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UBC Theses and Dissertations

Studies in the genus sporidiobolus Higham, June Gwendoline 1972

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STUDIES IN THE GENUS SPORIDIOBOLUS by JUNE GWENDOLINE HIGHAM B.Sc, University of Br i t i s h Columbia, 1970 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 September, 1972 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f BOTANY The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada OCTOBER 3 . 1 9 7 2 . Date - 1 1 -ABSTRACT The synthesis of Sporobolomyces odorus dikarya i s confirmed. Some of the morphological 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 of st r a i n s of Sporldlobolus and of Sporo-bolomyces dikarya were observed and compared. These included optimum temperatures f o r mycelial and t o t a l growth, standard c y t o l o g i c a l and c o l o n i a l descriptions, a b i l i t y to assimi-l a t e carbon compounds, and u l t r a v i o l e t s u r v i v a l curves. Attempts were made to dedlkaryotize mycelia by u l t r a v i o l e t i r r a d i a t i o n and by chemical means; these were unsuccessful. Various techniques were used i n an attempt to induce germin-a t i o n of r e s t i n g spores. Presoaking In d i s t i l l e d water followed by incubation on water agar at room temperature proved successful f o r Sporobolomyces odorus spores which germinated by the formation of ustilaginaceous promycelia. Primary s p o r i d i a of t h i s s t r a i n gave r i s e to non-mycelial colonies, thus completing the l i f e cycle of t h i s organism. A new species, Sporldlobolus mlcrosporus. i s described. The dimensions of the yeast c e l l s and b a l l i s t o s p o r e s average 2 X . The dikaryon i s almost i d e n t i c a l to that of other species, but the r e s t i n g spores are t y p i c a l l y i n t e r c a l a r y . Carbon source a s s i m i l a t i o n by the new species d i f f e r e d from that of other species of the genus. Attempts to germinate r e s t i n g spores of Sporldlobolus mlcrosporus were unsuccessful. - l l i -TABLE OF CONTENTS Introduction • • • 1 Materials and Methods ••••• 8 Results 20 Sporobolomyces odorus dikaryon .............. 20 Sporobolomyces sp. JGH #3 • 26 Sporldlobolus sp. UBC #5035 30 Sporldlobolus johnsonll •••• 37 "Sporldlobolus rulnenil" 41 Physiological tests 42 Mating studies 42 Single c e l l isolates • 43 Ultraviolet experiments • 44 Chemical mutagens ........................... 46 Chlamydospore germination •••••• • 46 Discussion . . . o * . . . * • • . • 53 Sporldlobolus mlcrosporus n. sp 54 Bibliography 63 Appendix 67 - i v LIST OF FIGURES Figure 1. Colony diameter of Sporobolomyces 25 odorus dikaryon at various temper-atures after 7 and 14 days on CJM. Figure 2 . Colony diameter of Sporobolomyces 28 sp. JGH #3 at various temperatures after 7 and l k days on CjM. Figure 3 . Carbon source u t i l i z a t i o n by Spor- 29 ldlobolus sp. UBC #5035 and strains of Sporobolomyces spp. Figure 4. Colony diameter of Sporldlobolus sp. 36 UBC #5035 at various temperatures after 7 and 14 days on CJM. Figure 5 . Colony diameter of Sporldlobolus 40 .lohnsonii at various temperatures after 7 and 14 days on CjM. Figure 6 . Ultraviolet survival curves for 4-5 Sporldlobolus t^ohnsonll. Spor-obolomyces odorus. and Sporldlobolus sp. UBC #5035. -V-LIST OF PLATES Plate 1 A - Colonies of Sporobolomyces 35 odorus, Sporobolomyces spp. Sporobolomyces salmonicolor, and Sporldlobolus sp. UBC #5035* B Yeast cells of Sporidlobolus  johnsonii. C - Yeast cells of Sporldlobolus sp. UBC #5035. D - G - Morphology of Sporldlobolus sp. UBC #5035. Plate 2 Sporldlobolus johnsonii and Sporldlobolus sp. UBC #5035 on CjM plus 2-deoxy-D-glucose. ^7 Plate 3 Germination of