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Monograph of the genus Tilletiopsis Gokhale , Atulchandra Anant 1971

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A MONOGRAPH OF THE GENUS TILLETIOPSIS By ATULCHANDRA ANANT GOKHALE B . S c , M.Sc. , U n i v e r s i t y of Poona A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science i n the Department of Botany We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA Ju l y , 1971 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Brit ish 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 i i ABSTRACT N u t r i t i o n a l and p h y s i o l o g i c a l studies were made on species of T i l l e t i o p s i s . The v a r i a t i o n i n a s s i m i l a t i o n of glucose, maltose, starch, sodium n i t r a t e , glutamic a c i d and phenylalanine proved to be useful i n d e l i m i t i n g the species. The other c r i t e r i a used i n the c l a s s i f i c a t i o n of T i l l e t i o p s i s were s p l i t t i n g of arbutin, pigment production, thiamine requirement and g e l a t i n l i q u e f a c t i o n . On the basis of these r e s u l t s , a key f o r i d e n t i f i -c a tion of species of T i l l e t i o p s i s has been proposed. Three previously unknown species of T i l l e t i o p s i s ; T_. albescens, T_. pallescens and T_. fulvescens have been described. These new species showed some v a r i a t i o n i n t h e i r morphology as w e l l as i n t h e i r physiology. i i i LIST OF CONTENTS PAGE INTRODUCTION AND REVIEW OF LITERATURE 1 Presentation of the problem 6 MATERIALS AND METHODS 8 I s o l a t i o n 8 Inoculum preparation 9 Morphology 9 N u t r i t i o n and physiology 12 E f f e c t s of pH on growth 12 E f f e c t s of temperature on growth 12 A s s i m i l a t i o n of carbon compounds 13 A s s i m i l a t i o n of nitrogen compounds 13 E f f e c t s of vitamins on growth 13 S p l i t t i n g of arbutin 14 Ethanol as a s o l e carbon source 14 Production of starch 15 Production of a c i d on chalk agar medium 15 A b i l i t y to l i q u i f y g e l a t i n 15 Hydrolysis of urea 16 Conjugation studies 16 OBSERVATIONS 18 Morphology 18 N u t r i t i o n and physiology 22 E f f e c t s of pH on growth 22 E f f e c t s of temperature on growth 22 A s s i m i l a t i o n of carbon and nitrogen compounds 22 E f f e c t s of vitamins on growth 23 Results of miscellaneous tests 24 Conjugation studies 24 Descriptions of the new species of T i l l e t i o p s i s 32 A key f o r the i d e n t i f i c a t i o n of species of T i l l e t i o p s i s 41 DISCUSSION 43 BIBLIOGRAPHY 49 APPENDIX 1 - Composition of the media 53 2 - Stains 55 3 - Cleaning s o l u t i o n 55 i v LIST OF TABLES Page TABLE 1. a) A l i s t of i s o l a t e s used i n morphological, n u t r i t i o n a l and p h y s i o l o g i c a l studies 10 b) A l i s t of i s o l a t e s used i n the conjugation studies 11 TABLE 2 E f f e c t of pH on growth of T i l l e t i o p s i s species.... 25 TABLE 3 A s s i m i l a t i o n of various carbon and nitrogen compounds by T i l l e t i o p s i s species. 27 TABLE 4 E f f e c t s of various vitamins on growth of T i l l e t i o p s i s species 28 TABLE 5 Results of miscellaneous tests on species of T i l l e t i o p s i s 29 TABLE 6 P h y s i o l o g i c a l c h a r a c t e r i s t i c s of taxonomic value f o r d e l i m i t i n g T i l l e t i o p s i s species 31 V . LIST OF PLATES Page PLATE 1: C u l t u r a l c h a r a c t e r i s t i c s of the species of T i l l e t i o p s i s 21 PLATE 2: E f f e c t s of d i f f e r e n t temperatures on growth of various species of T i l l e t i o p s i s 26 PLATE 3: Morphology of T i l l e t i o p s i s albescens 34 PLATE 4: Morphology of T i l l e t i o p s i s pallescens 36 PLATE 5: Morphology of T i l l e t i o p s i s fulvescens 39 PLATE 6: Figures 1 to 3: T i l l e t i o p s i s fulvescens: e f f e c t of yeast extract on pigment production. Figures 4 to 9: C u l t u r a l c h a r a c t e r i s t i c s of _T. albescens, _T. pallescens and T_. fulvescens Figure 10 to 12: nuclear s t a i n i n g 40 v i . ACKNOWLEDGEMENTS I am indebted to Dr. R.J. Bandoni who introduced me to t h i s f i e l d of research; h i s continuing i n t e r e s t was most valuable. Thanks are also due to Drs. K. Tubaki, K. Wells, W.B. Cooke and Mrs. S. Reid f o r providing c e r t a i n i s o l a t e s used i n t h i s study. It i s my pleasure to thank Dr. B.N J o h r i f o r many invaluable suggestions; discussions with him proved very useful i n carr y i n g out t h i s work. F i n a n c i a l assistance through a teaching A s s i s t a n t s h i p from the Department of Botany and a Research A s s i s t a n t s h i p through N.R.C. grants of Dr. Bandoni i s g r a t e f u l l y acknowledged. Thanks are due to Mrs. Pat Waldron f o r taking the enormous task of typing the manuscript. CHAPTER 1 INTRODUCTION The family Sporobolomycetaceae was defined (Derx, 1930) to include the genera Sporobolomyces, B u l l e r a and T i l l e t i o p s i s . Sporobolomyces was f i r s t described by Kluyver and van N i e l (1924) and B u l l e r a was described by Derx (1930). The name T i l l e t i o p s i s was p r o v i s i o n a l l y suggested f o r a fungus found on some leaves by Derx (1930) and he v a l i d a t e d i t with a short L a t i n diagnosis i n 1948. Derx (1948), described another new genus, I t e r s o n i l i a , p l a c i n g i t i n the family Sporobolomycetaceae. Nyland (1948) published an account of an unusual fungus which he l a t e r (1949) described as a new genus and species, Sporidiobolus Johnsonii. The genus was con-sidered by him to belong to the family Sporobolomycetaceae. A l l members of t h i s family produce a e r i a l c o n i d i a attached assyme-t r i c a l l y at the apex of sterigmata and discharged v i o l e n t l y . Kluyver and van N i e l (1924) observed that i n Sporobolomyces, each conidium was d i s -charged by a drop-excretion mechanism of the same nature as that found on b a s i d i a of Hymenomycetes and the Uredineae. Derx (1930) also pointed out that the spores of Sporobolomyces were projected at maturity i n the same manner as basidiospores and proposed the name 'b a l l i s t o s p o r e ' s to these c h a r a c t e r i s t i c c o n i d i a . B u l l e r a species normally do not produce mycelium and remain yeast-l i k e ; Sporobolomyces includes both y e a s t - l i k e and mycelial forms, but the 2. occurrence of -mycelium i s r e s t r i c t e d to very few species. The genera I t e r s o n i l i a , Sporidiobolus and T i l l e t i o p s i s , a l l produce mycelium. In I t e r s o n i l i a and Sporidiobolus, abundant clamp connections are formed on the mycelium; clamps are la c k i n g i n T i l l e t i o p s i s . The colonies of Sporobolomyces and Sporidiobolus are usually pink to salmon coloured because of the presence of carotenoid pigments; one species of Sporobolomyces, S_. s i n g u l a r i s , shows white colonies. In B u l l e r a and I t e r s o n i l i a pigments generally are l a c k i n g and the colonies are p a l l i d to y e l l o w i s h . T i l l e t i o p s i s colonies are generally cream to white though yellowish and l i l a c coloured colonies are found i n some species. In Sporidiobolus abundant chlamydospores are produced while i n I t e r s o n i l i a and T i l l e t i o p s i s , chlamydospores are few and generally are formed only i n o l d c u l t u r e s . L a f f i n and Cutter (1959) c a r r i e d out extensive studies on the genus Sporidiobolus; i n t h e i r proposed l i f e c y c l e , the d i p l o i d phase alternated with a d i c a r y o t i c phase and the two were shown to be of equal importance i n the l i f e c ycle of the organism. These authors also reported the occur-rence of sexual and asexual cycles i n Sporobolomyces and T i l l e t i o p s i s but t h e i r studies were never published. Sowell and Korf (1960) suggested merging T i l l e t i o p s i s and I t e r s o n i l i a and proposed that the name I t e r s o n i l i a be used f o r the combined genus. Their proposal was based upon observations on the budding monocaryophase i n I t e r s o n i l i a . They state " I t i s only i n the shape of the b a l l i s t o s p o r e s , 3. f a l c a t e i n the T i l l e t i o p s i s , lunate-reniform i n monocaryophase I t e r s o n i l i a , that a d i s t i n c t i o n can be drawn. The constancy of t h i s s i n g l e character has not been s u f f i c i e n t l y i n v e s t i g a t e d . " Sowell and Korf (I960), apparently f a i l e d to r e - e s t a b l i s h the dicaryophase from the monocaryophase i n I t e r s o n i l i a and therefore t h e i r suggestion has not been followed. A sexual phase was observed by van der Walt (1970) i n a s t r