A MONOGRAPH OF THE GENUS TILLETIOPSIS
By
ATULCHANDRA ANANT GOKHALE
B.Sc, M.Sc. , University of Poona
A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF
Master of Science
in the Department
of
Botany
We accept this thesis as conforming to the required standard
THE UNIVERSITY OF BRITISH COLUMBIA
July, 1971 In presenting this thesis in partial fulfilment of the requirements for
an advanced degree at the University of British Columbia, I agree that
the Library shall make it freely available for reference and study.
I further agree that permission for extensive copying of this thesis
for scholarly purposes may be granted by the Head of my Department or
by his representatives. It is understood that copying or publication
of this thesis for financial gain shall not be allowed without my
written permission.
Depa rtment
The University of British Columbia Vancouver 8, Canada ii
ABSTRACT
Nutritional and physiological studies were made on species of
Tilletiopsis.
The variation in assimilation of glucose, maltose, starch, sodium nitrate, glutamic acid and phenylalanine proved to be useful in delimiting the species. The other criteria used in the classification of Tilletiopsis were splitting of arbutin, pigment production, thiamine requirement and gelatin liquefaction. On the basis of these results, a key for identifi• cation of species of Tilletiopsis has been proposed.
Three previously unknown species of Tilletiopsis; T_. albescens,
T_. pallescens and T_. fulvescens have been described. These new species showed some variation in their morphology as well as in their physiology. iii
LIST OF CONTENTS PAGE
INTRODUCTION AND REVIEW OF LITERATURE 1 Presentation of the problem 6
MATERIALS AND METHODS 8 Isolation 8 Inoculum preparation 9 Morphology 9 Nutrition and physiology 12 Effects of pH on growth 12 Effects of temperature on growth 12 Assimilation of carbon compounds 13 Assimilation of nitrogen compounds 13 Effects of vitamins on growth 13 Splitting of arbutin 14 Ethanol as a sole carbon source 14 Production of starch 15 Production of acid on chalk agar medium 15 Ability to liquify gelatin 15 Hydrolysis of urea 16 Conjugation studies 16
OBSERVATIONS 18 Morphology 18 Nutrition and physiology 22 Effects of pH on growth 22 Effects of temperature on growth 22 Assimilation of carbon and nitrogen compounds 22 Effects of vitamins on growth 23 Results of miscellaneous tests 24 Conjugation studies 24 Descriptions of the new species of Tilletiopsis 32 A key for the identification of species of Tilletiopsis 41
DISCUSSION 43
BIBLIOGRAPHY 49
APPENDIX 1 - Composition of the media 53 2 - Stains 55 3 - Cleaning solution 55 iv
LIST OF TABLES
Page
TABLE 1. a) A list of isolates used in morphological, nutritional and physiological studies 10
b) A list of isolates used in the conjugation
studies 11
TABLE 2 Effect of pH on growth of Tilletiopsis species.... 25
TABLE 3 Assimilation of various carbon and nitrogen compounds by Tilletiopsis species. 27 TABLE 4 Effects of various vitamins on growth of Tilletiopsis species 28
TABLE 5 Results of miscellaneous tests on species of Tilletiopsis 29
TABLE 6 Physiological characteristics of taxonomic value for delimiting Tilletiopsis species 31 V.
LIST OF PLATES
Page
PLATE 1: Cultural characteristics of the species of Tilletiopsis 21
PLATE 2: Effects of different temperatures on growth
of various species of Tilletiopsis 26
PLATE 3: Morphology of Tilletiopsis albescens 34
PLATE 4: Morphology of Tilletiopsis pallescens 36
PLATE 5: Morphology of Tilletiopsis fulvescens 39 PLATE 6: Figures 1 to 3: Tilletiopsis fulvescens: effect of yeast extract on pigment production. Figures 4 to 9: Cultural characteristics of _T. albescens, _T. pallescens and T_. fulvescens Figure 10 to 12: nuclear staining 40 vi.
ACKNOWLEDGEMENTS
I am indebted to Dr. R.J. Bandoni who introduced me to this field of research; his continuing interest was most valuable.
Thanks are also due to Drs. K. Tubaki, K. Wells, W.B. Cooke and Mrs. S. Reid for providing certain isolates used in this study.
It is my pleasure to thank Dr. B.N Johri for many invaluable suggestions; discussions with him proved very useful in carrying out this work.
Financial assistance through a teaching Assistantship from the Department of Botany and a Research Assistantship through N.R.C. grants of Dr. Bandoni is gratefully acknowledged.
Thanks are due to Mrs. Pat Waldron for taking the enormous task of typing the manuscript. CHAPTER 1
INTRODUCTION
The family Sporobolomycetaceae was defined (Derx, 1930) to include
the genera Sporobolomyces, Bullera and Tilletiopsis. Sporobolomyces was
first described by Kluyver and van Niel (1924) and Bullera was described by Derx (1930). The name Tilletiopsis was provisionally suggested for a
fungus found on some leaves by Derx (1930) and he validated it with a
short Latin diagnosis in 1948. Derx (1948), described another new genus,
Itersonilia, placing it in the family Sporobolomycetaceae. Nyland (1948)
published an account of an unusual fungus which he later (1949) described
as a new genus and species, Sporidiobolus Johnsonii. The genus was con•
sidered by him to belong to the family Sporobolomycetaceae.
All members of this family produce aerial conidia attached assyme-
trically at the apex of sterigmata and discharged violently. Kluyver and
van Niel (1924) observed that in Sporobolomyces, each conidium was dis•
charged by a drop-excretion mechanism of the same nature as that found on
basidia of Hymenomycetes and the Uredineae. Derx (1930) also pointed out
that the spores of Sporobolomyces were projected at maturity in the same
manner as basidiospores and proposed the name 'ballistospore's to these
characteristic conidia.
Bullera species normally do not produce mycelium and remain yeast•
like; Sporobolomyces includes both yeast-like and mycelial forms, but the 2.
occurrence of -mycelium is restricted to very few species. The genera
Itersonilia, Sporidiobolus and Tilletiopsis, all produce mycelium. In
Itersonilia and Sporidiobolus, abundant clamp connections are formed on the mycelium; clamps are lacking in Tilletiopsis.
The colonies of Sporobolomyces and Sporidiobolus are usually pink
to salmon coloured because of the presence of carotenoid pigments; one species of Sporobolomyces, S_. singularis, shows white colonies. In Bullera and Itersonilia pigments generally are lacking and the colonies are pallid
to yellowish. Tilletiopsis colonies are generally cream to white though yellowish and lilac coloured colonies are found in some species.
In Sporidiobolus abundant chlamydospores are produced while in
Itersonilia and Tilletiopsis, chlamydospores are few and generally are
formed only in old cultures.
Laffin and Cutter (1959) carried out extensive studies on the genus
Sporidiobolus; in their proposed life cycle, the diploid phase alternated with a dicaryotic phase and the two were shown to be of equal importance
in the life cycle of the organism. These authors also reported the occur•
rence of sexual and asexual cycles in Sporobolomyces and Tilletiopsis but
their studies were never published.
Sowell and Korf (1960) suggested merging Tilletiopsis and Itersonilia
and proposed that the name Itersonilia be used for the combined genus.
Their proposal was based upon observations on the budding monocaryophase in
Itersonilia. They state "It is only in the shape of the ballistospores, 3. falcate in the Tilletiopsis, lunate-reniform in monocaryophase Itersonilia, that a distinction can be drawn. The constancy of this single character has not been sufficiently investigated." Sowell and Korf (I960), apparently failed to re-establish the dicaryophase from the monocaryophase in
Itersonilia and therefore their suggestion has not been followed.