chlamydospores of Sporobolomyces odorus at room temperature on water agar. *9 Plate k Germination of chlamydospores of Sporobolomyces odorus at room temperature and at 15°Co 52 - v i -ACKNOWLEDGMENT The author wishes to thank the following people f o r t h e i r assistance during the research reported i n t h i s t h e s i s , and during the preparation of the thesis I t s e l f . Dr. H.J. Bandoni, to whom, as supervisor of t h i s thesis, I am most indebted. Dr. K. Cole and Dr. G.C. Hughes I I I , who, upon reading the o r i g i n a l manuscript, made many usefu l suggestions. Michael Higham, whose moral support and a i d i n preparation of the plates were invaluable. And to the Faculty, Sta f f , and Students of the Botany Department who have a s s i s t e d the author during t h i s work. INTRODUCTION The g e n u s S p o r l d l o b o l u s was d e s c r i b e d by N y l a n d i n 1949. The g e n e r i c d e s c r i p t i o n s t r e s s e d t h e f o r m a t i o n o f s t e r i g m a t e b a l l i s t o s p o r e s c a p a b l e o f r e p r o d u c t i o n by r e p e t i t i o n o r by b u d d i n g , c o u p l e d w i t h t h e p r e s e n c e o f m y c e l i u m b e a r i n g b o t h c l a m p c o n n e c t i o n s a n d t h i c k - w a l l e d , b r o w n - p i g m e n t e d c h l a m y d o -s p o r e s . B a s e d u p o n t h e e v i d e n c e o f t h e b a l l i s t o s p o r e d i s c h a r g e m e c h a n i s m , t h e c l a m p c o n n e c t i o n s , a n d t h e mode o f g e r m i n a t i o n o f t h e r e s t i n g s p o r e s by s p o r i d l u m - b e a r i n g germ t u b e s , N y l a n d p l a c e d t h e g e n u s i n t h e H e t e r o b a s i d i o m y c e t i d a e . He h e s i t a t e d t o a s s i g n S p o r l d l o b o l u s t o t h e U s t i l a g i n a l e s b e c a u s e o f l a c k o f k n o w l e d g e o f t h e l i f e c y c l e (1949) a n d l a c k o f p r o o f o f t h e o r g a n i s m ' s p a r a s i t i s m (1948b). N y l a n d (1948a, 1948b, 1949) c o m p a r e d S p o r l d l o b o l u s w i t h t h e i m p e r f e c t g e n u s S p o r o b o l o m y c e s K l u y v e r a n d v a n N i e l (1925). The o n l y d i f f e r e n c e he n o t e d was t h a t t h e l a t t e r g e n u s p r o d u c e d no m y c e l i u m . The two g e n e r a were a p p a r e n t l y c l o s e l y r e l a t e d b u t o n l y S p o r l d l o b o l u s d e m o n s t r a t e d a p e r f e c t s t a t e ; a l t h o u g h N y l a n d h a d s e e n no s i g n s o f p l a smogamy o r r e d u c t i o n d i v i s i o n i n t h e g e n u s , he h a d o b s e r v e d k a r y o g a m y i n t h e r e s t i n g s p o r e . The a f f i n i t i e s o f S p o r o b o l o m y c e s have b e e n u n d e r d e b a t e since the genus was described. Based upon the similarity of the discharge mechanism of the ballistospores of this genus and that of Hymenomycete basidiospores, Kluyver and van Niel (1925» 1927) suggested that Sporobolomyoes was a Basidiomycete. Lohwag (I926) disputed this theory* al l y i n g the genus with the Asoomycetous yeasts. Guilliermond (1927) and Buller (1933) published detailed accounts of the cytology of Sporobolomyoes. showing a l l known stages of the organism to be monokaryotic, with no evidence of plasmogamy, karyogamy, reduction division, or a dlkaryo-phase. However, Buller did believe that the imperfect Sporob- olomyces had basidiomycetous a f f i n i t i e s . He based his conclusion upon ah extended comparative study of the discharge mechanism of both true basidiospores and of Sporobolomyces sporidia. Buller dismissed thelack of a known dikaryophase i n Sporob- olomyces as Insufficient cause to dispute the evidence of an identical discharge mechanism which he considered too complex to have