a i n of Sporobolomyces salmonicolor (Fischer et Brebeck) Kluyver et van N i e l , 1894; the name Aessosporon was proposed f o r t h i s perfect stage. The s t r a i n was found to form t e l i o s p o r e s which germinated u s u a l l y by the formation of a non-septate promycelium bearing 2-4 s p o r i d i a . This new basidiomycete yeast genus was assigned to family T i l l e t i a c e a e . Recently, i n Sporobolomyces  odorus Derx, 1930; Bandoni et a l . (1971) observed conjugation, d i c a r y o t i c hyphae with clamp connections and chlamydospores. The s i m i l a r i t i e s i n t h i s d i c a r y o t i c phase and that of Sporidiobolus j o h n s o n i i were s t r i k i n g and i t was suggested that they could be the same. The family Sporobolomycetaceae i s generally included i n the Basidiomycetes, although i t s a f f i n i t i e s are not c l e a r . Kluyver and van N i e l (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 t h e i r accept-ance of t h i s view ( B u l l e r , 1933; Martin, 1952; S a i n c l i v i e r , 1952). Alexopoulos (1962) stated that some of the characters of the Sporobolomyce- taceae point convincingly to a basidiomycetous r e l a t i o n s h i p and he includes them with Basidiomycetes. A d i f f e r e n t view was taken by Lowag (1926); he 4. did not consider Sporobolomyces as of basidiomycete o r i g i n . Bessey (1950) included the family Sporobolomycetaceae with non-ascosporogenous yeasts. Nyland (1950) c a r r i e d out a comparative study of two species of T i l l e t i o p s i s and a species of Entyloma. He pointed out a d i f f e r e n c e i n cultures of Entyloma and T i l l e t i o p s i s i n that the b a l l i s t o s p o r e s of Entyloma did not bud i n a y e a s t - l i k e manner. Nyland suggested that the T i l l e t i o p s i s may represent n o n - p a r a s i t i c forms of the 'white-smuts.' The name T i l l e t i o p s i s was p r o v i s i o n a l l y suggested by Derx (1930) because of the morphological resemblance to cultures of T i l l e t i a spp. His f i v e i s o l a t e s were r e a d i l y d i s t i n g u i s h a b l e on the basis of b a l l i s t o s p o r e s i z e . The spore si z e s of h i s s t r a i n s 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 L a t i n diagnosis given by Derx (1948) i s as follows: Mycelium hyalinum, septatum, repens, sterigmata i n aera ascendentia formans. Sporae s o l i t a r i a e , f a l c a t e , leves, hyalinae. Species t i p i c a : T i l l e t i o p s i s spec. No. 4 Derx. Nyland (1950) c o l l e c t e d two i s o l a t e s of T i l l e t i o p s i s from a v a r i e t y of hosts during the summer of 1947 i n Washington s t a t e , U.S.A. His two i s o l a t e s had spore s i z e s averaging 9 x 1.8 u and 14 x 2.4 u. The f i r s t i s o l a t e resembled Derx's i s o l a t e no. 1 and second resembled Derx's i s o l a t e s no.s 3 and 4. Nyland proposed the name T i l l e t i o p s i s washingtonensis f o r h i s large-spored species and considered i t as the type species of the genus; Derx's "type species" ( s t r a i n 4) was no longer a v a i l a b l e at the 'Centraalbureau voor Schimmelcultures'. For the i s o l a t e with small spores, 5. Nyland proposed the name T i l l e t i o p s i s minor; i t d i f f e r e d from the _T. washingtonensis by i t s smaller spores, darker colony colour and the c a r t i -laginous consistency. During the summer of 1951, Tubaki (1952) i s o l a t e d 16 s t r a i n s of T i l l e t i o p s i s from leaves of 13 d i f f e r e n t hosts i n the v i c i n i t y of Tokyo, Japan. According to him, eight s t r a i n s were d i f f e r e n t from the T i l l e t i o p s i s species already described by Nyland. One s t r a i n resembled T_. minor but was more yel l o w i s h , d i d not produce soluble pigments, and formed f l a t colonies on agar media; Tubaki proposed the name T i l l e t i o p s i s minor var f l a v a f o r thi s i s o l a t e . The colonies of two s t r a i n s were s o f t i n texture and l i l a c i n colour; Tubaki described these i s o l a t e s as T i l l e t i o p s i s l i l a c i n a . He described another new species, T i l l e t i o p s i s cremea, f o r f i v e s t r a i n s with cream coloured colonies and smaller spores than those of T. washingtonensis. L i t t l e i s known about the n u t r i t i o n and physiology of T i l l e t i o p s i s species. Tubaki (1952) reported that 25 C was the optimum temperature f o r growth of T_. minor, T. minor var f l a v a , _T. cremea and _T. l i l a c i n a . Sundstrom (1964) c a r r i e d out a comparative study of carbon a s s i m i l a t i o n by three s t r a i n s of T i l l e t i o p s i s and a species of Exobasidium; he also i n v e s t i g a t e d e f f e c t s of vitamins on growth of these organisms. However, he did not i d e n t i f y the species of T i l l e t i o p s i s he studied nor did he i n -clude a d e s c r i p t i o n of the s t r a i n s . 6. Presentation of the Problem Tubaki (1952) published a key f o r i d e n t i f i c a t i o n of a l l known species of T i l l e t i o p s i s . His key i s based e n t i r e l y on the colour and consistency of colonies as follows: KEY TO SPECIES (Tubaki, 1952) Colony y e l l o w i s h colony yellow, then brown T\ minor colony b r i g h t yellow _T. minor var f l a v a Colony cream coloured surface tough T_. washingtonensis surface s o f t T_. cremea Colony l i l a c coloured T_. l i l a c i n a In the summer of 1970, I obtained s e v e r a l i s o l a t e s of T i l l e t i o p s i s from Dr. .R.J. Bandoni and attempted to i d e n t i f y them using Tubaki's key. I t was observed that the characters such as colour and consistency of colonies were v a r i a b l e and were influenced by c u l t u r a l conditions, medium composition and age of the cu l t u r e . Some i s o l a t e s showed d i s t i n c t colony colours as described by Tubaki, but others v a r i e d i n colour, e s p e c i a l l y between yellowish brown and b r i g h t yellow. I also found many colonies with consistencies varying from s o f t to tough. Some colonies appeared to be s o f t when young, but they toughened with age. Because of the in c o n s i s t e n -c i e s i n colour and texture of colonies, i t was found to be d i f f i c u l t to place these 'intermediate' i s o l a t e s i n the described species. 7. B a l l i s t o s p o r e s i z e was used by Derx (1930) and to some extent by Nyland (1950) to d i f f e r e n t i a t e between the species of T i l l e t i o p s i s . During my preliminary studies b a l l i s t o s p o r e measurements were made and diff e r e n c e s did not appear to be c h a r a c t e r i s t i c . In general, the morphology of b a l l i s t o s p o r e s did not reveal d i s t i n c t characters which could be used i n i d e n t i f i c a t i o n of the species. The i n i t i a l studies on i s o l a t e s from the U.B.C. culture c o l l e c t i o n suggested the presence of some undescribed species. I n a b i l i t y to adequately describe and d i s t i n g u i s h among species on the basis of c h a r a c t e r i s t i c s r e -ported i n the l i t e r a t u r e l e d to attempts to use n u t r i t i o n a l and p h y s i o l o g i c a l c h a r a c t e r i s t i c s . These features are already i n use i n i d e n t i f i c a t i o n of species of Sporobolomyces, Sporidiobolus and B u l l e r a . This study was undertaken (1) to examine the p o s s i b i l i t y of using n u t r i t i o n a l and p h y s i o l o g i c a l c h a r a c t e r i s t i c s i n i d e n t i f i c a t i o n , (2) to make test crosses i n an attempt to f i n d the sexual stage and (3) to prepare a monograph of the genus T i l l e t i o p s i s . 8. CHAPTER 2 MATERIALS AND METHODS I s o l a t i o n ; Derx's (1930) "spore f a l l " method was used f o r i s o l a t i o n of the s t r a i n s of T i l l e t i o p s i s . Leaves or plant parts were f i x e d with I masking tape i n s i d e a s t e r i l e P e t r i p l a t e l i d and i t was inver t e d over Malt extract-Yeast extract-Peptone (MYP) agar medium (Appendix 1). B a l l i s -tospores were discharged v i o l e n t l y and landed on agar l a y e r where they germinated and grew into colonies. A large number of b a l l i s t o s p o r e s was discharged w i t h i n 2 to 3 hours. By turning the l i d around at short i n t e r -v a l s during discharge of the b a l l i s t o s p o r e s , a greater d i s p e r s a l was ob-tained. A f t e r microscopic examination through the bottom of the P e t r i p l a t e , a s i n g l e colony was i s o l a t e d . In doubtful cases, the i s o l a t e d colo-nies were tra n s f e r r e d to n u t r i e n t s o l u t i o n s . A f t e r 48 hours of incubation, suspensions were plated out on an MYP agar medium and s o l i t a r y colonies were i s o l a t e d . Stock cultures were maintained on MYP agar s l a n t s and were stored i n a r e f r i g e r a t o r at 4 C. The composition of media used during t h i s study are given i n Appendix 1. A l i s t of the s t r a i n s studied, year and place of i s o l a t i o n and t h e i r sources i s given i n Table 1. The numbers given to the various s t r a i n s are the U.B.C. culture c o l l e c t i o n numbers. 