A sexual phase was observed by van der Walt (1970) in a strain of
Sporobolomyces salmonicolor (Fischer et Brebeck) Kluyver et van Niel, 1894; the name Aessosporon was proposed for this perfect stage. The strain was found to form teliospores which germinated usually by the formation of a non-septate promycelium bearing 2-4 sporidia. This new basidiomycete yeast genus was assigned to family Tilletiaceae. Recently, in Sporobolomyces odorus Derx, 1930; Bandoni et al. (1971) observed conjugation, dicaryotic hyphae with clamp connections and chlamydospores. The similarities in this dicaryotic phase and that of Sporidiobolus johnsonii were striking and it was suggested that they could be the same.
The family Sporobolomycetaceae is generally included in the
Basidiomycetes, although its affinities are not clear. Kluyver and van
Niel (1924) suggested that Sporobolomyces species are Basidiomycetes which may be included in"the Hemibasidii whereas Derx (1930) considered them as reduced hymenomycetes. Since then many workers have expressed their accept• ance of this view (Buller, 1933; Martin, 1952; Sainclivier, 1952).
Alexopoulos (1962) stated that some of the characters of the Sporobolomyce• taceae point convincingly to a basidiomycetous relationship and he includes them with Basidiomycetes. A different view was taken by Lowag (1926); he 4.
did not consider Sporobolomyces as of basidiomycete origin. Bessey (1950)
included the family Sporobolomycetaceae with non-ascosporogenous yeasts.
Nyland (1950) carried out a comparative study of two species of
Tilletiopsis and a species of Entyloma. He pointed out a difference in
cultures of Entyloma and Tilletiopsis in that the ballistospores of Entyloma
did not bud in a yeast-like manner. Nyland suggested that the Tilletiopsis may represent non-parasitic forms of the 'white-smuts.'
The name Tilletiopsis was provisionally suggested by Derx (1930)
because of the morphological resemblance to cultures of Tilletia spp. His
five isolates were readily distinguishable on the basis of ballistospore
size. The spore sizes of his strains were, 9 x 2 u; 7 x 1.6 u; 15 x 2 u;
13.4 x 2.3 u and 12 x 3 u. The Latin diagnosis given by Derx (1948) is as
follows:
Mycelium hyalinum, septatum, repens, sterigmata in aera ascendentia
formans. Sporae solitariae, falcate, leves, hyalinae. Species tipica:
Tilletiopsis spec. No. 4 Derx.
Nyland (1950) collected two isolates of Tilletiopsis from a variety
of hosts during the summer of 1947 in Washington state, U.S.A. His two
isolates had spore sizes averaging 9 x 1.8 u and 14 x 2.4 u. The first
isolate resembled Derx's isolate no. 1 and second resembled Derx's isolates
no.s 3 and 4. Nyland proposed the name Tilletiopsis washingtonensis for his
large-spored species and considered it as the type species of the genus;
Derx's "type species" (strain 4) was no longer available at the
'Centraalbureau voor Schimmelcultures'. For the isolate with small spores, 5.
Nyland proposed the name Tilletiopsis minor; it differed from the _T. washingtonensis by its smaller spores, darker colony colour and the carti• laginous consistency.
During the summer of 1951, Tubaki (1952) isolated 16 strains of
Tilletiopsis from leaves of 13 different hosts in the vicinity of Tokyo,
Japan. According to him, eight strains were different from the Tilletiopsis species already described by Nyland. One strain resembled T_. minor but was more yellowish, did not produce soluble pigments, and formed flat colonies on agar media; Tubaki proposed the name Tilletiopsis minor var flava for
this isolate. The colonies of two strains were soft in texture and lilac
in colour; Tubaki described these isolates as Tilletiopsis lilacina. He
described another new species, Tilletiopsis cremea, for five strains with
cream coloured colonies and smaller spores than those of T. washingtonensis.
Little is known about the nutrition and physiology of Tilletiopsis
species. Tubaki (1952) reported that 25 C was the optimum temperature for
growth of T_. minor, T. minor var flava, _T. cremea and _T. lilacina.
Sundstrom (1964) carried out a comparative study of carbon assimilation by three strains of Tilletiopsis and a species of Exobasidium; he also
investigated effects of vitamins on growth of these organisms. However, he did not identify the species of Tilletiopsis he studied nor did he in•
clude a description of the strains. 6.
Presentation of the Problem
Tubaki (1952) published a key for identification of all known species of Tilletiopsis. His key is based entirely on the colour and
consistency of colonies as follows:
KEY TO SPECIES (Tubaki, 1952)
Colony yellowish
colony yellow, then brown T\ minor
colony bright yellow _T. minor var flava
Colony cream coloured
surface tough T_. washingtonensis
surface soft T_. cremea
Colony lilac coloured T_. lilacina
In the summer of 1970, I obtained several isolates of Tilletiopsis
from Dr. .R.J. Bandoni and attempted to identify them using Tubaki's key.
It was observed that the characters such as colour and consistency of
colonies were variable and were influenced by cultural conditions, medium
composition and age of the culture. Some isolates showed distinct colony
colours as described by Tubaki, but others varied in colour, especially between yellowish brown and bright yellow. I also found many colonies with
consistencies varying from soft to tough. Some colonies appeared to be
soft when young, but they toughened with age. Because of the inconsisten•
cies in colour and texture of colonies, it was found to be difficult to
place these 'intermediate' isolates in the described species. 7.
Ballistospore size was used by Derx (1930) and to some extent by
Nyland (1950) to differentiate between the species of Tilletiopsis. During my preliminary studies ballistospore measurements were made and differences did not appear to be characteristic. In general, the morphology of ballistospores did not reveal distinct characters which could be used in identification of the species.
The initial studies on isolates from the U.B.C. culture collection suggested the presence of some undescribed species. Inability to adequately describe and distinguish among species on the basis of characteristics re• ported in the literature led to attempts to use nutritional and physiological characteristics. These features are already in use in identification of species of Sporobolomyces, Sporidiobolus and Bullera.
This study was undertaken (1) to examine the possibility of using nutritional and physiological characteristics in identification, (2) to make test crosses in an attempt to find the sexual stage and (3) to prepare a monograph of the genus Tilletiopsis. 8.
CHAPTER 2
MATERIALS AND METHODS
Isolation; Derx's (1930) "spore fall" method was used for isolation
of the strains of Tilletiopsis. Leaves or plant parts were fixed with I
masking tape inside a sterile Petri plate lid and it was inverted over
Malt extract-Yeast extract-Peptone (MYP) agar medium (Appendix 1). Ballis-
tospores were discharged violently and landed on agar layer where they
germinated and grew into colonies. A large number of ballistospores was
discharged within 2 to 3 hours. By turning the lid around at short inter•
vals during discharge of the ballistospores, a greater dispersal was ob•
tained. After microscopic examination through the bottom of the Petri
plate, a single colony was isolated. In doubtful cases, the isolated colo•
nies were transferred to nutrient solutions. After 48 hours of incubation,
suspensions were plated out on an MYP agar medium and solitary colonies
were isolated. Stock cultures were maintained on MYP agar slants and were
stored in a refrigerator at 4 C.
The composition of media used during this study are given in
Appendix 1. A list of the strains studied, year and place of isolation
and their sources is given in Table 1. The numbers given to the various
strains are the U.B.C. culture collection numbers. 9.
Inoculum preparation; (Lilly and Barnett, 1951) Each strain was grown on a MYP plate for 7 days at room temperature. A disc (14 mm-diam.) of mycelium and agar was transferred to a sterile blender containing 80 ml of sterile distilled water. The fungus was blended at low speed and was allowed to settle for about an hour; the supernatant served as the inoculum
(approximately 35 ug/ml of spore suspension).
Morphology
Petri plates with MYP agar medium were inoculated with a cell sus• pension and incubated at 23 C for 2 days. The plate with fungus was in• verted over a dry microscopic slide and discharged ballistospores were collected on the slide. The width and length of ballistospores were mea• sured, fifteen to thirty spores were examined per spore print. Using the same technique, ballistospores of all species and strains were collected on MYP plates and their modes of germination and growth were studied. The line drawings were made by use of the Camera lucida.