evolved more than onoe. Additional evidence for Sporobolomyces being a l l i e d with the Basidiomycetes has since been provided by comparative studies of the base composition of DNA (Nakase and Komagata, I968; Storck et a l , I969) and of urease activity (Nakase and Komagata, I968) . - 3 -Nyland (1948b) observed that a l l yeast c e l l s of Sporldlobolus were uninucleate, yet a l l gave r i s e - with no apparent intervening plasmogamy - to dl k a r y o t i c mycelium. He concluded that these c e l l s must be d i p l o i d . This hypothesis was supported by the f a c t that karyogamy, but not reduction d i v i s i o n , had been observed i n the chlamydospores. L a f f i n and Cutter (1959 s ) observed that single c e l l Isolates of Sporldlobolus .johnsonll i n v a r i a b l y gave r i s e to clamped mycelium and that s u r v i v a l curves following u l t r a v i o l e t i r r a d i a t i o n showed the plateau c h a r a c t e r i s t i c of d i p l o i d or polypoid organisms. They concluded that meiosis must occur i n the formation of the dl k a r y o t i c mycelium from d i p l o i d yeast c e l l s . By microsurgical and micromanipulatory techniques, L a f f i n and Cutter (1959*>) were able to force a very small number of matings between c e l l s of Sporldlobolus Johnsonll. Whereas the re s u l t s were not highly successful i n number, the use of colour and vitamin-deficiency markers d i d show that Sporldlobolus Johnsonl! i s normally heterokaryotic. This f a c t suggests that the organism, at some point i n i t s ancestry, must have undergone sexual contact of some kind. Sporldlobolus .lohngnnit i s the only v a l i d l y described species of the genus, and Nyland*s s t r a i n i s the only - 4 -publlshed isolate. Phaff (in Lodder, 1970) described "Sporld-lobolus rulnenll" but has delayed publication of a Latin diagnosis pending results of DNA base composition and DNA-DNA homology studies (personal communication, 1972). This precaution i s necessary since the two "species" are morphol-ogically almost identical, differing only i n the a b i l i t y to assimilate certain carbon sources, and may therefore be two strains of the same species. "Sporldlobolus rulnenll* i s thus a nomen nudum. Van der Walt (1970) presented a perfect stage (Aesso-sporon) of Sporobolomyoes salmonlcolor (Fischer and Brebeck. 1894) Kluyver and van Niel. Aessosporon lacks mycelium and consists of a simple alternation of haploid and diploid yeast c e l l s . Diploidization i s probably achieved by "•..somatogamous autogamy i.e. by the fusion of the haploid nucleus of a c e l l with that of i t s bud" (Van der Walt, 1970). Heiosis was observed i n the germination of teliospores to form non-septate promycella from which budded one to four sporidla. The ploldy of the yeast c e l l s and sporidla was determined by DNA analysis (Van der Walt and Pitout, I 9 6 9 ) . The genus Aessosporon was assigned to the Tilletiaceae (Van der Walt, 1970). - 5 -Bandoni et a l (1971) published the description of a perfect stage of Sporobolomyces odorus Derx. In this species monokaryotic yeast c e l l s of different strains v i s i b l y conjugated under laboratory conditions. The dlkaryotic phase thus produced was morphologically almost identical to that of Sporldlobolus Johnsonll. AB i n S. Johnsonll karyogamy was observed i n the chlamydospores; the chiamydo-spores also germinated i n a similar fashion with the product-ion of promycelium-like structures or of dlkaryotic mycelium. The authors also noted that the basidia produced appeared to be tilletiaceous (like those of Aessosporon) but might, due to the conditions of germination, be abnormal. No attempts were made to determine the ploldy of the yeast c e l l s and no name was proposed for the new perfect