9. Inoculum preparation; ( L i l l y and Barnett, 1951) Each s t r a i n was grown on a MYP pl a t e f o r 7 days at room temperature. A d i s c (14 mm-diam.) of mycelium and agar was t r a n s f e r r e d to a s t e r i l e blender containing 80 ml of s t e r i l e d i s t i l l e d water. The fungus was blended at low speed and was allowed to s e t t l e f o r about an hour; the supernatant served as the inoculum (approximately 35 ug/ml of spore suspension). Morphology P e t r i plates with MYP agar medium were inoculated with a c e l l sus-pension and incubated at 23 C f o r 2 days. The plate with fungus was i n -verted over a dry microscopic s l i d e and discharged b a l l i s t o s p o r e s were c o l l e c t e d on the s l i d e . The width and length of b a l l i s t o s p o r e s were mea-sured, f i f t e e n to t h i r t y spores were examined per spore p r i n t . Using the same technique, b a l l i s t o s p o r e s of a l l species and s t r a i n s were c o l l e c t e d on MYP plates and t h e i r modes of germination and growth were studied. The l i n e drawings were made by use of the Camera l u c i d a . Nuclear s t a i n i n g was performed with Heidenhain's 'iron hematoxylin' s t a i n (Johansen, 1940) ( f o r d e t a i l s , see Appendix 2). The i s o l a t e s were grown on MYP, Czapek's Dox (CZD), malt extract (MEA) and minimal medium (MM) agar and observations were made of t h e i r com-parative growth patterns. TABLE 1 10. (a) A) A l i s t of i s o l a t e s used i n morphological, n u t r i t i o n a l and p h y s i o l o g i c a l studies UBC species substrate or year and culture or source place of number s t r a i n i s o l a t i o n 912 925 915 916 917 907 926 (b) 8007 8006 (c) (d) c r e m e a Tubaki NI-3131 T. l i l a c i n a Tubaki NI-3114 T_. minor Nyland NI-3064 T. minor Nyland NI-6905 Dr. K. Tubaki, I n s t i t u t e of Fermentation, Osaka, Japan T_. minor var f l a v a Tubaki NI-3112 ) ) T. washingtonensis Nyland undescribed species undescribed species undescribed species Dr. K. Wells, Dept. of Botany, U n i v e r s i t y of C a l i f o r n i a , Davis, C a l i f . , U.S.A. filamentous organisms i n sewage Sirobasidium sp. f r u i t i n g body F o r s y t h i a sp. l e a f 1968, U.S.A. 1968 Japan 1968, B.C. Canada 11. B) A l i s t of i s o l a t e s used i n the conjugation studies (The i s o l a t e s were t e n t a t i v e l y i d e n t i f i e d ) UBC culture number species or s t r a i n substrate or source year and place of i s o l a t i o n 801 914 8030 8031 8032 8022 8029 927 T. l i l a c i n a Acer sp. l e a f 975 (e) 952 T. minor var f l a v a T. cremea undescribed species ( s i m i l a r to UBC #926) T. washingtonensis Asarum sp. l e a f Rubus sp. l e a f Rumex sp. l e a f filamentous or-ganisms from sewage Quercus sp. l e a f 1971, Hope, B.C. 1968, Parks-v i l l e , B.C. 1968, White Rock, B.C. 1970, Vane. B.C. 1970, Carbon-dale, I l l i n o i s U.S.A. 1968, C i n c i n -n a t i , Ohio, U.S.A. 1970, Missouri U.S.A. 1967, Iowa, U.S.A. a) With the exception of T_. minor, a l l other species are represented by one i s o l a t e . b) UBC i s o l a t e 926 was received from Dr. W.B. Cooke, Mycologist, B i o l o g i c a l Treatment Research Program, U.S. Dept. of the I n t e r i o r , C i n c i n n a t i , Ohio, U.S.A. c) Sirobasidium f r u i t i n g body was c o l l e c t e d i n Japan (Nov. 1968) by Mr. T. F l e g e l and the s t r a i n was i s o l a t e d by Mrs. S. Reid i n Vancouver (Dec. 1968) by 'Spore f a l l method'. d) I s o l a t e 8006 was obtained from Mrs. S. Reid, Botany Department, Un i v e r s i t y of B r i t i s h Columbia, Vancouver, B.C. e) UBC #975 was obtained from Mr. John N o e l l , Dept. of Botany, Washington "University, Missouri, U.S.A. 12. N u t r i t i o n and Physiology E f f e c t of pH on growth: L i q u i d CZD medium was used to determine e f f e c t s of d i f f e r e n t pH values on growth. F i f t y ml of the medium was d i s -pensed i n t o each Erlenmeyer f l a s k (125 ml). The pH of the medium was ad-just e d by adding 1 N HC1 or 1 N NaOH s o l u t i o n s ; 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 i n i t i a l pH values; the pH values were 4.0, 5.0, 5.6, 6.0, 6.5, 7.0, 7.5 and 8.0. The fl a s k s were inoculated with 1 ml of c e l l suspension and incubated f o r 14 days on a r e c i p r o c a t i n g shaker at 84 strokes/minute. The fungus from each f l a s k was c o l l e c t e d on a pre-weighed f i l t e r paper (Whatman no. 1). I t was washed, dr i e d at 80 C f o r about 24 hours and weighed. The pH of the f i l t r a t e was checked. No b u f f e r was added to the medium i n t h i s study. E f f e c t s of temperature on growth: The inoculum f o r temperature studies was prepared as described by L i l l y and Barnett (1951). Cultures were tr a n s f e r r e d from MP sla n t s to CZD agar medium and were incubated at room temperature f o r 4 days. Uniform discs (3 mm i n diam.) of mycelium and agar were cut from P e t r i dishes; each d i s c served as the inoculum source f o r 1 te s t c u l t u r e . The inoculum was placed i n the center of a P e t r i p l a t e 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 f o r 14 days i n the dark and the l i n e a r growth was recorded by measuring colony diameters ( i n c l u d i n g colony diameter of the inoculum). Each te s t was made i n t r i p l i c a t e . 13. A s s i m i l a t i o n tests and vitamin studies: In these studies, chemi-c a l l y cleaned glassware was used. A l l f l a s k s and pipettes were cleaned with a potassium dichromate-sulphuric a c i d cleaning s o l u t i o n (Appendix 3), and r i n s e d thoroughly i n glass d i s t i l l e d water before use. A s s i m i l a t i o n of carbon compounds: A number of compounds were se-l e c t e d from the l i s t of compounds given by Wickerham (1951); glucose, galactose, maltose, l a c t o s e , sucrose, mannitol and starch. Each compound was autoclaved separately and added a s e p t i c a l l y to the carbon-free CZD medium. The f i n a l concentration of each carbon compound was 3 percent (wt/. v o l . ) . Erlenmeyer f l a s k s (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 r e c i p r o c a t i n g shaker f o r 14 days i n the dark. The dry weights were determined as described f o r pH study. Flasks were prepared i n t r i p l i -cate f o r each compound and controls were used that gave e i t h e r optimal or no growth. By comparing the growth of the controls with growth i n the t e s t medium, information was obtained about the a b i l i t y of the species and s t r a i n s to u t i l i z e various compounds. A s s i m i l a t i o n of nitrogen compounds: The following compounds were employed f o r nitrogen a s s i m i l a t i o n tests (Wickerham, 1951); sodium n i t r a t e , potassium n i t r a t e , ammonium sulphate, glutamic a c i d , phenylalanine and asparagine. The f i n a l concentration of each nitrogen compound was 0.2 per-cent (wt./vol.). The experimental procedure was as described f o r the carbon a s s i m i l a t i o n t e s t s . E f f e c t s of vitamins on growth: The following vitamins were tested f o r t h e i r e f f e c t on growth: thiamine (5 ug), b i o t i n (0.25 ug), i n o s i t o l 14. (0.25 ug) and pyridoxine (5 mg). The figures i n the brackets i n d i c a t e the f i n a l concentration (wt./vol.) of each vitamin. F i f t y ml of l i q u i d CZD medium was dispensed i n Erlenmeyer f l a s k s (125 ml) and s t e r i l e vitamin solutions were added a s e p t i c a l l y to each f l a s k . The fl a s k s were inoculated with 1 ml of spore suspension and the cultures were incubated at 23 C for 14 days i n the dark. Dry weights were determined as described previously. S p l i t t i n g of arbutin: (Lodder and Kreger-van R i j , 1952). The composition of medium was as follows: arbutin (NBC), 5.0 g.; yeast extract ( D i f c o ) , 2.0 g.; agar, 12 g.; d i s t i l l e d water, 1 l i t e r . A drop of s t e r i l e f e r r i c c h l o r i d e s o l u t i o n (0.6 percent; w/v) was placed i n the center of a s t e r i l e P e t r i p l a t e . The arbutin yeast agar medium was poured i n the P e t r i p late and the l i q u i d s were thoroughly mixed and allowed to cool. The plat e s were inoculated and incubated at room temperature. A p o s i t i v e r e a c t i o n was i n d i c a t e d by a dark brown coloured zone* around colonies a f t e r 5 to 7 days. Ethanol as sole carbon source: (Lodder and Kreger-van R i j , 1952). The b a s i c medium contained the follo w i n g : (NH^) 2 S0^, 1.0 g. ; KIL^PO^, 1.0 g. ; MgSO^. 