Nuclear staining was performed with Heidenhain's 'iron hematoxylin' stain (Johansen, 1940) (for details, see Appendix 2).
The isolates were grown on MYP, Czapek's Dox (CZD), malt extract
(MEA) and minimal medium (MM) agar and observations were made of their com• parative growth patterns. 10. (a) TABLE 1
A) A list of isolates used in morphological, nutritional and physiological studies
UBC species substrate or year and culture or source place of number strain isolation
912 cremea Tubaki NI-3131
925 T. lilacina Tubaki NI-3114
915 T_. minor Nyland Dr. K. Tubaki, Institute NI-3064 of Fermentation, Osaka, Japan 916 T. minor Nyland NI-6905
917 T_. minor var flava Tubaki NI-3112 ) ) 907 T. washingtonensis Nyland Dr. K. Wells, Dept. of Botany, University of California, Davis, Calif., U.S.A.
(b) 926 undescribed species filamentous organisms 1968, U.S.A. in sewage (c) 8007 undescribed species Sirobasidium sp. 1968 Japan fruiting body (d) 8006 undescribed species Forsythia sp. leaf 1968, B.C. Canada 11.
B) A list of isolates used in the conjugation studies (The isolates were tentatively identified)
UBC species substrate or year and culture or source place of number strain isolation
801 T. lilacina Acer sp. leaf 1971, Hope, B.C. 914
8030 1968, Parks- ville, B.C.
8031 Asarum sp. leaf 1968, White Rock, B.C.
8032 Rubus sp. leaf
8022 T. minor var flava 1970, Vane. B.C.
T. cremea Rumex sp. leaf 1970, Carbon- 8029 dale, Illinois U.S.A.
undescribed species filamentous or• 1968, Cincin• 927 (similar to UBC #926) ganisms from nati, Ohio, sewage U.S.A.
(e) 975 T. washingtonensis Quercus sp. leaf 1970, Missouri U.S.A.
952 1967, Iowa, U.S.A. a) With the exception of T_. minor, all other species are represented by one isolate. b) UBC isolate 926 was received from Dr. W.B. Cooke, Mycologist, Biological Treatment Research Program, U.S. Dept. of the Interior, Cincinnati, Ohio, U.S.A. c) Sirobasidium fruiting body was collected in Japan (Nov. 1968) by Mr. T. Flegel and the strain was isolated by Mrs. S. Reid in Vancouver (Dec. 1968) by 'Spore fall method'. d) Isolate 8006 was obtained from Mrs. S. Reid, Botany Department, University of British Columbia, Vancouver, B.C. e) UBC #975 was obtained from Mr. John Noell, Dept. of Botany, Washington "University, Missouri, U.S.A. 12.
Nutrition and Physiology
Effect of pH on growth: Liquid CZD medium was used to determine
effects of different pH values on growth. Fifty ml of the medium was dis•
pensed into each Erlenmeyer flask (125 ml). The pH of the medium was ad• justed by adding 1 N HC1 or 1 N NaOH solutions; a "Radiometer" pH-meter
(PMH 28) was used to determine the pH values. The medium was autoclaved
and the pH values checked. These values were considered as initial pH values;
the pH values were 4.0, 5.0, 5.6, 6.0, 6.5, 7.0, 7.5 and 8.0. The flasks were inoculated with 1 ml of cell suspension and incubated for 14 days on a reciprocating shaker at 84 strokes/minute. The fungus from each flask was collected on a pre-weighed filter paper (Whatman no. 1). It was washed,
dried at 80 C for about 24 hours and weighed. The pH of the filtrate was
checked. No buffer was added to the medium in this study.
Effects of temperature on growth: The inoculum for temperature
studies was prepared as described by Lilly and Barnett (1951). Cultures were
transferred from MP slants to CZD agar medium and were incubated at room
temperature for 4 days. Uniform discs (3 mm in diam.) of mycelium and agar were cut from Petri dishes; each disc served as the inoculum source for 1
test culture.
The inoculum was placed in the center of a Petri plate containing
CZD agar medium. The growth of each fungus was studied at 4 C, 10 C, 15 C,
20 C, 20 C, 23 C, 25 C, 30 C and 36 C. The cultures were incubated for 14
days in the dark and the linear growth was recorded by measuring colony
diameters (including colony diameter of the inoculum). Each test was made
in triplicate. 13.
Assimilation tests and vitamin studies: In these studies, chemi• cally cleaned glassware was used. All flasks and pipettes were cleaned with a potassium dichromate-sulphuric acid cleaning solution (Appendix 3), and rinsed thoroughly in glass distilled water before use.
Assimilation of carbon compounds: A number of compounds were se• lected from the list of compounds given by Wickerham (1951); glucose, galactose, maltose, lactose, sucrose, mannitol and starch. Each compound was autoclaved separately and added aseptically to the carbon-free CZD medium. The final concentration of each carbon compound was 3 percent (wt/. vol.). Erlenmeyer flasks (125 ml) containing 50 ml of the CZD medium were inoculated with 1 ml of the spore suspension. The cultures were incubated at 23 C on a reciprocating shaker for 14 days in the dark. The dry weights were determined as described for pH study. Flasks were prepared in tripli• cate for each compound and controls were used that gave either optimal or no growth. By comparing the growth of the controls with growth in the test medium, information was obtained about the ability of the species and strains to utilize various compounds.
Assimilation of nitrogen compounds: The following compounds were employed for nitrogen assimilation tests (Wickerham, 1951); sodium nitrate, potassium nitrate, ammonium sulphate, glutamic acid, phenylalanine and asparagine. The final concentration of each nitrogen compound was 0.2 per•
cent (wt./vol.). The experimental procedure was as described for the carbon assimilation tests.
Effects of vitamins on growth: The following vitamins were tested
for their effect on growth: thiamine (5 ug), biotin (0.25 ug), inositol 14.
(0.25 ug) and pyridoxine (5 mg). The figures in the brackets indicate the final concentration (wt./vol.) of each vitamin. Fifty ml of liquid
CZD medium was dispensed in Erlenmeyer flasks (125 ml) and sterile vitamin solutions were added aseptically to each flask. The flasks were inoculated with 1 ml of spore suspension and the cultures were incubated at 23 C for
14 days in the dark. Dry weights were determined as described previously.
Splitting of arbutin: (Lodder and Kreger-van Rij, 1952). The composition of medium was as follows: arbutin (NBC), 5.0 g.; yeast extract
(Difco), 2.0 g.; agar, 12 g.; distilled water, 1 liter.
A drop of sterile ferric chloride solution (0.6 percent; w/v) was placed in the center of a sterile Petri plate. The arbutin yeast agar medium was poured in the Petri plate and the liquids were thoroughly mixed and allowed to cool. The plates were inoculated and incubated at room temperature. A positive reaction was indicated by a dark brown coloured zone* around colonies after 5 to 7 days.
Ethanol as sole carbon source: (Lodder and Kreger-van Rij, 1952).
The basic medium contained the following: (NH^)2 S0^, 1.0 g. ; KIL^PO^, 1.0 g. ;
MgSO^. 7H20, 0.5 g.; distilled water, 1 liter.
Five ml of the basic medium was dispensed in each test tube (16 mm).
The medium was autoclaved in the tubes and 3 percent ethanol was added, aseptically to the basic medium. The tubes were inoculated with a spore suspension and were incubated at room temperature. Blank tubes, without
* DBC #8006 was found to produce yellowish brown pigment in the presence of yeast extract and therefore yeast extract was substituted by sodium nitrate to avoid confusion. 15. ethanol, were used for comparison. A positive ethanol utilization was indicated by growth of the organism after 21 days.
Production of starch: (Mager and Aschner, 1947).
The composition of the medium was as follows: (NH^^ SO^, 1.0 g. ;
MgS04.7H20, 0.5 g.; KH^O^, 1.0 g. ; glucose, 10 g.; thiamine, 100 ug.; agar, 12 g.; distilled water, 1 liter. Lugol's iodine solution was prepared as follows: iodine, 1.0 g.; KI, 2.0 g.; distilled water, 300 ml.