state of Sporobolo-myces odorus. It was suggested, however, that possibly this was a synthetic version of Sporldlobolus johnsonll. Dedikaryotization of mycelial Basidiomycetes which bear clamp connections has been attempted by several researchers. Fries and Aschan (1952) and Papazian (1955) used a micro-surgical technique. A micromanipulator was used to puncture the terminal c e l l and the hook c e l l of a young unfused clamp connection. The resulting uninucleate penultimate c e l l was then severed from the hypha and cultured to produce a monokaryotic mycelial culture. This method was laborious -6-and time consuming, and was not marked by a h i g h r a t e o f s u c c e s s . M i l e s and Raper (1956) grew Schizophyllum commune I n media c o n t a i n i n g sodium t a u r o c h o l a t e (0.15$) o r c h o l i c a c i d (0.12$). The e f f e c t of these compounds upon the organism was to d i s r u p t the f u s i o n o f the clamp c o n n e c t i o n s , thereby r e s u l t i n g i n an abundance of simple septa, f a l s e (unfused) clamps, and *bow-tie" clamps ( M i l e s and Raper, 1956). M a c e r a t i o n o f the c u l t u r e i n a Waring b l e n d e r severed the monokaryotic hook c e l l s from the normal d i k a r y o t i c mycelium. When the macerate was p l a t e d onto n u t r i e n t medium a p r o p o r t i o n of the r e s u l t i n g c o l o n i e s had been d e r i v e d from these u n i n u c l e a t e c e l l s and were t h e r e f o r e monokaryotic. The method of M i l e s and Raper (1956) has s i n c e been used by s e v e r a l a u t h o r s , u t i l i z i n g v a r i o u s chemicals as d e d i k a r y o t i z i n g agents. Da Costa and K e r r u i s h (1962) and K e r r u i s h and Da Costa ( I 9 6 3 ) used s e v e r a l c h e m i c a l s , i n c l u d i n g c h o l i c a c i d and a r s e n a t e s , to d e d l k a r y o t i z e L e n z l t e s t r a b e a and o t h e r Basidiomycetes. McClaren ( I 9 7 O ) used sodium a r s e n a t e (0.0k2%) and c h o l i c a c i d (0.5%) to d e d l k a r y o t i z e Coprlnus  m y c e l i o c e p h a l u s . Moore and Stewart ( I 9 7 I ) used the same method to de--7-dikaryotize Coprlnus lasopus. substituting 2-deoiy-D-glucose as the dedikaryotizing agent. This compound disrupts glucan synthesis i n oel l walls (Johnson, 1968a, 1968b; Farkas et a l , I969) thereby preventing the successful fusion of clamp hook cells with the penultimate hyphal c e l l . This results, as with other chemical dedlkaryotlzing agents, i n numerous monkaryotlc branches forming from hook c e l l s and bow-tie clamps. In 1971 Bandoni (unpublished) isolated both Sporobolo-myces and Sporldlobolus forms of a fungus from a Jamaican source. The present study sought to characterize these iso-lates, and to investigate the nature of the genus Sporldlo-bolus. - 8 -MATERIALS AND METHODS The fungi studied i n this work were: Sporidlobolus  .lohnsonll Nyland, Sporldlobolus rulnenll Phaff, Sporobolo-myces odorus Derx, Sporobolomyces salmonlcolor (Fischer & Brebeck) Kluyver & van Niel, and new isolates of Sporobolo-myces and Sporldlobolus form, studied were as follows: Sporldlobolus .lohnsonli "Sporldlobolus rulnenll' Sporobolomyces odorus The origins of the strains - University of Br i t i s h Columbia Culture Collection (UBC) #865 (= #304, least Culture Collection; Davis, California. - UBC #5039 (* #67-67, Yeast Culture Collection, Davis, Calif.) - UBC #948 - isolated by J . Higham from collection of Hellcoma monlllpes (collected by E. Bandoni, UBC Endow-ment Lands, Jan. 1970). Compatible with UBC # 9 4 9 . - UBC #949 - isolated by M. Rafiq from s o i l from Harrapa, West Pakistan, Jan. 1970. - UBC #981 = #483, Centraal bureau voor Schimmeloulture, Baarn, Netherlands (type culture). Sporobolomyces salmonlcolor- UBC #988 = CBS # 4 9 6 . - UBC #989 = CBS #1013. Compatible with UBC # 9 8 1 . - 9 -Sporldiobolus sp. - UBC #5035 - isolated by E. Bandoni from herbaceous culm, collected Runaway Bay, Jamaica, Nov. 1971. Sporobolomyces sp. - JGH #1, 2, 3» 5, 6. Isolated by J.Eigham from same culm as UBC #5035. - SF #251. 