7H 20, 0.5 g.; d i s t i l l e d water, 1 l i t e r . F ive ml of the b a s i c medium was dispensed i n each t e s t tube (16 mm). The medium was autoclaved i n the tubes and 3 percent ethanol was added, a s e p t i c a l l y to the b a s i c 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 i n the presence of yeast extract and therefore yeast extract was sub s t i t u t e d by sodium n i t r a t e to avoid confusion. 15. ethanol, were used f o r comparison. A p o s i t i v e ethanol u t i l i z a t i o n was ind i c a t e d by growth of the organism a f t e r 21 days. Production of starch: (Mager and Aschner, 1947). The composition of the medium was as follows: (NH^^ SO^, 1.0 g. ; MgS0 4.7H 20, 0.5 g.; KH^O^, 1.0 g. ; glucose, 10 g.; thiamine, 100 ug.; agar, 12 g.; d i s t i l l e d water, 1 l i t e r . Lugol's iodine s o l u t i o n was prepared as follows: iodine, 1.0 g.; KI, 2.0 g.; d i s t i l l e d water, 300 ml. Erlenmeyer flasks (125 ml) containing 25 ml of the medium were inoculated with a c e l l suspension and incubated at room temperature on a r e c i p r o c a t i n g shaker. A f t e r 24 days of incubation, the fungus from each f l a s k was separated from the t e s t medium. To the t e s t medium, 2 drops of Lugol's iodine s o l u t i o n were added. A deep blue colour i n d i c a t e d a p o s i t i v e r e a c t i o n . Production of a c i d on chalk agar medium: (van der Walt, 1970). The medium was prepared with following constituents: glucose, 50 g.; CaCO^ ( p r e c i p i t a t e d ) , 5.0 g.; yeast extract ( D i f c o ) , 5.0 g.; agar, 12 g.; d i s t i l l e d water, 1 l i t e r . F ive ml of the ba s i c medium was dispensed i n 16 mm t e s t tubes and autoclaved. The s l a n t s were inoculated with a spore suspension and incubated at room temperature f o r 14 days. I f s u f f i c i e n t a c i d was produced to c l a r i f y the opaque medium, the r e a c t i o n was reported as p o s i t i v e . A b i l i t y to l i q u i f y g e l a t i n : (Wickerham, 1951) The b a s i c medium was prepared as follows: glucose, 0.5 g. ; (NH^^SO^ 0.5 g.; NaN0 3, 0.2 g.; MgS04 7H 20, 0.05 g.; Ca(N0 3) 2, 0.05 g.; KCI, 0.05 g.; d i s t i l l e d water, 100 ml. 16. Ten grams of gelatin (Kno^were dissolved in 90 ml of hot d i s t i l l e d 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 liq u i d layer, i f 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.; d i s t i l l e d water, 1 l i t e r . A quantity of 4.5 ml of medium was dispensed into each 16 mm test tube and st e r i l i z e d . Immediately after s t e r i l i z a t i o n , 0.5 ml of 20 percent f i l t e r - s t e r i l i z e d 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 i f a deep pink colour appeared within 5 days. Conjugation tests: In this study, several other isolates were used i n addition to those l i s t e d in Table 1(A); the isolates were tentatively identified using Tubaki's key. A l i s t of the isolates used, year and place of isolation and their sources is given in Table 1(B). 17. Erlenmeyer f l a s k s (125 ml) containing 25 ml of l i q u i d MYP medium were inoculated with b a l l i s t o s p o r e suspension and incubated at room tempera-ture on a r e c i p r o c a t i n g shaker f o r 2 days. A l o o p f u l of suspension from two i s o l a t e s was placed on MYP agar plates and was mixed thoroughly; sev e r a l p o s s i b l e combinations were t r i e d f o r mating reactions. The plates were examined m i c r o s c o p i c a l l y and the method f o r observation of p l a t e s was as described by Bandoni et a l . (1971). In a d d i t i o n to MYP medium, the follow-ing media were also employed i n t h i s study; Corn-meal (CMA), MEA, MM, Potato-dextrose (PDA), water agar (WA) medium. In some instances, the i s o l a t e s were mixed and grown e i t h e r (1) i n l i q u i d media (2) under anaerobic conditions or (3) i n the dark. 18. CHAPTER 3 OBSERVATIONS Morphology The morphology of the described species of T i l l e t i o p s i s was reported by Nyland (1950) and Tubaki (1952). The observations of my study are given below and i n Plate 1. T i l l e t i o p s i s 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 s o f t when young, then tough. Hyphae d e l i c a t e , septate, branched, 1.0-2.5 u i n diam. B a l l i s t o s p o r e s curved, hyaline, f i n e l y granular, 6.0-14.0 x 1.0-2.0 u., germinating by r e p e t i t i o n or by germ tube. Chlamydospores terminal or i n t e r c a l a r y , hyaline, globose, obovate to clavate, produced s i n g l y or i n chains, 4.0-14.0 u i n diam. T i l l e t i o p s i s minor Nyland var f l a v a Tubaki (1952) On CZD medium: colonies white, no soluble pigments. On MYP medium: colonies 'Pale orange yellow', no soluble pigments. Hyphae d e l i c a t e , septate, branched, 1.0-2.0 u i n diam. B a l l i s t o s p o r e s curved, hyaline, f i n e l y granular, 6.0-17.0 x 1.0-2.5 u.; germinating by r e p e t i t i o n or by germ tube. Budded c e l l s f i l i f o r m , 7.0-19.0 x 1.0-3.0 u. 19. Chlamydospores terminal or i n t e r c a l a r y , hyaline, globose to obovate, produced s i n g l y or i n chains, 4.0-11.0 u i n diam. T i l l e t i o p s i s washingtonensis Nyland (1950) On CZD medium: colonies white to cream; s o f t when young, then tough. On MYP medium: colonies cream to 'Naples y e l l o w 1 ; s o f t when young then tough. Hyphae d e l i c a t e , septate, branched, 1.5-3.0 u i n diam. B a l l i s t o s p o r e s hyaline, curved, f i n e l y granular, 7.0-18.0 x 1.5-3.0 u.; germinate by r e p e t i t i o n or by germ tube. Budded c e l l s f i l i f o r m or i r r e g u l a r , l a r g e r than b a l l i s t o s p o r e s . Chlamydospores terminal or i n t e r c a l a r y , h yaline, globose to obovate, produced s i n g l y or i n chains, 10.0-16.0 u i n diam. T i l l e t i o p s i s cremea Tubaki (1952) On CZD medium: colonies white to cream, s o f t . On MYP medium: colonies cream to ' l i g h t b u f f , s o f t , f i n e l y wrinkled. Hyphae d e l i c a t e , septate, branched, 1.0-3.0 u i n diam. B a l l i s t o -spores hyaline, curved, f i n e l y granular, 8.0-18.0 x 1.0-3.0 u, germinating by r e p e t i t i o n or by germ tube. Budded c e l l s s t r a i g h t , f i l i f o r m , u s u a l l y l a r g e r than b a l l i s t o s p o r e s , 9.0-20.0 x 1.5-3.0 u. Chlamydospores terminal or i n t e r c a l a r y , hyaline, globose, o c c a s i o n a l l y i r r e g u l a r i n shape, produced s i n g l y or i n chains, 4.0-11.0 u i n diam. T i l l e t i o p s i s l i l a c i n a Tubaki (1952) On CZD medium: colonies white to cream, s o f t . On MYP medium: colonies ' l i g h t p i n k i s h l i l a c ' , s o f t , f i n e l y wrinkled. 20. Hyphae usually d e l i c a t e , septate, branched, 1.0-3.0 u i n diam. B a l l i s t o s p o r e s hyaline, curved, f i n e l y granular, 6.0-18.0 x 1.0-3.0 u., germinating by r e p e t i t i o n or by germ tube. Budded c e l l s s t r a i g h t or i r r e g u l a r i n shape. Chlamydospores r a r e l y produced, but r e a d i l y produced under anaerobic conditions, terminal or i n t e r c a l a r y , hyaline, globose to obovate, 6.0-11.0 u i n diam. The following i s o l a t e s have been described as new species of T i l l e t i o p s i s , f u r t h e r i n the text (see page 32). (a) I s o l a t e UBC #926 ( T i l l e t i o p s i s albescens Gokhale, 1971) (b) I s o l a t e UBC #8007 ( T i l l e t i o p s i s pallescens Gokhale, 1971) (c) I s o l a t e UBC #8006 ( T i l l e t i o p s i s fulvescens Gokhale, 1971) In a l l s t r a i n s and species, the b a l l i s t o s p o r e s and the mycelium were found to be uninucleate. PLATE I C u l t u r a l c h a r a c t e r i s t i c s of the species of T i l l e t i o p s i s (On CZD medium; incubation period-14 days) Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 T_. minor T. minor var f l a v a T. washingtonensis T\ cremea T. l i l a c i n a T. albescens (UBC #926) T. pallescens (UBC #8007) T. fulvescens (UBC #8006) P L A T E I 8 22. N u t r i t i o n and Physiology The purpose of these studies was to search for n u t r i t i o n a l or p h y s i o l o g i c a l characters which could be used i n d i f f e r e n t i a t i o n of species of T i l l e t i o p s i s . E f f e c t of pH on growth; This i n v e s t i g a t i o n was c a r r i e d out to determine i f optimum pH f o r T i l l e t i o p s i s species v a r i e d . Maximum growth was obtained i n a l l the species, when the medium was n e u t r a l or s l i g h t l y a l k a l i n e . At pH 4, there was no growth i n 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). E f f e c t of temperature on growth: Growth of d i f f e r e n t species at elevated temperatures was studied and the r e s u l t s are given i n Plate 2. The l i n e a r growth i n each species was maximum at temperatures 23 and 25 C. There was no v i s i b l e growth at 4 and 36 C except i n T_. albescens, where some growth was observed at both these temperatures. With the exception °f l i l a c i n a , the remainder of the species showed scanty growth at 30 C. The optimum temperature f o r growth of most of the T i l l e t i o p s i s species was between 23-25 C. A s s i m i l a t i o n of carbon and nitrogen compounds: Starch and sucrose were r e a d i l y u t i l i z e d whereas galactose and l a c t o s e were poor sources of carbon f o r most of the species of T i l l e t i o p s i s . Glucose and mannitol were as s i m i l a t e d only by J_. washingtonensis, T_. albescens, and T. pallescens where-as maltose was a s s i m i l a t e d only by T. minor, T. washingtonensis, T. l i l a c i n a and T. albescens (Table 3). 