Erlenmeyer flasks (125 ml) containing 25 ml of the medium were inoculated with a cell suspension and incubated at room temperature on a reciprocating shaker. After 24 days of incubation, the fungus from each flask was separated from the test medium. To the test medium, 2 drops of
Lugol's iodine solution were added. A deep blue colour indicated a positive reaction.
Production of acid on chalk agar medium: (van der Walt, 1970).
The medium was prepared with following constituents: glucose, 50 g.;
CaCO^ (precipitated), 5.0 g.; yeast extract (Difco), 5.0 g.; agar, 12 g.; distilled water, 1 liter.
Five ml of the basic medium was dispensed in 16 mm test tubes and autoclaved. The slants were inoculated with a spore suspension and incubated at room temperature for 14 days. If sufficient acid was produced to clarify the opaque medium, the reaction was reported as positive.
Ability to liquify gelatin: (Wickerham, 1951)
The basic medium was prepared as follows: glucose, 0.5 g. ; (NH^^SO^
0.5 g.; NaN03, 0.2 g.; MgS04 7H20, 0.05 g.; Ca(N03)2, 0.05 g.; KCI, 0.05 g.; distilled water, 100 ml. 16.
Ten grams of gelatin (Kno^were dissolved in 90 ml of hot distilled water. The solution was pipetted in 4.5 ml quantities into 16 mm test tubes and was autoclaved. The tubes were allowed to cool to about 40 C and 0.5 ml of the basic medium was added aseptically to each tube. The tubes were gently shaken to disperse the basic medium in the gelatin solu• tion and the tubes were allowed to stand in a vertical position. A drop of spore suspension was spread over the surface of the medium and tubes were incubated at room temperature. At 7 and 24 days, the depth of the liquid layer, if any, was measured.
Hydrolysis of urea: (Seeliger, 1956).
The basic medium contained the following: peptone (Difco), 1.0 g.;
glucose, 1.0 g.: NaCl, 5.0 g.: KH2P0^, 2.0 g.; phenol red, 0.012 g.; agar,
12.0 g.; distilled water, 1 liter.
A quantity of 4.5 ml of medium was dispensed into each 16 mm test tube and sterilized. Immediately after sterilization, 0.5 ml of 20 percent filter-sterilized urea solution was added to each tube and tubes were allowed to cool. The slants were inoculated with a spore suspension and the tubes were incubated at room temperature. The reaction was recorded as positive if a deep pink colour appeared within 5 days.
Conjugation tests:
In this study, several other isolates were used in addition to
those listed in Table 1(A); the isolates were tentatively identified using
Tubaki's key. A list of the isolates used, year and place of isolation
and their sources is given in Table 1(B). 17.
Erlenmeyer flasks (125 ml) containing 25 ml of liquid MYP medium were inoculated with ballistospore suspension and incubated at room tempera•
ture on a reciprocating shaker for 2 days. A loopful of suspension from
two isolates was placed on MYP agar plates and was mixed thoroughly;
several possible combinations were tried for mating reactions. The plates
were examined microscopically and the method for observation of plates was
as described by Bandoni et al. (1971). In addition to MYP medium, the follow•
ing media were also employed in this study; Corn-meal (CMA), MEA, MM,
Potato-dextrose (PDA), water agar (WA) medium. In some instances, the
isolates were mixed and grown either (1) in liquid media (2) under anaerobic
conditions or (3) in the dark. 18.
CHAPTER 3
OBSERVATIONS
Morphology
The morphology of the described species of Tilletiopsis was reported by Nyland (1950) and Tubaki (1952). The observations of my study are given below and in Plate 1.
Tilletiopsis minor Nyland (1950)
On CZD medium: colonies white, no soluble pigments.
On MYP medium: colonies 'Sanford's brown', producing 'Sudan brown'
coloured soluble pigment; colonies soft when young, then tough.
Hyphae delicate, septate, branched, 1.0-2.5 u in diam. Ballistospores curved, hyaline, finely granular, 6.0-14.0 x 1.0-2.0 u., germinating by repetition or by germ tube. Chlamydospores terminal or intercalary, hyaline, globose, obovate to clavate, produced singly or in chains, 4.0-14.0 u in diam.
Tilletiopsis minor Nyland var flava Tubaki (1952)
On CZD medium: colonies white, no soluble pigments.
On MYP medium: colonies 'Pale orange yellow', no soluble pigments.
Hyphae delicate, septate, branched, 1.0-2.0 u in diam. Ballistospores
curved, hyaline, finely granular, 6.0-17.0 x 1.0-2.5 u.; germinating by repetition or by germ tube. Budded cells filiform, 7.0-19.0 x 1.0-3.0 u. 19.
Chlamydospores terminal or intercalary, hyaline, globose to obovate,
produced singly or in chains, 4.0-11.0 u in diam.
Tilletiopsis washingtonensis Nyland (1950)
On CZD medium: colonies white to cream; soft when young, then tough.
On MYP medium: colonies cream to 'Naples yellow1; soft when young
then tough.
Hyphae delicate, septate, branched, 1.5-3.0 u in diam. Ballistospores
hyaline, curved, finely granular, 7.0-18.0 x 1.5-3.0 u.; germinate by
repetition or by germ tube. Budded cells filiform or irregular, larger than ballistospores. Chlamydospores terminal or intercalary, hyaline, globose
to obovate, produced singly or in chains, 10.0-16.0 u in diam.
Tilletiopsis cremea Tubaki (1952)
On CZD medium: colonies white to cream, soft.
On MYP medium: colonies cream to 'light buff, soft, finely wrinkled.
Hyphae delicate, septate, branched, 1.0-3.0 u in diam. Ballisto•
spores hyaline, curved, finely granular, 8.0-18.0 x 1.0-3.0 u, germinating
by repetition or by germ tube. Budded cells straight, filiform, usually
larger than ballistospores, 9.0-20.0 x 1.5-3.0 u. Chlamydospores terminal
or intercalary, hyaline, globose, occasionally irregular in shape, produced
singly or in chains, 4.0-11.0 u in diam.
Tilletiopsis lilacina Tubaki (1952)
On CZD medium: colonies white to cream, soft.
On MYP medium: colonies 'light pinkish lilac', soft, finely wrinkled. 20.
Hyphae usually delicate, septate, branched, 1.0-3.0 u in diam.
Ballistospores hyaline, curved, finely granular, 6.0-18.0 x 1.0-3.0 u., germinating by repetition or by germ tube. Budded cells straight or irregular in shape. Chlamydospores rarely produced, but readily produced under anaerobic conditions, terminal or intercalary, hyaline, globose to obovate, 6.0-11.0 u in diam.
The following isolates have been described as new species of
Tilletiopsis, further in the text (see page 32).
(a) Isolate UBC #926 (Tilletiopsis albescens Gokhale, 1971)
(b) Isolate UBC #8007 (Tilletiopsis pallescens Gokhale, 1971)
(c) Isolate UBC #8006 (Tilletiopsis fulvescens Gokhale, 1971)
In all strains and species, the ballistospores and the mycelium were found to be uninucleate. PLATE I
Cultural characteristics of the species of Tilletiopsis (On CZD medium; incubation period-14 days)
Figure 1 T_. minor
Figure 2 T. minor var flava
Figure 3 T. washingtonensis
Figure 4 T\ cremea Figure 5 T. lilacina
Figure 6 T. albescens (UBC #926)
Figure 7 T. pallescens (UBC #8007)
Figure 8 T. fulvescens (UBC #8006) PLATE I
8 22.
Nutrition and Physiology
The purpose of these studies was to search for nutritional or physiological characters which could be used in differentiation of species of Tilletiopsis.
