89:
551-556.

Gunsalus, I. C. 1955. Extraction of enzymes from micro-
organism (bacteria and yeast).. Methods of
'Enzymology. Academic Press, New York, Vol. 1.,
p. 51-64.

124

Gunsalus, I. C. and R. Y. Stanier. 1962. Exoenzymes.
The Bacteria: A Treatise on Structure and Function.
The Physiology of Growth. Academic Press, New York,
Vol. IV, p. 1212170.

Jensen, W. A. Botanical Histochemistry. W. H. Freeman
and Co., San Francisco.

Jotisankasa, V.,H. S. Nielson Jr. and N. F. Conant. 1970.
Phialophora dermatitidis, it's morphology and
biology. Sabouraudia 8: 98-107.

Kano, K. 1937. Uber die chromoblastomykose durch einen
noch nicht als pathogen beschriebenen pliz Hormiscum
dermatitidis n. cp. Arch. Dermat. Syph. 176: 282.

Lehninger, A. L. 1970. Proteins: Behavior in Solution.
Biochemistry. lst Ed. Wortthublishers, Inc.,
New York, p. 129-146.

Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J.
Randall. 1951. Protein measurement with the folin
phenol reagent. .J. Biol. Chem. 193: 165-175.

Luck, H. 1963. Method of enzymatic analysis. Edited by
Hans-Ulrich Bergmeyer. Academic Press, New York,
p. 895-897..

Macko, V., G. R. Honold and M. A. Tahman. 1967. Soluble
proteins and multiple enzyme forms in early growth
of wheat. Phytochemistry 6: 465-471.

Martin, D. S. 1938.- The antigenic similarity of a fungus
Cladospora americana isolated from wood pulp to
Phialophora verrucosa isolated from patients with
dermatitis verrucosa (chromoblastomycosis). Amer.
J. TrOp. Med. 18: 421-426.

Martin, D. S., R. D. Baker and N. F. Conant. 1936. A
case of verrucous dermatitis caused by Hormodendrum
pedrosoi (chromoblastomycosis) in North Carolina.
Amer. J. Trop. Med. 16: 593-607.

Martin, G. W. and C. J. Alexopoulos. .1969. The Myxomycetes.
University of Iowa Press, Iowa City, p. 561.

Medlar, E. M. 1915: ‘A new fungus Rhinophora verrucosa
pathogenic for man. Mycologia 7: 200-203.

Medlar, E. M. 1915. A cutaneous infection caused by a
new fungus Phialophora verrucosa with a study of the
fungus. J. Med. Res. 32: 507-522.

125

Miller, G. L. 1959. Protein determination for large
number of samples. Anal. Chem. 31: 964.

Mitchell, P. and J. Moyle. 1959.- Permeability of the
envelopes of-StaphyZococcus-aureus to some salts,
amino acid and non-electrolyte. J. Gen. Microbiol.
20: 434-441.

Montemayer, L. de. 1949. Studies de 1as propiedades
biologicas de varias cepas de hongos patogenos
causates_de-la eromomicosis y de especias vecinas
saprofitas y patogenas. Mycopath. Mycol. Appl.
4: 379-382. -

Moore, M. and F. P. de Almeida. 1935. -Etiologic agents
Chromomycosis (chromoblastomycosis) of Terra Torres,
Fonseca and Leao, 1922 of North and South America.
Rev. Biol. Hyge..6: 94.

Moore, M. 1941. The.chorio-a11antoic membrane of the
developing chick as a.medium for the cultivation
and histo-pathologic study of pathogenic fungi.
Amer. J. Path. 17: 103-120.

Moore, M. and E. Landecker. 1972. The Fundamentals of
the Fungi. Prentice-Hall, Inc., Englewood Cliffs,
N.J., p. 205-211.

Nielson, H. 8. Jr. and N. F. Conant. 1968. A new human
pathogenic PhiaZophora. Sabouraudia 6: 228-231.

Ornstein, L. 11964. Disc electrophoresis. Ann. N.Y.
Acad. Sci. 121: 321-349.

Pollard, C. J. and B. N. Singh. 1968. Early effects of
Gibberellic acid on barley aleurone layers. Bio-
chem. Biophy. Res. Commu. 33(2): 321-326.

Pollock, M. R. 1962. -Exoenzymes. The Bacteria: A
Treatise on Structure.and Function. Vol. IV.
The Physiology of Growth. Academic Press, New
York.

Reisfeld, R. A., V. J..Lewis and D. E. Williams. 1962.
Disk electrophoresis of proteins and peptides on
polyacrylamide gels. Nature 195: 281-283.

Rosenthal, S. A. .1964. Enzymatic studies with pathogenic
fungi. .Newsletter of the Medical Mycol. Soc., N.Y.
3: 1-4.