23. N i t r a t e s appeared to be r e a d i l y u t i l i z a b l e source of nitrogen f o r most of the species, however _T. minor var f l a v a and _T. cremea did not a s s i m i l a t e sodium n i t r a t e and potassium n i t r a t e , r e s p e c t i v e l y . Ammonium sulphate, glutamic a c i d , phenylalanine and asparagine were found to be poor sources of nitrogen f o r most of the species, though these compounds were r e a d i l y u t i l i z e d by _T. albescens and _T. fulvescens. T_. pallescens d i f f e r e d from these species as i t did not u t i l i z e phenylalanine. In T_. albescens, there was a d i s t i n c t pattern of u t i l i z a t i o n when the following compounds were provided as carbon and nitrogen sources; glucose, maltose, starch, mannitol, sucrose and sodium n i t r a t e , potassium n i t r a t e , ammonium sulphate, glutamic a c i d , phenylalanine and asparagine. The biomass production was about 10 times greater than that i n the remainder of the species. In _T. pallescens, s i m i l a r growth was observed when glucose, mannitol, sodium n i t r a t e , potassium n i t r a t e , ammonium sulphate, glutamic acid and asparagine were provided as carbon and nitrogen sources. There appeared to be a d i s t i n c t v a r i a t i o n i n the a b i l i t y of various species to a s s i m i l a t e d i f f e r e n t carbon and nitrogen compounds; t h i s v a r i a -t i o n i s being used i n d e l i m i t i n g the species of T i l l e t i o p s i s . E f f e c t s of vitamins on growth: The growth of T. minor, T_. minor var f l a v a , T_. washingtonensis, T. cremea, T_. l i l a c i n a and T_. fulvescens was stimulated by a d d i t i o n of thiamine to the medium (Table 4). However, growth of J_. albescens and _T. pallescens was unaffected a f t e r a d d i t i o n of thiamine. The vitamins b i o t i n , i n o s i t o l and pyridoxine had no stimulatory e f f e c t on growth of any of the species studied. 24. The results of the tests: s p l i t t i n g 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 s p l i t t i n g of arbutin were given by TP. washingtonensis, _T. l i l a c i n a , ]T_. cremea and T\ fulvescens. Ethanol was u t i l i z e d only by T_. albescens as a sole source of carbon; no v i s i b l e 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. l i l a c i n a (5 mm). The two species, _T. albescens, and T. pallescens did not l i q u i f y gelatin. A l l 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 E f f e c t of pH on growth of T i l l e t i o p s i s species UBC//926 UBC//8007 UBC//8006 I n i t i a l T. T. T. T. T. T. T. T. pH minor minor var washing- cremea l i l a c i n a albescens pallescens f u l v e -f l a v a tonensis scens 4.0 - - _ _ _ _ 5.0 + + + + +;.• + + + 5.6 + + + + + + + + 6.0 + + + + + + + + + + 6.5 + + 4 + + + + + + + + + + + ++ 7.0 + + + + ++ ++ ++ ++ ++ ++ 7.5 + + + + + + + + + + 4 + + + ++ 8.0 + + + + + + + + no growth f a i r growth maximum growth 26. PLATE II E f f e c t s of d i f f e r e n t temperatures on growth of various species of T i l l e t i o p s i s P L A T E II 30-20-10-E E <r'30-LU r-20-LU 2 1 0 -< Q i — 30-20-4 O O 10-30-20-10-T. albescens(926) T. pallescens(8007) T. cremea T. I i l ac ina T. washingtonensis T. fulvescens(8006) T. mi nor T. minor var. f l a v a I JliL - r 4 10 15 20 23 25 T E M P E R A T U R E S -Bp p -30 36 27. TABLE 3 As s i m i l a t i o n of various carbon and nitrogen compounds by T i l l e t i o p s i s species UBC//926 #8007 #8006 T. T. minor T. T. T. T. T. T. minor var. washing- cremea l i l a c i n a albe- p a l l e - f u l v e -f l a v a tonensis scens scens scens Carbon compounds glucose w - + - - + + galactose - - + - - - - -maltose + - + - + + - -l a c t o s e - - - - - + sucrose + - + + + + + + mannitol - - + - w + + w starch + + + + + + - -Nitrogen compounds NaN03 + - + + + + + + KN03 + + + - w + + w (NH 4) 2S0 4 - - w - - + + w glut, a c i d W - + - w + + + phen. alanine + - - - w + - + asparagine w - - - - + + w Test medium: CZD l i q u i d medium Incubation period: 14 days - = not u t i l i z e d + = u t i l i z e d W = weak u t i l i z a t i o n * = In carbon a s s i m i l a t i o n t e s t s , NaN0 3 was the sole nitrogen source; and i n nitrogen a s s i m i l a t i o n t e s t s , sucrose was the sole carbon source. 28. TABLE 4 E f f e c t s of various vitamins on growth of T i l l e t i o p s i s species vitamins T. T. minor added minor var f l a v a T. T. T. washing- cremea l i l a c i n a tonensis UBC//926 UBC//8007 UBC//8006 T. T. _T. albe- p a l l e - f u l v e -scens scens scens Thiamine + + + + + - - + Bio t i n - - _ _ _ _ I n o s i t o l - - _ _ _ _ Pyridox-ine - _ _ _ _ Th+Bi+In +Py + + + + ' + - - + Control - - - - - - -Test medium: CZD l i q u i d medium Incubation period: 14 days - = c o n t r o l growth + = growth stim u l a t i o n 29. TABLE 5 Results of miscellaneous tests on species of T i l l e t i o p s i s UBC//926 UBC//8007 UBC//8006 Tests T. T. T. T. _T. T. T. T. minor minor v a r . W a s h i n g t o n - ere m e a l i l a - a l b e - p a l l e - f u l v e -f l a v a nensis cina scens scens scens s p l i t t i n g of arbutin + ethanol as a carbon source production of s t a r c h g e l a t i n l i q u e f a c -tionem.m. ) production of a c i d 7-12 6-8 6-10 7-11 1-5 5-6 hyd r o l y s i s of urea + W + * = the height of l i q u i f i e d g e l a t i n was measured i n 16 mm test tubes, at 7 and 26 days. - = negative r e a c t i o n + = p o s i t i v e r e a c t i o n W = weak re a c t i o n 30. The results of morphological, nutritional and physiological studies indicated that the isolated UBC #926, 8007 and 8006 were different from the species of T i l l e t i o p s i s 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 latt e r two isolates, the b a l l i -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 i n isolates 926 and 8006. The isolates 926 and 8007 were easy to separate from other species of T i l l e t i o p s i s 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 u t i l i z e ethanol, maltose and starch as sole source of carbon. Phenylalanine was u t i l i z e d by isolate 926 whereas isolate 8007 did not u t i l i z e i t 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 P h y s i o l o g i c a l c h a r a c t e r i s t i c s of taxonomic value f o r d e l i m i t i n g T i l l e t i o p s i s spp. UBC//926 UBC//8007 UBC//8006 T. T. T. T. T. T. T. T. "minor minor washing- "cremea T i l a- albe- p~alle- Tulve-var f l a v a tonensis cina scens scens scens A s s i m i l a t i o n of Glucose W - + - - + + Maltose + - + - + + Starch + + + + + . + Sodium n i -t r a t e + - + - + + + + Glutamic a c i d W - + - W + + + Phenyl-alanine + - - - W + - + S p l i t t i n g of arbutin - - + + + - - + Ethanol as a carbon source - - - - - + Hydrolysis of Urea + + + + + W + + Thiamine dependency + + + + + - - + Ge l a t i n l i -quefaction + + + + + - - + Pigment formation + - - _ _ _ _ + + = p o s i t i v e a s s i m i l a t i o n - = negative a s s i m i l a t i o n W = weak a s s i m i l a t i o n 32. The production of pigment was observed only i n the presence of yeast extract. There was a d i s t i n c t increase i n pigment production when the concentration of yeast e x t r a c t was r a i s e d from 0.05 to 1.5 percent (Plate VI). From the observations presented, i s o l a t e s 926, 8007 and 8006 appear to be new species of T i l l e t i o p s i s . The d e s c r i p t i o n of the new species i s given i n the following pages. T i l l e t i o p s i s albescens Gokhale sp. n. Coloniae c a r t i l a g i n o s a e , durae, albae, v