Effect of pH on growth; This investigation was carried out to determine if optimum pH for Tilletiopsis species varied. Maximum growth was obtained in all the species, when the medium was neutral or slightly alkaline. At pH 4, there was no growth in any of the species while there was some growth when pH of the medium was 5.0, 5.6, 6.0 or 8.0 (Table 2).
Effect of temperature on growth: Growth of different species at elevated temperatures was studied and the results are given in Plate 2.
The linear growth in each species was maximum at temperatures 23 and 25 C.
There was no visible growth at 4 and 36 C except in T_. albescens, where some growth was observed at both these temperatures. With the exception
°f lilacina, the remainder of the species showed scanty growth at 30 C.
The optimum temperature for growth of most of the Tilletiopsis species was between 23-25 C.
Assimilation of carbon and nitrogen compounds: Starch and sucrose were readily utilized whereas galactose and lactose were poor sources of carbon for most of the species of Tilletiopsis. Glucose and mannitol were assimilated only by J_. washingtonensis, T_. albescens, and T. pallescens where• as maltose was assimilated only by T. minor, T. washingtonensis, T. lilacina and T. albescens (Table 3). 23.
Nitrates appeared to be readily utilizable source of nitrogen for most of the species, however _T. minor var flava and _T. cremea did not assimilate sodium nitrate and potassium nitrate, respectively. Ammonium sulphate, glutamic acid, phenylalanine and asparagine were found to be poor sources of nitrogen for most of the species, though these compounds were readily utilized by _T. albescens and _T. fulvescens. T_. pallescens differed from these species as it did not utilize phenylalanine.
In T_. albescens, there was a distinct pattern of utilization when the following compounds were provided as carbon and nitrogen sources; glucose, maltose, starch, mannitol, sucrose and sodium nitrate, potassium nitrate, ammonium sulphate, glutamic acid, phenylalanine and asparagine. The biomass production was about 10 times greater than that in the remainder of the species. In _T. pallescens, similar growth was observed when glucose, mannitol, sodium nitrate, potassium nitrate, ammonium sulphate, glutamic acid and asparagine were provided as carbon and nitrogen sources.
There appeared to be a distinct variation in the ability of various species to assimilate different carbon and nitrogen compounds; this varia• tion is being used in delimiting the species of Tilletiopsis.
Effects of vitamins on growth: The growth of T. minor, T_. minor var flava, T_. washingtonensis, T. cremea, T_. lilacina and T_. fulvescens was stimulated by addition of thiamine to the medium (Table 4). However, growth of J_. albescens and _T. pallescens was unaffected after addition of thiamine.
The vitamins biotin, inositol and pyridoxine had no stimulatory effect on growth of any of the species studied. 24.
The results of the tests: splitting of arbutin, ethanol as a sole carbon source, production of acid, gelatin liquefaction and hydrolysis of urea are presented in Table 5.
The positive reactions for splitting of arbutin were given by
TP. washingtonensis, _T. lilacina, ]T_. cremea and T\ fulvescens. Ethanol was utilized only by T_. albescens as a sole source of carbon; no visible growth was observed in any of the remaining species. None of the species gave positive reactions for either the starch test or acid production on chalk
agar. The liquefaction of gelatin varied within a wide range; maximum
liquefaction was observed with _T. minor (12 mm) and the minimum was with
T. lilacina (5 mm). The two species, _T. albescens, and T. pallescens did not liquify gelatin. All species hydrolyzed urea, but T. albescens showed
a weak reaction.
Conjugation studies: The mating studies were not successful.
There was no positive indication of conjugation under any of the conditions
tested. 25.
TABLE 2
Effect of pH on growth of Tilletiopsis species
UBC//926 UBC//8007 UBC//8006 Initial T. T. T. T. T. T. T. T. pH minor minor var washing- cremea lilacina albescens pallescens fulve- flava tonensis scens
4.0 - - ____
5.0 + + + + +;.• + + +
5.6 + + + ++ ++ +
6.0 + + + + + ++++ +
6.5 ++4+ ++++++++++ ++
7.0 ++++ ++ ++ ++ ++ ++ ++
7.5 ++++ ++++++4+++ ++
8.0 + + + + + + + +
no growth
fair growth
maximum growth 26.
PLATE II
Effects of different temperatures on growth of various species of Tilletiopsis PLATE II
T. albescens(926) T. pallescens(8007) 30-
20- I 10- E E T. cremea 210- < Q i— T. washingtonensis 30- T. fulvescens(8006) 20-4 10- O O JliL T. mi nor 30- T. minor var. flava 20- 10- -Bp p- -r 10 15 20 23 25 30 36 4 TEMPERATURES 27. TABLE 3 Assimilation of various carbon and nitrogen compounds by Tilletiopsis species UBC//926 #8007 #8006 T. T. minor T. T. T. T. T. T. minor var. washing- cremea lilacina albe- palle- fulve- flava tonensis scens scens scens Carbon compounds glucose w - + - - + + galactose - - + - - - - - maltose + - + - + + - - lactose - - - - - + - sucrose + + + + + + + mannitol - - + - w + + w starch + + + + + + - - Nitrogen compounds NaN03 + - + + + + + + KN03 + + + - w + + w (NH4)2S04 - - w - - + + w glut, acid W - + - w + + + phen. alanine + - - - w + - + asparagine w - - - - + + w Test medium: CZD liquid medium Incubation period: 14 days - = not utilized + = utilized W = weak utilization * = In carbon assimilation tests, NaN03 was the sole nitrogen source; and in nitrogen assimilation tests, sucrose was the sole carbon source. 28. TABLE 4 Effects of various vitamins on growth of Tilletiopsis species UBC//926 UBC//8007 UBC//8006 vitamins T. T. minor T. T. T. T. T. _T. added minor var flava washing- cremea lilacina albe- palle- fulve- tonensis scens scens scens Thiamine + + + + + - - + Bio tin - - ____ Inositol -- ____ Pyridox- ine - ____ Th+Bi+In +Py + + ++'+- - + Control ------ Test medium: CZD liquid medium Incubation period: 14 days - = control growth + = growth stimulation 29. TABLE 5 Results of miscellaneous tests on species of Tilletiopsis UBC//926 UBC//8007 UBC//8006 Tests T. T. T. T. _T. T. T. T. minor minor var. Washington- ere me a lila- albe- palle- f ulve- flava nensis cina scens scens scens splitting of arbutin + ethanol as a carbon source production of starch gelatin liquefac• tionem.m. ) 7-12 6-8 6-10 7-11 1-5 5-6 production of acid hydrolysis of urea + W + * = the height of liquified gelatin was measured in 16 mm test tubes, at 7 and 26 days. - = negative reaction + = positive reaction W = weak reaction 30. The results of morphological, nutritional and physiological studies indicated that the isolated UBC #926, 8007 and 8006 were different from the species of Tilletiopsis described by Nyland (1950) and Tubaki (1952). The results of comparative studies are shown in Table 6. In isolate 926, the ballistospores were found to germinate by repetition as well as by producing germ tubes. But in isolates 8007 and 8006, ballistospores were observed to germinate by producing germ tubes which in turn produced new ballistospores. In the latter two isolates, the balli• stospores were seen to be produced only from the mycelium. In isolates 926 and 8007, growth was comparatively faster than the isolate 8006 and mycelium was also more extensive in the former two isolates. The hyphae of isolate 8006 were narrow (1.0-2.5 u) whereas in the isolates 926 and 8007, they were comparatively broader (2.0-3.5 u; 2.0-3.5 u). Chlamydospores were not observed in isolate 8007 whereas they were formed only under anaerobic conditions in isolates 926 and 8006. The isolates 926 and 8007 were easy to separate from other species of Tilletiopsis on the basis of assimilation tests, effect of thiamine on growth and gelatin liquefaction. The differentiation between isolates 926 and 8007 was based upon the assimilation tests. Isolate 926 was found to utilize ethanol, maltose and starch as sole source of carbon. Phenylalanine was utilized by isolate 926 whereas isolate 8007 did not utilize it as a sole source of nitrogen. The separation of isolate 8006 was based upon the pigment production. This isolate was found to produce a yellowish brown pigment on MYP medium. 