254, 255. 258, 261. Isolated by R. Bandoni from same culm as UBC #5035. Compatible isolates : SF #251 X SF #261, JGH #5 X SF #261. A l l media used in these studies are l i s t e d i n the appendix, together with their contents and, where necessary, notes on the mode of preparation. Routine maintenance cultures were kept on Malt Yeast Soytone (MYS) agar plates at room temperature. Morphological and physiological studies were carried on according to the techniques of Phaff and Lodder (in Lodder, 1970); modifications i n the procedures are stated below. Colony and c e l l morphology were observed i n 3% Malt Extraot (ME), and i n Glucose Yeast Peptone (GYP), and on the corresponding solid media (MA and GYP agar). Cultures were incubated at 2 0°C; observations were made 3 days and -10-one month a f t e r I n o c u l a t i o n . C e l l s i z e s were determined by measuring a t l e a s t 20 c e l l s a t random w i t h a p r e v i o u s l y c a l i b r a t e d o p t i c a l micrometer. Dalmau p l a t e s were I n o c u l a t e d a c c o r d i n g to the method d e s c r i b e d i n Lodder (1970) and i n Lodder and Kreger-van R i J (1952). They were i n c u b a t e d i n an i n v e r t e d p o s i t i o n f o r 7 days a t room temperature. The media used f o r these p l a t e s were: D i f c o Potato Dextrose Agar (PDA), D i f c o Corn Meal Agar (CMA), D i f c o Yeast Morphology Agar (YMA), and C o n j u g a t i o n Medium (CJM). A r b u t i n s l a n t s were i n o c u l a t e d w i t h the organisms to be t e s t e d f o r the a b i l i t y to hydrolize/3 - g l u c o s i d e s . Incub-a t i o n was a t room temperature, w i t h o b s e r v a t i o n s made a f t e r 7 and 14 days. Fermentative a b i l i t y was t e s t e d by i n o c u l a t i n g tubes o f 0.5% Yeast E x t r a c t and 2% d e x t r o s e i n water, each c o n t a i n i n g a Durham tube. Tubes were observed a f t e r 7 and 14 days. The a b i l i t y to grow a t 37°C. was t e s t e d by i n o c u l a t i o n of MYS and YMA p l a t e s w i t h the organisms to be s t u d i e d . A f t e r - l i -the se had been Incubated at 37°C for 7 days, a subculture was made to a fresh plate of the same medium. This was then incubated at 37°C. for an additional 7 days. Growth i n vitamin-free medium was tested i n the following manner. A l l glassware was cleaned with chromic acid over-night, then thoroughly rinsed i n d i s t i l l e d water. Tubes of st e r i l e Vitamin-free Medium were then inoculated with the strains to be studied. These were incubated at room temperature for 7 days. A subculture was then made to fresh tubes of Vitamin-free Medium, which were then incubated for an addit-ional 7 days before observations were made. Nitrate assimilation was tested i n an identical manner but Difco Yeast Carbon Base plus 0,5% potassium nitrate was substituted for the Vitamin-free Medium. Carbon source u t i l i z a t i o n was observed on solid medium, using a modification of the technique of Shrlfine et a l (1954). A l l glassware was cleaned i n chromic a d d over-night, then thoroughly rinsed with d i s t i l l e d water. Agar was washed i n a series of solvents by suspending i t for several hours by means of a magnetic s t i r r e r . The agar was -12-then recovered by Buchner funnel with Watman #1 f i l t e r paper. The solvents used, i n order, were: d i s t i l l e d water - 3 washes; petroleum ether - one wash; ethyl acetate - one wash; acetone - one wash; d i s t i l l e d water - one wash; acetone - one wash. After the f i n a l wash and f i l t e r i n g , the agar was allowed to dry for one week to insure com-plete evaporation of the solvents used. Preliminary tests using Difco Yeast Nitrogen Base and varying concentrations of dextrose (0, 0.1, 0.5» 1» 2, 4, and 5%) showed that growth of the organisms to be tested was no heavier at 5% dextrose than at 0.5% (see also Shrifine et a l , 1954). Therefore the medium for car-bon assimilation was prepared using the equivalent of 0.5% dextrose of each carbon source. The carbon sources were st e r i l i z e d separately from the medium. A l l disaccharides and heat l a b i l e compounds were dissolved i n d i s t i l l e d water and f i l t e r s t e r i l i z e d by means of a Hetricel f i l t e r (pore size * 0.2 ) i n a Swinny Millipore F i l t e r attachment to a standard hypodermic needle. A l l other carbon sources were st e r i l i z e d by autoclaving at 10 l b . pressure for one hour. -13-The medium used was Difco least Nitrogen Base plus 1.5# agar. This was autoclaved for one hour at 10 l b . pressure i n double strength aliquots i n screw cap tubes. After s t e r i l i z a t i o n * the double strength carbon sources were mixed with the hot medium by gentle shaking, and the resulting complete medium was poured into s t e r i l e plastic petri dishes. The pl&t«s were allowed to dry for 3 days prior to inoculation. Inoculation of the carbon source medium was by very fine wire needle, and consisted of the smallest possible number of c e l l s ; inoculum was from a 48 hour room tempera-ture MYS culture. One point inoculation of each strain being tested was made per dish ( Plate 1A duplicates the arrangement of colonies); two plates of each carbon source were used to give two replications of each strain. Controls consisted of 6 similarly inoculated plates of the same medium with no added carbon source. Incubation was i n an inverted position at 15°C. for 14 days with observations being made every 3 days. After observations had been made on the above carbon source experiment, doubtful results were checked by using Lodder 1s (1970) technique i n l i q u i d medium. This involved -14-the carbon sources which produced positive results for a l l strains, suggesting possible cross-feeding of simple carbon compounds amongst the strains (Shrifine et a l , 1954). Results which contradicted previously published data were also double-ohecked by this method. The medium was prepared i n the same way as above except that the agar was omitted and a l l carbon sources were f i l t e r s t e r i l i z e d (with the exoeption of ln u l l n ) . The resulting medium was pipetted into s t e r i l e acid-cleaned test tubes; after inoculation these were kept at room temperature for 14 days, with observations being made every three days. Controls consisted of two tubes of each strain i n s t e r i l e Difco Yeast Nitrogen Base with no added carbon* Hating experiments were based upon the techniques of Bandonl et a l ( I 9 7 I ) . One loopful of each of the strains to be mated was transferred to a plate of nutrient medium and the two were thoroughly mixed by gentle s t i r r i n g with a transfer loop. Four orosses were made per plate for i n i t i a l crosses, followed by one cross per plate where i t was desirable to repeat crosses to verify results. Routine matings were made on CjM; other media used were MDA (Laffln and Cutter, 1959), MYS, MYP (Bandoni et a l , 1971). CjM plus 1% activated -15-charcoal, water agar* Minimal Medium (MIN), and Glucose Asparagine Medium (GA). Incubation was at 15°C, 20°C, or at room temperature, i n an inverted position, for 7 to 14 days. Observation was made directly on the