e t e r i o r e s extense myceliales. Hyphae plerumque t e n e l l a e , septatae, ramosae, i u n i o r e s hyalinae, v e t u s t i o r e s fuscantes, 2.0-3.5 u i n diam. B a l l i s t o s p o r a e hyalinae, curvatae, minute granulosae, plerumque 2 v e l 3 g l o b u l i s o l e o s i s armatae, uninucleatae, 2.0-3.5 x 11.5-19.5 u i n diam. r e p e t i t i o n e aut tubulo germinali germinantes, excentrice sterigmatibus hypharum v e l ballistosporarum a f f i x a e . C e l l u l a e gemmiparae b a l l i s t o s p o r i s maiores, 2.0-3.0 x 16.0-22.0 u. Chlamydosporae non observatae. Growth on Malt extract agar: Colonies white, tough and c a r t i l a g i -nous i n texture and r e l a t i v e l y l i t t l e mycelium i s produced. Dalmau plate cultures on YMA: The aerobic growth i s s i m i l a r to that on MEA medium. Under anaerobic conditions; b a l l i s t o s p o r e s or budded c e l l s are r a r e l y produced, most of the growth i s i n mycelial form, empty c e l l s are frequently found. Chlamydospores are observed, mostly i n t e r c a l a r y and empty c e l l s occur on e i t h e r side of a chlamydospore. The chlamydospores measure 6.0-14.0 u i n diameter and t h e i r shape va r i e s 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 o i l 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 L i b e r t y v i l l e , I l l i n o i s , 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 B r i t i s h Columbia, Vancouver 8, B.C. Etymology: albescens - becoming white; from Latin, albus and suffix-scens (becoming). L i f e history: Ballistospores are produced on sterigmata which arise from other ballistospores or from the mycelium. No clamps are formed. T i l l e t i o p s i s 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 I I I T i l l e t i o p s i s albescens Figure 1: B a l l i s t o s p o r e s Figure 2: Budded c e l l s Figure 3: B a l l i s t o s p o r e s germinating by r e p e t i t i o n Figure 4: B a l l i s t o s p o r e s produced from hyphae Figure 5: Chlamydospores (under anaerobic conditions, on YMA medium) P L A T E M l T . a l b e s c e n s 35. Growth on Malt extract agar: Relatively l i t t l e 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 i s 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 f r u i t body was collected in Shamoda, Japan in November, 1968 and the strain was isolated i n Vancouver, B.C. in December, 1968. It is preserved i n Mycology Herbarium, U.B.C. , Vane, 8, B.C. Etymology: pallescens- becoming white (pale); from Latin, pallidus and suffix-scens (becoming). Lif e 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 T i l l e t i o p s i s pallescens Figure 1: B a l l i s t o s p o r e s Figure 2: B a l l i s t o s p o r e s germinating by germ tube Figure 3: Hyphal swellings (under anaerobic conditions, on YMA medium) P L A T E I V 3 T i l l e t i o p s i s fulvescens Gokhale sp. n. Coloniae iuniores saccharomycetarum s i m i l e s , v e t u s t i o r e s myceliales, iuniores ochraceo-croceae, v e t u s t i o r e s fuscatae ("Sudan Brown-Dresden Brown). In medio saccharomycetarum c e r e v i s i a e extracto adjecto. Hyphae t e n e l l a e , septatae, ramosae, iuniores hyalinae, 1.0-2.5 u i n diam. B a l l i s t o s p o r a e hyalinae, curvatae, minute granulosae, uninucleatae, 1.0-2.5 x 8.0-15.0 u., excentrice sterigmatibus mycelio ortae a f f i x a e . Chlamydosporae non observatae. Growth on Malt extract agar: Colonies are white to "Buff yellow" and s o f t i n consistency. There i s no pigment production. Dalmau plate culture on YMA: Colonies are "Ochraceous orange" to "Sudan Brown" and a soluble "Sudan Brown" to "Dresden Brown" pigment i s produced. The aerobic growth, i n general, i s s i m i l a r to that on MEA. Anaerobic growth: B a l l i s t o s p o r e s or budded c e l l s are r a r e l y observed and most of the growth i s i n mycelial form. Chlamydospores are produced, e i t h e r i n t e r c a l a r y or terminal and usually show dense contents. They are smooth walled, clavate to obovate i n shape and measure 5.0-12.0 i n diam. Colonies y e a s t - l i k e when young, l a t e r m y c e l i a l ; when young "Ochraceous orange", then "Sudan Brown", produce soluble "Sudan Brown" to "Dresden Brown" pigment i n presence of yeast e x t r a c t . Hyphae d e l i c a t e , septate, branched, hyaline when young, 1.0-2.5 u i n diameter. B a l l i s t o -spores hyaline, curved, f i n e l y granular, uninucleate, 1.0-2.5 x 8.0-15.0 u., e c c e n t r i c a l l y attached to sterigmata which a r i s e only from the mycelium. Budded c e l l s r a r e l y formed. Chlamysospores not observed. 38. Type c u l t u r e : UBC //8006, i s o l a t e d from F o r s y t h i a l e a f which was c o l l e c t e d i n North Vancouver, B.C., i n Sept., 1968. I t Is preserved i n the Mycology Herbarium, U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, B.C. Etymology: fulvescens - becoming yellowish brown; from L a t i n , fulvus and su f f i x - s c e n s (becoming). L i f e - h i s t o r y : B a l l i s t o s p o r e s germinate by germ tubes which i n turn produce new b a l l i s t o s p o r e s , without mating; clamps are not formed. PLATE V T i l l e t i o p s i s fulvescens Figure 1: B a l l i s t o s p o r e s Figure 2: B a l l i s t o s p o r e s germinating by germ tube Figure 3: B a l l i s t o s p o r e s produced from hyphae Figure 4: Chlamydospores (under anaerobic conditions, on YMA medium) P L A T E V T. fu l vescens AO. PLATE VI Figures 1, 2 and 3: Figures 4 and 5: Figures 6 and 7: Figures 8 and 9: Figures 10, 11 and 12: T i l l e t i o p s i s fulvescens: e f f e c t 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 r a i s e d (on MYP medium) Cu l t u r a l c h a r a c t e r i s t i c s of T_. albescens Figure 4: on CZD medium Figure 5: on MYP medium Cu l t u r a l c h a r a c t e r i s t i c s of T. pallescens Figure 6: on CZD medium Figure 7: on MYP medium Cu l t u r a l c h a r a c t e r i s t i c s of _T. fulvescens Figure 8: on CZD medium (no yeast extract) Figure 9: on MYP medium (with yeast extract) Nuclear s t a i n i n g (Iron-haematoxylin stain) Figure 10: T_. albescens- uninucleate b a l l i s t o s p o r e s Figure 11: T?. pallescens- uninucleate b a l l i s t o s p o r e s Figure 12: T. fulvescens- uninucleate b a l l i s t o s p o r e s P L A T E V I 41. Tubaki (1952) presented a key to i d e n t i f y four described species and a v a r i e t y of T i l l e t i o p s i s spp. His i d e n t i f i c a t i o n was based on colour and texture of the colonies. During these studies, several other i s o l a t e s were c o l l e c t e d from the v i c i n i t y of Vancouver, B.C. and some also were obtained from UBC culture c o l l e c t i o n . The colonies of these i s o l a t e s showed a v a r i e t y of colours ranging from whi t i s h cream to yellowish brown. I t was observed that the p l a c i n g of the i s o l a t e s into described species only on the basis of colour and the texture of the colonies i s d i f f i c u l t . Therefore, a key f o r i d e n t i f i c a t i o n of T i l l e t i o p s i s species i s prepared to include the previously described as w e l l as the species mentioned herein. The key, presented below, i s based on t h e i r n u t r i t i o n a l , p h y s i o l o g i c a l and morphological c h a r a c t e r i s t i c s observed during t h i s i n v e s t i g a t i o n . "A Key f o r the i d e n t i f i c a t i o n of species of T i l l e t i o p s i s " l a . Arbutin s p l i t t i n g p o s i t i v e 2. l b . Arbutin s p l i t t i n g negative 3. 2a. Sodium n i t r a t e , glutamic a c i d a s s i m i l a t e d 4. 2b. Sodium n i t r a t e , glutamic a c i d not a s s i m i l a t e d 5. 4a. No pigment production Glucose, maltose, starch assimilated, phenylalanine not a s s i m i l a t e d T_. washingtonensis 4b. Soluble yellowish brown pigment produced Glucose, maltose, starch not assimilated, phenylalanine a s s i m i l a t e d T_. fulvescens 5a. Maltose assi m i l a t e d T_. l i l a c i n a 5b. Maltose not assimilated T. cremea 42. 3a. Thiamine dependent, g e l a t i n l i q u e f a c t i o n p o s i t i v e chlamydospores produced L i t t l e mycelium produced 6. 3b. Thiamine independent, g e l a t i n l i q u e f a c t i o n negative chlamydospores not produced extensive mycelium produced 7. 