31. TABLE 6 Physiological characteristics of taxonomic value for delimiting Tilletiopsis spp. UBC//926 UBC//8007 UBC//8006 T. T. T. T. T. T. T. T. "minor minor washing- "cremea Til a- albe- p~alle- Tulve- var flava tonensis cina scens scens scens Assimilation of Glucose W - + - - + + Maltose + - + - + + Starch + + + + +.+ Sodium ni• trate + - + - + + + + Glutamic acid W - + -W + + + Phenyl• alanine + - - - W + - + Splitting of arbutin - - + + +- - + Ethanol as a carbon source -- - - - + Hydrolysis of Urea + + + + +W + + Thiamine dependency + + + + +- - + Gelatin li• quefaction + + + + +- - + Pigment formation + - - ____ + + = positive assimilation - = negative assimilation W = weak assimilation 32. The production of pigment was observed only in the presence of yeast extract. There was a distinct increase in pigment production when the concentration of yeast extract was raised from 0.05 to 1.5 percent (Plate VI). From the observations presented, isolates 926, 8007 and 8006 appear to be new species of Tilletiopsis. The description of the new species is given in the following pages. Tilletiopsis albescens Gokhale sp. n. Coloniae cartilaginosae, durae, albae, veteriores extense myceliales. Hyphae plerumque tenellae, septatae, ramosae, iuniores hyalinae, vetustiores fuscantes, 2.0-3.5 u in diam. Ballistosporae hyalinae, curvatae, minute granulosae, plerumque 2 vel 3 globulis oleosis armatae, uninucleatae, 2.0- 3.5 x 11.5-19.5 u in diam. repetitione aut tubulo germinali germinantes, excentrice sterigmatibus hypharum vel ballistosporarum affixae. Cellulae gemmiparae ballistosporis maiores, 2.0-3.0 x 16.0-22.0 u. Chlamydosporae non observatae. Growth on Malt extract agar: Colonies white, tough and cartilagi• nous in texture and relatively little mycelium is produced. Dalmau plate cultures on YMA: The aerobic growth is similar to that on MEA medium. Under anaerobic conditions; ballistospores or budded cells are rarely produced, most of the growth is in mycelial form, empty cells are frequently found. Chlamydospores are observed, mostly intercalary and empty cells occur on either side of a chlamydospore. The chlamydospores measure 6.0-14.0 u in diameter and their shape varies from globose to obovate. 33. Colonies cartilaginous and tough, white, older colonies extensive• ly mycelial. Hyphae usually delicate, septate, branched, hyaline when young, darker with age, 2.0-3.5 u in diam. Ballistospores hyaline, curved, finely granular, usually with 2 to 3 oil globules, uninucleate, 2.0-3.5 x 11.5-19.5 u., germinating by repetition or by germ tube, eccentrically attached to sterigmata of hyphae or ballistospores. Budded cells larger than the ballistospores, 2.0-3.0 x 16.0-22.0 u. Chlamydospores not ob• served. Type culture: UBC # 926, isolated from filamentous organisms from sewage in Libertyville, Illinois, U.S.A. It was recovered by pour plate method on neopeptone-dextrose agar at 1:10000, in July, 1968. It is preserved in the Mycology Herbarium, University of British Columbia, Vancouver 8, B.C. Etymology: albescens - becoming white; from Latin, albus and suffix-scens (becoming). Life history: Ballistospores are produced on sterigmata which arise from other ballistospores or from the mycelium. No clamps are formed. Tilletiopsis pallescens Gokhale sp. n. Coloniae pellescenti-albicantes, veteriores durae, cartilaginosae. Hyphae tenellae, septatae, ramosae, ivniores hyalinae, vetustiores fuscantes, 2.0-3.5 u in diam. Ballistosporae hyalinae, curvatae, minute granulosae, uninucleatae, 2.0-3.5 x 10.5-19.0 u., sempere mycelio productae, excentrice sterigmatibus affixae. Ballistosporae tubulo germinali germi- nantes. Cellulae gemmiparae raro praesentes. Chlamydosporae non observa- tae. 34. PLATE III Tilletiopsis albescens Figure 1: Ballistospores Figure 2: Budded cells Figure 3: Ballistospores germinating by repetition Figure 4: Ballistospores produced from hyphae Figure 5: Chlamydospores (under anaerobic conditions, on YMA medium) PLATE Ml T. albescens 35. Growth on Malt extract agar: Relatively little mycelium is produced and colonies are pale white and not so cartilaginous. Mycelium is septate, branched and measures 2.0-3.5 u in diameter. Dalmau plate culture on YMA: The aerobic growth is similar to that on MEA. Anaerobic growth: Ballistospores or budded cells are rarely produced. Most of the growth is mycelial; some of the inflated cells show dense contents. Chlamydospores are not observed. Hyphae delicate, septate, branched, hyaline when young, becomes dark with age, 2.0-3.5 u in diameter. Ballistospores hyaline, curved, finely granular, uninucleate, 2.0-3.5 x 10.5-19.0 u., always produced from the mycelium, eccentrically attached to sterigmata; ballistospores germinate by germ tube. Budded cells rarely formed. Chlamydospores not observed. Type culture: UBC #8007, Sirobasidium fruit body was collected in Shamoda, Japan in November, 1968 and the strain was isolated in Vancouver, B.C. in December, 1968. It is preserved in Mycology Herbarium, U.B.C. , Vane, 8, B.C. Etymology: pallescens- becoming white (pale); from Latin, pallidus and suffix-scens (becoming). Life history: Ballistospores are always produced from the mycelium without mating, clamps are not formed. Ballistospores germinate giving rise to mycelium; budded cells are rarely formed. PLATE IV Tilletiopsis pallescens Figure 1: Ballistospores Figure 2: Ballistospores germinating by germ tube Figure 3: Hyphal swellings (under anaerobic conditions, on YMA medium) PLATE IV 3 Tilletiopsis fulvescens Gokhale sp. n. Coloniae iuniores saccharomycetarum similes, vetustiores myceliales, iuniores ochraceo-croceae, vetustiores fuscatae ("Sudan Brown-Dresden Brown). In medio saccharomycetarum cerevisiae extracto adjecto. Hyphae tenellae, septatae, ramosae, iuniores hyalinae, 1.0-2.5 u in diam. Ballistosporae hyalinae, curvatae, minute granulosae, uninucleatae, 1.0-2.5 x 8.0-15.0 u., excentrice sterigmatibus mycelio ortae affixae. Chlamydosporae non observatae. Growth on Malt extract agar: Colonies are white to "Buff yellow" and soft in consistency. There is no pigment production. Dalmau plate culture on YMA: Colonies are "Ochraceous orange" to "Sudan Brown" and a soluble "Sudan Brown" to "Dresden Brown" pigment is produced. The aerobic growth, in general, is similar to that on MEA. Anaerobic growth: Ballistospores or budded cells are rarely observed and most of the growth is in mycelial form. Chlamydospores are produced, either intercalary or terminal and usually show dense contents. They are smooth walled, clavate to obovate in shape and measure 5.0-12.0 in diam. Colonies yeast-like when young, later mycelial; when young "Ochraceous orange", then "Sudan Brown", produce soluble "Sudan Brown" to "Dresden Brown" pigment in presence of yeast extract. Hyphae delicate, septate, branched, hyaline when young, 1.0-2.5 u in diameter. Ballisto• spores hyaline, curved, finely granular, uninucleate, 1.0-2.5 x 8.0-15.0 u., eccentrically attached to sterigmata which arise only from the mycelium. Budded cells rarely formed. Chlamysospores not observed. 