 

126

Rudolph, M. 1914. Uber die brasilianische "figueira".
Arch. f. schiffs u. Tropen. Hyg. 18: 498.

Salfelder, K., J..Schway and.N. A. Romero et al. 1968.
Habitat de Nocardia asteroides, Phialophora

pedrosoi.y. Cryptococcus neoformans en Venezuela.
Mycopath. Mycol. Appl. 34: 144-154.

Schechter, Y., J. W. Laudau, N..Dabrowa and V. D. Newcamer.
1966. Comparative disc electrophoresis studies of
proteins from dermatophytes. Sabouraudia 5: 144-149.

Schechter, Y., J. W. Laudau.and N. Dabrowa. 1972. Compara-
tive electrophoresis and numerical taxonomy of some
Candida species. .Mycologia 64: 841-853.

Schwartz, I. S. and C. W. Emmons. .1968. Subcutaneous
cystic granuloma caused by a fungus of wood pulp,
Phialophora richardsiae. Amer. J. Clin. Path. 49:
500-509.

Seeliger, H. P. R. 1968.. Serology as an aid to taxonomy.
The Fungi. Vol. III. Academic Press, New York,
p. 597-619.

Silva, M. 1957. The parasitic phase of the fungi of
chromoblastomycosis: Development of sclerotic
cells in vitro and in viva. Mycologia 49: 318-331.

Silva, M. 1958. The saprophytic phase of the fungi of
chromoblastomycosis: Effect of nutrients and
temperature upon growth and morphology. Trans.
N.Y. Acad. Sci. 21: 46-57.

Silva, M. 1960. .Growth characteristics of the fungi of
chromoblastomycosis. Ann. N.Y. Acad. Sci. 89:
17-29. .

Simson, W. 1946. Chromoblastomycosis. Some observations
on the type of disease in South Africa. Mycologia
38: 432-449.

Steward, F. C., R. F. Lyndon and J. T..Barber. 1965.
Acrylamide gel electrophoresis of soluble plant
proteins: A study on pea seedlings in relation to
development. Amer. J. Bot. 52: 155-164.

Stipes, R.J. 1970.. Comparativemycelial protein and
enzyme patterns in four species of Ceratocystis.
Mycologia 62: 987-995.

Stone, K. 1930. A study.of yeasts: By the complement
fixation test. Lancet 291: 577-578.

127

Szaniszlo, P. J., B. H. Cooperand H. S. Voges. 1972.
Chemical compositions of the hyphal walls of three
chromomycotic.agents. Sabouraudia 10: 94-102.

Terra, F., M. Torres, 0. Da Fonseca and A. E. Area-Leao.
1922. Nova typo dermatite verucosa por Acrotheca
com associacao de leishmaniosa. Brazil-med. 36:
363-367.

Thomas, D. L. and R. M. Brown. ,1970(a). New taxonomic
criteria in the classification of ChZococcum species.
111. Isozyme analysis. J. Phycol. 6: 293-299.

Thomas, D. L. and R. M. Brown. 1970(b). Isoenzyme analysis
and morphological variation of thirty-two isolates
of Protosiphon. Phycologia 9: 285-292.

Trejos, A. 1954.1 Cladosporium carrionii n. sp. and the.
pathogenicity of Cladosporia isolated from chromo-
blastomycosis. Rev. Biol. Trop. 2: 75-112.

Tsai, C. Y. and Y. C. Lu, L. T. Wang, T. L. Hsu and J. L.
Sung. 1966. Systemic chromoblastomycosis due to
Hormodendrum dermatitidis (Kano) Conant. Amer. J.
Clin. Path. 46: 103-113.

Tyrrell, D. 1969. Biochemical systematics and fungi.
Bot. Rev. 35: 305—361.

Vaughan, J. G., A. Waite, D. Boutter and S. Walters.
1965. Taxonomic investigation of several Brasaica
species using serology and the separation of protein
by electrOphoresis on gel. Nature 208: 704-712.

Webb, H. M., A..Gafoor and J. B. Heale. 1972. Protein and
enzyme patterns in strains of Verticillium. Trans.
Brit. Mycol. Soc. 59: 393-402.

West, B. 1967. Nutrition of Phialophora verrucosa A 126.
Mycopath. Mycol. Appl. 31: 12-16.

Whitney, P. J., G. Vaughan and J. B. Heale. 1968. A disc
electrophoresis study of the proteins of Verticillium
aZbO-atrum, Verticillium dahliae and Fusarium
oxyeporum with reference to their taxonomy. J.

Exp. Bot. 19: 415-426.

Wilson, J. W. and O. A. Plunkett. 1965. The fungous
diseases of man.. Univ. of California Press,
Berkeley, p. 179-189.

MW

8825

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