6a. Maltose, phenylalanine assimilated T. minor 6b. Maltose, phenylalanine not assi m i l a t e d T. minor var f l a v a 7a. Ethanol, st a r c h , maltose, phenylalanine assim i l a t e d T. albescens 7b. Ethanol, starch, maltose, phenylalanine not assi m i l a t e d _T. pallescens The v a l i d i t y of a c l a s s i f i c a t i o n system, based on n u t r i t i o n a l and p h y s i o l o g i c a l characters, i s often questioned by taxonomists. To check the v a l i d i t y of the present key, seven t e n t a t i v e l y i d e n t i f i e d i s o l a t e s of T i l l e t i o p s i s (Table 1-B) were run through the n u t r i t i o n a l and p h y s i o l o g i c a l t e s t s . On the bas i s of these t e s t s , i s o l a t e 8030 was placed i n _T. l i l a c i n a whereas i s o l a t e 8033 was i d e n t i f i e d as T_. minor. The key was found to be s a t i s f a c t o r y i n i d e n t i f i c a t i o n of the remaining f i v e i s o -l a t e s of T i l l e t i o p s i s . These r e s u l t s suggest that the proposed key could be used i n i d e n t i f i c a t i o n of the species of T i l l e t i o p s i s 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 a l l 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 f a c i l i t i e s . " There are" many workers who consider that morphological characters are more variable than the physiological ones (Wickerham and Rettger, 1939; Skinner, 1947; G r i f f i t h s , 1958) and the latter should be used in the clas s i f i c a t i o n . In some cases, classifications based on morphology are of limited value because of a lack of suitable diagnostic features (Parmeter, 1965) and i n some, special conditions are needed to allow the development of the morphological features on which the system is based. A good example of this l a t t e r is in Phytophthora (Erwin et a l . , 1963), where extensive cultural and mating tests are often necessary for satisfactory determination of species. In summarizing the d i f f i c u l t i e s 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 c l a s s i f i c a -tion systems. Barnett (1960,1961) suggested that yeasts should be c l a s s i -fied on purely biochemical c r i t e r i a 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 u n t i l a systematic study has been made of i t s 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 c r i t e r i a are used to differentiate the lower taxa, species in particular." The a b i l i t y to u t i l i z e 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 a l l the biochemical procedures used in the c l a s s i f i c a -tion of yeasts. The taxonomic value of various c r i t e r i a such as arbutin s p l i t t i n g , 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 f o r i d e n t i f i c a t i o n of yeasts (Wickerham, 1951; Lodder and Kreger-van M j , 1952; Hasegawa and Banno, 1970). Amongst the members of family Sporobolomycetaceae, Phaff (1970) presented keys f o r i d e n t i f i c a t i o n of species of 3 genera; Sporobolomyces, B u l l e r a and Sporidiobolus. His keys are p r i m a r i l y based on n u t r i t i o n a l and morphological c h a r a c t e r i s t i c s of these organisms. Pr e v i o u s l y , Tubaki (1952) proposed a key f o r i d e n t i f i c a t i o n of the species of T i l l e t i o p s i s based e n t i r e l y on colour and consistency of the c o l o n i e s . As pointed out e a r l i e r , these features were found to be v a r i a b l e and therefore i t was found d i f f i c u l t to place several i s o l a t e s i n the described species. In the present study, three previously unknown species of T i l l e t i o p s i s have been described and a key has been proposed to i d e n t i f y the species of T i l l e t i o p s i s . The separation of the species and the pro-posed key are mainly based on t h e i r n u t r i t i o n a l , p h y s i o l o g i c a l and mor-ph o l o g i c a l c h a r a c t e r i s t i c s . The features such as a s s i m i l a t i o n of carbon and nitrogen compounds, s p l i t t i n g of arbutin, pigment production, mode of germination of b a l l i s t o s p o r e s , the production of chlamydospores have been used i n d e l i m i t i n g the species of T i l l e t i o p s i s . T i l l e t i o p s i s spp. have been divided into two groups on the basis of t h e i r a r b u t i n s p l i t t i n g a b i l i t y . This property i s commonly used as one of the c h a r a c t e r i s t i c s to d i s t i n g u i s h the genus Hansenula (arbutin p o s i t i v e ) from the genus P i c h i a (arbutin negative). Barnett et a l . (1956) and Cook (1958) have also used t h i s character f o r taxonomic purposes. Further separation of T i l l e t i o p s i s species i s based on t h e i r a b i l i t i e s to u t i l i z e 46. various carbon and nitrogen sources. Such a s s i m i l a t i o n tests have long been used i n yeast taxonomy (Wickerham, 1951, Lodder and Kreger-van R i j , 1952). Phaff (1970) has made use of these a s s i m i l a t i o n tests i n d i s -t i n g u i s h i n g species of Sporobolomyces, B u l l e r a and Sporidiobolus. T^ . fulvescens was found to produce a soluble yellowish brown pigment i n the presence of yeast extract; t h i s character alone separates T_. fulvescens from other species of T i l l e t i o p s i s . The u t i l i z a t i o n of carbon and nitrogen compounds was c h a r a c t e r i s t i c i n _T. albescens and T\ pallescens; the biomass production was about 10 to 15 times greater than that observed i n any other species. Also, T_. albescens and T_. pallescens were found to be thiamine independent, they did not l i q u i f y g e l a t i n and produced extensive mycelium; these features separated them from T_. minor and T. minor var f l a v a . Vitamin requirement has been used as a taxonomic c r i t e r i o n by Schultz and Atkin (1947) f o r Saccharomyces c e r e v i s i a e , Wickerham (1951) f o r Hansenula spp. and by Hasegawa and Banno (1963) f o r species of Rhodotorula. G e l a t i n l i q u e f a c t i o n as a taxonomic c r i t e r i o n has been found to be of l i m i t e d value (Wickerham, 1951; van der Walt, 1970); i n the present scheme g e l a t i n l i q u e -f a c t i o n has been used along with other characters. Attempts were also made to search f o r the sexual phase i n T i l l e t i o p s i s although they did not prove s u c c e s s f u l . In recent years, several workers have reported the occurrence of sexua l i t y i n yeasts and y e a s t - l i k e organisms. The mating reactions were observed i n Rhodotorula by Banno (1967) and i n Candida-like species by F e l l et a l . (1969). I t i s noteworthy that the four species of Rhodosporidium (perfect stage of Rhodotorula), a l l had the same imperfect state on the basis of n u t r i t i o n a l 47. and physiological studies. Banno (1967) and F e l l et a l . (1969) have clearly shown that these yeast genera belong to the order Ustilaginales. In the present study, the failure i n obtaining the sexual phase in T i l l e t i o p s i s eliminated the important feature which has often been used in the taxonomy of many fungi. In general, most of the c r i t e r i a which have been used here are often employed in taxonomy of yeasts and yeast-like organisms. The keys (Lodder and Kreger-van R i j , 1952; Phass, 1970) based on such c r i t e r i a have proved very useful for identification purposes. The author believes that the present key w i l l also prove useful in routine work and i d e n t i f i -cation of species of T i l l e t i o p s i s . 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 a l . , 1955, Capriotti, 1961 b). Also, in keys presented by various workers, two species are differentiated on the basis of one or two nutritional d i f f e r -ences (Uden and Buckley, 1970; Phaff and Ahearn, 1970). There is d i f f e r -ence of opinion regarding the number of nutritional characters which would be sufficient to delimit a species. This judgement solely rests on the 48. i n d i v i d u a l and to date there i s no standardization f o r the number/s of n u t r i t i o n a l characters which should be used i n d e l i m i t i n g the species. In several instances, new species have been described on the basis of a s i n g l e i s o l a t e (Phaff and do Carmo-Sousa, 1962; van der Walt, 1970). With the above mentioned fa c t s i n mind, three new species of T i l l e t i o p s i s have been described. However, i n w r i t e r ' s opinion, i t would be worthwhile to study a l a r g e r number of i s o l a t e s and to explore biochemical and physi o l o -g i c a l characters which might provide a b e t t e r understanding f o r c l a s s i f i -c ation of the genus T i l l e t i o p s i s . 49. BIBLIOGRAPHY Ainsworth, C.G. 1961. Dictionary of the fungi. Commonwealth Mycologi-c a l I n s t i t u t e , 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 i n c l a s s i f y i n g yeasts. J. Gen. M i c r o b i o l . 15: 529-555. Barnett, J.A. 1960. Comparative studies of yeasts. Nature, 186: 449-451, Barnett, J.A. 1961. Biochemical c l a s s i f i c a t i o n of yeasts. Nature. 189: 76. Bandoni, R.J., K.J. Lobo and S.A. Brezden. 