38. Type culture: UBC //8006, isolated from Forsythia leaf which was collected in North Vancouver, B.C., in Sept., 1968. It Is preserved in the Mycology Herbarium, University of British Columbia, Vancouver 8, B.C. Etymology: fulvescens - becoming yellowish brown; from Latin, fulvus and suffix-scens (becoming). Life-history: Ballistospores germinate by germ tubes which in turn produce new ballistospores, without mating; clamps are not formed. PLATE V Tilletiopsis fulvescens Figure 1: Ballistospores Figure 2: Ballistospores germinating by germ tube Figure 3: Ballistospores produced from hyphae Figure 4: Chlamydospores (under anaerobic conditions, on YMA medium) PLATE V T. fulvescens AO. PLATE VI Figures 1, 2 and 3: Tilletiopsis fulvescens: effect of yeast extract on pigment production Figure 1: No yeast extract Figure 2: 0.05 percent yeast extract Figure 3: 0.15 percent yeast extract Note that the pigment production increased when the concentration of yeast extract was raised (on MYP medium) Figures 4 and 5: Cultural characteristics of T_. albescens Figure 4: on CZD medium Figure 5: on MYP medium Figures 6 and 7: Cultural characteristics of T. pallescens Figure 6: on CZD medium Figure 7: on MYP medium Figures 8 and 9: Cultural characteristics of _T. fulvescens Figure 8: on CZD medium (no yeast extract) Figure 9: on MYP medium (with yeast extract) Figures 10, 11 Nuclear staining (Iron-haematoxylin stain) and 12: Figure 10: T_. albescens- uninucleate ballistospores Figure 11: T?. pallescens- uninucleate ballistospores Figure 12: T. fulvescens- uninucleate ballistospores PLATE VI 41. Tubaki (1952) presented a key to identify four described species and a variety of Tilletiopsis spp. His identification was based on colour and texture of the colonies. During these studies, several other isolates were collected from the vicinity of Vancouver, B.C. and some also were obtained from UBC culture collection. The colonies of these isolates showed a variety of colours ranging from whitish cream to yellowish brown. It was observed that the placing of the isolates into described species only on the basis of colour and the texture of the colonies is difficult. Therefore, a key for identification of Tilletiopsis species is prepared to include the previously described as well as the species mentioned herein. The key, presented below, is based on their nutritional, physiological and morphological characteristics observed during this investigation. "A Key for the identification of species of Tilletiopsis" la. Arbutin splitting positive 2. lb. Arbutin splitting negative 3. 2a. Sodium nitrate, glutamic acid assimilated 4. 2b. Sodium nitrate, glutamic acid not assimilated 5. 4a. No pigment production Glucose, maltose, starch assimilated, phenylalanine not assimilated T_. washingtonensis 4b. Soluble yellowish brown pigment produced Glucose, maltose, starch not assimilated, phenylalanine assimilated T_. fulvescens 5a. Maltose assimilated T_. lilacina 5b. Maltose not assimilated T. cremea 42. 3a. Thiamine dependent, gelatin liquefaction positive chlamydospores produced Little mycelium produced 6. 3b. Thiamine independent, gelatin liquefaction negative chlamydospores not produced extensive mycelium produced 7. 6a. Maltose, phenylalanine assimilated T. minor 6b. Maltose, phenylalanine not assimilated T. minor var flava 7a. Ethanol, starch, maltose, phenylalanine assimilated T. albescens 7b. Ethanol, starch, maltose, phenylalanine not assimilated _T. pallescens The validity of a classification system, based on nutritional and physiological characters, is often questioned by taxonomists. To check the validity of the present key, seven tentatively identified isolates of Tilletiopsis (Table 1-B) were run through the nutritional and physiological tests. On the basis of these tests, isolate 8030 was placed in _T. lilacina whereas isolate 8033 was identified as T_. minor. The key was found to be satisfactory in identification of the remaining five iso• lates of Tilletiopsis. These results suggest that the proposed key could be used in identification of the species of Tilletiopsis with a good deal of success. 43. CHAPTER 4 DISCUSSION Morphological characters have traditionally been used to classify fungi. The use of morphological characters has been defended by several workers; Snyder and Tousson (1965) suggested that only morphological characters should be used to delimit species and all higher categories. Hall (1969) pointed out that the morphological approach has several out• standing values; he stated that "Provided the appropriate morphologic stages are present, the fungus, whether alive or dead, can be identified on any substrate by biologists with access to normal biological laboratory facilities." There are" many workers who consider that morphological characters are more variable than the physiological ones (Wickerham and Rettger, 1939; Skinner, 1947; Griffiths, 1958) and the latter should be used in the classification. In some cases, classifications based on morphology are of limited value because of a lack of suitable diagnostic features (Parmeter, 1965) and in some, special conditions are needed to allow the development of the morphological features on which the system is based. A good example of this latter is in Phytophthora (Erwin et al., 1963), where extensive cultural and mating tests are often necessary for satisfactory determination of species. In summarizing the difficulties in using morphological charac• ters in classification systems, Hall (1969) stated that "We have situations 44. where suitable morphological features are rare, sporadic or lacking. We have situations in which similarities in morphology do not necessarily indicate genetic relationship. We have situations where weighting of characters by different individuals is such that confusion and controversy cloud the true genetic relationships." Hall further suggested that in such instances taxanomists should look to new methods to supplement or supplant the old ones. Recently, Lodder (1970) reviewed the taxonomy of yeasts and yeast-like organisms and pointed out some new trends in their classifica• tion systems. Barnett (1960,1961) suggested that yeasts should be classi• fied on purely biochemical criteria although the suggestion has not been accepted (Roberts and Thome, 1960; van der Walt, 1970). van der Walt (1970) indicated that no yeast can be identified or classified with confi• dence until a systematic study has been made of its morphological, cultural, sexual and physiological characteristics. He stated that "Morphological and reproductive characteristics are employed to decide the main taxonomy i.e. to demarcate the higher taxa, while physiological criteria are used to differentiate the lower taxa, species in particular." The ability to utilize different sources of carbon and nitrogen has often been used in the classification of yeasts and yeast-like organ• isms. Wickerham (1951) suggested that the assimilation tests would prove the most valuable of all the biochemical procedures used in the classifica• tion of yeasts. The taxonomic value of various criteria such as arbutin splitting, growth in vitamin free media, gelatin liquefaction etc. has been recently summarized by van der Walt (1970). 45. Several attempts have been made to present keys for identification of yeasts (Wickerham, 1951; Lodder and Kreger-van Mj, 1952; Hasegawa and Banno, 1970). Amongst the members of family Sporobolomycetaceae, Phaff (1970) presented keys for identification of species of 3 genera; Sporobolomyces, Bullera and Sporidiobolus. His keys are primarily based on nutritional and morphological characteristics of these organisms. Previously, Tubaki (1952) proposed a key for identification of the species of Tilletiopsis based entirely on colour and consistency of the colonies. As pointed out earlier, these features were found to be variable and therefore it was found difficult to place several isolates in the described species. In the present study, three previously unknown species of Tilletiopsis have been described and a key has been proposed to identify the species of Tilletiopsis. The separation of the species and the pro• posed key are mainly based on their nutritional, physiological and mor• phological characteristics. The features such as assimilation of carbon and nitrogen compounds, splitting of arbutin, pigment production, mode of germination of ballistospores, the production of chlamydospores have been used in delimiting the species of Tilletiopsis. Tilletiopsis spp. have been divided into two groups on the basis of their arbutin splitting ability. This property is commonly used as one of the characteristics to distinguish the genus Hansenula (arbutin positive) from the genus Pichia (arbutin negative). Barnett et al. (1956) and Cook (1958) have also used this character for taxonomic purposes. Further separation of Tilletiopsis species