1971. Conjugation and chlamydo-spores i n Sporobolomyces odorus. Can. J . Bot. 49: 683-686. Banno, I. 1967. Studies on the s e x u a l i t y of Rhodotorula. J. Gen. Appl. M i c r o b i o l . 13: 167-196. Bessey, E.A. 1950. Morphology and taxonomy of fungi. The B l a k i s t o n Co., P h i l a d e l p h i a . B u l l e r , A.H.R. 1933. Researches on f u n g i . 5: 171-206. Longmans, Green and Co., London. C a p r i o t t i , A. 1961b. Debaryomyces c a n t a r e l l i i nova species, a new yeast i s o l a t e d from F i n n i s h s o i l . Arch. M i c r o b i o l . 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. Etudes sur l e s sporobolomycetes. Ann. Mycol. 28: 1-23 1948. I t e r s o n i l i a . nouveau genre de sporobolomycetes a mycelium boucle. B u l l . Bot. Gard. Buitenzorg, ser. 3. 17: 465-472. Di f c o manual f o r dehydrated media and reagents. 1964. Difco l a b o r a t o r i e s . D e t r o i t 1, Michigan. 50. Erwin, D.C., G.A. Zentmyer, J . Galindo and J.S. Niedenhauser. 1963. V a r i a t i o n i n the genus Phytophthora. Ann. Rev. Phytopathol. 1: 375-396. F e l l , J.W., A.C. S t a t z e l l , I.L. Hunter and H.J. Phaff. 1969. Leucosporidium gen. n., the heterobasidiomycetous stage of sev e r a l yeasts of the genus Candida. Antonie van Leeuwenhoek. 35: 433-462. G r i f f i t h s , E. 1958. Sexual reproduction and v a r i a t i o n i n G l o e o t i n i a temulenta ( P r i l l and Delaer) Wilson and Gray. Trans. B r i t . Myc. Soc. 41: 461-482. Hasegawa, T. and I. Banno. 1963. Vitamin requirement as a taxonomic key f o r Rhodotorula sp. J . Gen. Appl. M i c r o b i o l . 9: 279-285. H a l l , R. 1969. Molecular approaches to taxonomy of fungi. Bot. Rev. 35: 285-304. Johansen, D.A. 1940. Plant Microtechnique. McGraw-Hill Book Co., Inc. New York. Kluyver, A.J. and C.B. van N i e l . 1924. Uber S p i e g e l b i l d e r erzeugende Hefenarten und die neue Hefengattung Sporobolomyces. Centr. Bakt. Parasitenk., Zweite Abt. 63: 1-20. L a f f i n , R.J. and V.M. Cutter, J r . 1959. Investigations on the l i f e c y c l e of Sporidiobolus j o h n s o n i i , I I I . J . E. Mitchel S c i . Soc. 75: 89-96, 97-100. L i l l y , V.G. and H.L. Barnett. 1951. Physiology of the fungi. McGraw-Hill Book Co., Inc. New York. Lodder, J. and N.J.W. Kreger-van R i j . 1952. The yeasts. A taxonomic study. N. Holland Publ. Co. Amsterdam. Lodder, J . (ed.) 1970. The yeasts. A taxonomic study. N. Holland Publ. Co. Amsterdam. Lowag, H. 1926. Sporobolomyces-Kein Basidiomyzet. Ann. Mycol. 24: 194-202. Mager, J . and M. Aschner. 1946. Starch r e a c t i o n as a i d i n i d e n t i f i c a t i o n of causative agent of "European Blastomycosis." Soc. Expt. B i o l , and Med. Proc. 62: 71-72. Martin, G.W. 1952. Revision of the North Central Tremellales. Univ. Iowa. Stud. Nat. H i s t . 19: 1-122. Nyland, G. 1948. Preliminary observat ions on the morphology and cytology of an undescribed heterobasidiomycete from Washington State. Mycologia. 40: 478-481. 51. Nyland, G. 1949. Studies on some unusual heterobasidiomycetes from Washington s t a t e . Mycologia. 41: 686-701. 1950. The genus T i l l e t i o p s i s . Mycologia. 42: 487-496. Parmeter, J.R. 1965. The taxonomy of s t e r i l e fungi. Phytopathology. 55: 826-828. Phaff, H.J. 1970. Discussion of the y e a s t - l i k e genera belonging to the Sporobolomycetaceae. Chapter 6. In: The yeasts. A taxonomic approach, (ed.) Lodder, N. Holland Publ. Co. Amsterdam. 815-862 pp. and D.G. Ahearn. 1970. Discussion of the genera of asporogenous yeasts not belonging to the Sporobolomycetaceae; Genus 7: Rhodotorula Harrison. In: The yeasts. A taxonomic approach, (ed.) Lodder, N. Holland Publ. Co., Amsterdam. and L. do Carmo-Sousa. 1962. Four new species of yeast i s o l a -ted from i n s e c t Frass i n bark of Tsuga heterophylla (Raf.) Sargent. Antonie van Leeuwenhoek. 28: 193-207. Ridgway, R. 1912. Colour standards and color nomenclature. Published by the author. Washington, D.C. Riker, A.J. and R.S. Riker. 1936. Introduction to research on plant diseases. John S. Swift Co.., Inc. St. Louis. Roberts, C. and R.S.W. Thome. 1960. Biochemical c l a s s i f i c a t i o n of yeasts. Nature. 188: 872-873. S a i n c l i v i e r , M. 1952. Caryologie des Sporobolomyces. B u l l . Soc. Bot. France. 99: 147-149. Schultz, A.S. and L. At k i n . 1947. The a b i l i t y of response i n yeast c l a s s i f i c a t i o n and nomenclature. Arch. Biochem. 14: 369-380. Seelinger, H.P.R. 1956. Use of a urease t e s t f o r the screening and i d e n t i f i c a t i o n of c r y p t o c o c i i . J. B a c t e r i o l . 72: 127-131. Shehata, A.M. El-Tabey, E.M. Mrak and H.J. Phaff. 1955. Yeasts i s o l a t e d from t h e i r suspected feeding places i n Southern and Central C a l i f o r n i a . Mycologia. 47: 799-811. Skinner, C.E. 1947. The y e a s t - l i k e fungi: Candida and Brettanomyces. B a c t e r i o l . Rev. 11: 227-274. Snyder, W.C. and T.A. Toussoun. 1965. Current status of taxonomy i n Fusarium species and t h e i r p e rfect stages. Phytopathology. 55 833-837. 52. Sowell, G. J r . and R.P. Korf. 1960 (1962). An emendation of the genus I t e r s o n i l i a based on studies of morphology and pathogenicity. Mycologia. 52: 934-945. Sundstrom, K.R. 1964. Studies of the physiology, morphology and serology of Exobasidium. Symbolae botan. U p s a l i e n s i s . 17: 1-100. Tubaki, K. 1952. Studies on the Sporobolomycetaceae i n Japan. I. On T i l l e t i o p s i s . Nagaoa. 1: 26-31. Uden, N. van and H. Buckley. 1970. Discussion of the genera of asporogenous yeasts not belonging to the Sporobolomycetaceae; Genus 2: Candida Berkhout. In: The yeasts. A taxonomic approach (ed.) Lodder, N. Holland Publ. Co., Amsterdam. 893-1087 pp. van der Walt, J.P. 1957. Three new sporogenous yeasts from s o i l . Antonie van Leeuwenhoek. 23: 23-29. 1970. The pe r f e c t and imperfect s t a t e of Sporobolomyces salmonicolor. Antonie van Leeuwenhoek. 36: 49-55. 1970. C r i t e r i a and methods used i n c l a s s i f i c a t i o n . 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 M o n i l i a albicans with s p e c i a l emphasis on morphology and morphological v a r i a t i o n . J . Trop. Med. Hyg. 42: 174-177, 187-192, 204-216. Wickerham, L.J. 1951. Taxonomy of yeasts. Technical b u l l e t i n no. 1029. V.S. Dept. of A g r i c u l t u r e . 1-56 pp. 53. APPENDIX NO. 1 MEDIA: The concentration of agar i n s o l i d media was 1.2 percent due to i t s 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. D i s t i l l e d H o0 1 l i t e r While preparing CZD medium, ferrous and magnesium s a l t s were autoclaved separately and added a s e p t i c a l l y to the remainder of the medium, thus preventing the formation of p r e c i p i t a t e s . 2) Malt Yeast extract Peptone medium: (MYP) Difco malt extract 15.0 g. Difco yeast extract.... 0.5 g. Difco peptone 2.5 g. D i s t i l l e d H 20 1 l i t e r 3) Malt E x t r a c t Agar medium: (MEA) Maltose 12.75 g. Dextrin 2.75 g. Glycerol 2.35 g. Difco peptone 0.78 g. D i s t i l l e d H 20 1 l i t e r 4) Water Agar: (WA) Bacto agar 12.0 g. D i s t i l l e d H 20 1 l i t e r 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. C a C l 2 0.1 g. Difco yeast extract 2.5 g. vitamin s o l . (con) 10.0 ml D i s t i l l e d H 20 1 l i t e r Appendix No. 1 (cont'd) 54. 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. D i s t i l l e d H o0 1 l i t e r Difco dehydrated media were used to prepare Potato dextrose agar (PDA) Corn meal agar (CMA) and Yeast morphology agar (YMA). Their preparation was as d i r e c t e d i n Difco manual (1964) . In a d d i t i o n to these general media, some s p e c i a l media were also used i n t h i s study. The composition of these media i s given at appropriate places i n the text. 55. APPENDIX NO. 2 STAINS: 1) Iron-haematoxylin s t a i n (Johansen, 1940) a) Stock s o l u t i o n : To 100 ml of d i s t i l l e d water, I added 0.05 g. of sodium bicarbonate and b o i l e d the s o l u t i o n . A quantity of 0.5 g. of dye was then added and the s o l u t i o n was allowed to cool . The s t a i n was stored i n a r e f r i g e r a t o r at 4 C. b) Mordant: 4 percent i r o n alum ( f e r r i c ammonium 500 ml. sulphate) G l a c i a l a c e t i c a c i d 6 ml. Concentrated sulphuric a c i d 0.6 ml. c) Destaining agent: Mordant (above described) d i l u t e d with an equal volume of d i s t i l l e d water. d) F i x a t i v e : Weak chromic-acetic a c i d s o l u t i o n 10 percent aqueous chromic a c i d 2.5 ml 10 percent aqueous a c e t i c a c i d 5.0 ml D i s t i l l e d water 100 ml APPENDIX 3 (Riker and Riker, 1936) CLEANING SOLUTION: Procedure: The potassium dichromate was dissolv e d i n the d i s t i l l e d water and the ac i d was added slowly to the s o l u t i o n . 

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