is based on their abilities to utilize 46. various carbon and nitrogen sources. Such assimilation tests have long been used in yeast taxonomy (Wickerham, 1951, Lodder and Kreger-van Rij, 1952). Phaff (1970) has made use of these assimilation tests in dis• tinguishing species of Sporobolomyces, Bullera and Sporidiobolus. T^. fulvescens was found to produce a soluble yellowish brown pigment in the presence of yeast extract; this character alone separates T_. fulvescens from other species of Tilletiopsis. The utilization of carbon and nitrogen compounds was characteristic in _T. albescens and T\ pallescens; the biomass production was about 10 to 15 times greater than that observed in any other species. Also, T_. albescens and T_. pallescens were found to be thiamine independent, they did not liquify gelatin and produced extensive mycelium; these features separated them from T_. minor and T. minor var flava. Vitamin requirement has been used as a taxonomic criterion by Schultz and Atkin (1947) for Saccharomyces cerevisiae, Wickerham (1951) for Hansenula spp. and by Hasegawa and Banno (1963) for species of Rhodotorula. Gelatin liquefaction as a taxonomic criterion has been found to be of limited value (Wickerham, 1951; van der Walt, 1970); in the present scheme gelatin lique• faction has been used along with other characters. Attempts were also made to search for the sexual phase in Tilletiopsis although they did not prove successful. In recent years, several workers have reported the occurrence of sexuality in yeasts and yeast-like organisms. The mating reactions were observed in Rhodotorula by Banno (1967) and in Candida-like species by Fell et al. (1969). It is noteworthy that the four species of Rhodosporidium (perfect stage of Rhodotorula), all had the same imperfect state on the basis of nutritional 47. and physiological studies. Banno (1967) and Fell et al. (1969) have clearly shown that these yeast genera belong to the order Ustilaginales. In the present study, the failure in obtaining the sexual phase in Tilletiopsis eliminated the important feature which has often been used in the taxonomy of many fungi. In general, most of the criteria which have been used here are often employed in taxonomy of yeasts and yeast-like organisms. The keys (Lodder and Kreger-van Rij, 1952; Phass, 1970) based on such criteria have proved very useful for identification purposes. The author believes that the present key will also prove useful in routine work and identifi• cation of species of Tilletiopsis. The author is aware of certain limitations of the present study. There are several other nutritional and physiological properties which have not been studied and some have not been fully investigated. The use of large numbers of isolates generally provides the range of variation within a species. By altering some media employed here, or using large numbers of isolates, some variation might be obtained. In the literature one may find that new species have been described on the basis of only one or two nutritional differences (Shehata et al., 1955, Capriotti, 1961 b). Also, in keys presented by various workers, two species are differentiated on the basis of one or two nutritional differ• ences (Uden and Buckley, 1970; Phaff and Ahearn, 1970). There is differ• ence of opinion regarding the number of nutritional characters which would be sufficient to delimit a species. This judgement solely rests on the 48. individual and to date there is no standardization for the number/s of nutritional characters which should be used in delimiting the species. In several instances, new species have been described on the basis of a single isolate (Phaff and do Carmo-Sousa, 1962; van der Walt, 1970). With the above mentioned facts in mind, three new species of Tilletiopsis have been described. However, in writer's opinion, it would be worthwhile to study a larger number of isolates and to explore biochemical and physiolo• gical characters which might provide a better understanding for classifi• cation of the genus Tilletiopsis. 49. BIBLIOGRAPHY Ainsworth, C.G. 1961. Dictionary of the fungi. Commonwealth Mycologi- cal Institute, Kew, Surrey. Alexopoulos, C.J. 1962. Introductory Mycology (Ed. 2). John Wiley and Sons, Inc., New York. Barnett, J.A. , M. Ingram and T. Swain. 1956. The use of B-glucosides in classifying yeasts. J. Gen. Microbiol. 15: 529-555. Barnett, J.A. 1960. Comparative studies of yeasts. Nature, 186: 449-451, Barnett, J.A. 1961. Biochemical classification of yeasts. Nature. 189: 76. Bandoni, R.J., K.J. Lobo and S.A. Brezden. 1971. Conjugation and chlamydo• spores in Sporobolomyces odorus. Can. J. Bot. 49: 683-686. Banno, I. 1967. Studies on the sexuality of Rhodotorula. J. Gen. Appl. Microbiol. 13: 167-196. Bessey, E.A. 1950. Morphology and taxonomy of fungi. The Blakiston Co., Philadelphia. Buller, A.H.R. 1933. Researches on fungi. 5: 171-206. Longmans, Green and Co., London. Capriotti, A. 1961b. Debaryomyces cantarellii nova species, a new yeast isolated from Finnish soil. Arch. Microbiol. 39: 123-129. Cochrane, V.W. 1958. Physiology of fungi. John Wiley and Sons, Inc., New York. Cook, A.H. 1958. The chemistry and biology of yeasts. Academic Press. London. Derx, H.G. 1930. 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Chapter 2. In: The yeasts. A taxonomic approach, (ed.) Lodder, N. Holland Publ. Co. Amsterdam. 34-113 pp. Wickerham, L.J. and L.F. Rettger, 1939. A taxonomic study of Monilia albicans with special emphasis on morphology and morphological variation. J. Trop. Med. Hyg. 42: 174-177, 187-192, 204-216. Wickerham, L.J. 1951. Taxonomy of yeasts. Technical bulletin no. 1029. V.S. Dept. of Agriculture. 1-56 pp. 53. APPENDIX NO. 1 MEDIA: The concentration of agar in solid media was 1.2 percent due to its high gel strength. 1) Czapek's Dox medium: (CZD) NaNOa 2.0 g. K2HP0, 1.0 g. KC1..7 0.5 g. MgS0,.7H?0 0.5 g. FeS0£...7 0.01 g. Sucrose 30.0 g. Distilled Ho0 1 liter While preparing CZD medium, ferrous and magnesium salts were autoclaved separately and added aseptically to the remainder of the medium, thus preventing the formation of precipitates. 2) Malt Yeast extract Peptone medium: (MYP) Difco malt extract 15.0 g. Difco yeast extract.... 0.5 g. Difco peptone 2.5 g. Distilled H20 1 liter 3) Malt Extract Agar medium: (MEA) Maltose 12.75 g. Dextrin 2.75 g. Glycerol 2.35 g. Difco peptone 0.78 g. Distilled H20 1 liter 4) Water Agar: (WA) Bacto agar 12.0 g. Distilled H20 1 liter 5) Minimal medium: (MM) Glucose 50.0 g. (NH ) SO, 3.0 g. KH PO^. .7 1.0 g. MgSO .7H 0 0.5 g. NaCl; 0.1 g. CaCl2 0.1 g. Difco yeast extract 2.5 g. vitamin sol. (con) 10.0 ml Distilled H20 1 liter 54. Appendix No. 1 (cont'd) 6) Malt Yeast extract Peptone Glucose medium: (MYP) Difco malt extract 3.0 g. Difco yeast extract 3.0 g. Difco peptone 5.0 g. Glucose 50.0 g. Distilled Ho0 1 liter Difco dehydrated media were used to prepare Potato dextrose agar (PDA) Corn meal agar (CMA) and Yeast morphology agar (YMA). Their preparation was as directed in Difco manual (1964) . In addition to these general media, some special media were also used in this study. The composition of these media is given at appropriate places in the text. 55. APPENDIX NO. 2 STAINS: 1) Iron-haematoxylin stain (Johansen, 1940) a) Stock solution: To 100 ml of distilled water, I added 0.05 g. of sodium bicarbonate and boiled the solution. A quantity of 0.5 g. of dye was then added and the solution was allowed to cool. The stain was stored in a refrigerator at 4 C. b) Mordant: 4 percent iron alum (ferric ammonium 500 ml. sulphate) Glacial acetic acid 6 ml. Concentrated sulphuric acid 0.6 ml. c) Destaining agent: Mordant (above described) diluted with an equal volume of distilled water. d) Fixative: Weak chromic-acetic acid solution 10 percent aqueous chromic acid 2.5 ml 10 percent aqueous acetic acid 5.0 ml Distilled water 100 ml APPENDIX 3 (Riker and Riker, 1936) CLEANING SOLUTION: Procedure: The potassium dichromate was dissolved in the distilled water and the acid was added slowly to the solution.