plate. A coverslip was used to scrape the surface growth from the culture; a fresh coverslip was then placed on the agar surface and the plate was placed directly on the microscope stage. Presence of mycelium bearing both clamp connections and ohlamydospores was required for the cross to be considered positive. Colony diameter was measured to determine the effeot of temperature on growth of certain strains. Inoculation of ce l l s from a young, vigorously growing MYS culture was made by a very fine transfer needle. Care was taken to make the inoculations of each strain of a standard size, but because of differences i n texture, this was impossible amongst strains. For each strain five point inoculations were made on each of two plates of CJM. Two such plates were incubated i n an inverted position at each of the following temperatures: 5°C., 1 0°C, 15°C., 2 0°C, room temperature, 3 0°C, and 37°C. -16-Each of the 10 r e s u l t a n t c o l o n i e s o f each s t r a i n a t each temperature was measured a f t e r 7 and 14 days. C o l o n i e s w i t h a m y c e l i a l f r i n g e were measured t w i c e : once f o r t o t a l growth and once f o r diameter minus the m y c e l i a l f r i n g e . Thus a n o t a t i o n o f the q u a l i t y of growth was p r o v i d e d . S e v e r a l techniques were used i n a t t e m p t i n g to germinate chlamydospores (Duran and S a f e e u l l a , I968; Jones, 1923; Lowther, 1948; Meiners and Waldher, 1859; N i e l s o n , I 9 6 6 ) . The s t a n d a r d p r e l i m i n a r y treatment was to macerate the agar c o n t a i n i n g the chlamydospores i n a s m a l l amount of s t e r i l e d i s t i l l e d water, u s i n g e i t h e r a Waring b l e n d e r o r a Pyrex homogenizer. The r e s u l t i n g macerate was p l a t e d onto n u t r i e n t and n o n - n u t r i e n t media immediately, o r was f i r s t d i l u t e d w i t h l a r g e amounts o f s t e r i l e d i s t i l l e d water. The spores were a l l o w e d to soak from 12 hours t o s e v e r a l weeks, w i t h the water being changed s e v e r a l times ( F i s c h e r and Holton, 1957). At i n t e r v a l s samples o f chlamydospores were t r a n s f e r r e d to p l a t e s o f v a r i o u s media. H e a t i n g o f the chlamydospores In a water bath (50°C. to 55°C.) f o r from 5 to 10 minutes was another' treatment used b e f o r e p l a t i n g out some of the s p o r e s . The media used i n the g e r m i n a t i o n experiments were -17-varied: water agar, CJM, GA, GA+, CMA, water agar plus 0.0$% potassium phosphate (pH 4 .7), and water agar plus 0.1% ammonium sulphate (pH 4.9). The chlamydospores were Incubated at 10, 15, 20, 30, and 37°C, as well as at room temperature. Flasks of PDA inoculated with S. Johnsonll and with UBC #5035 were incubated alternate days at 35°C. and at room temperature, according to the method of Nyland (1948a). Samples were taken from the flasks, crushed, and streaked out on water agar i n an attempt to duplicate Ny-land* s technique for germinating chlamydospores. Cultures were grown on CjM plus 1% activated char-coal for two to four weeks, then macerated and the chlamy-dospores plated out on water agar. Tubes of chlamydospore macerate were frozen (0 + 1°C.) for 48 hours, thawed, and plated onto water agar. Single c e l l isolates were made by plating out a series of diluted c e l l or macerated mycelial suspensions. At least 40 colonies were transferred from these original MIS plates to fresh ones. Eaoh resulting colony was considered to have been derived from a single c e l l . -18-Experiments requiring ultraviolet radiation u t i l i z e d a lamp emitting li g h t at a principal (94$) wavelength of 2537 £. The procedure for Irradiation was as follows: the organism to be studied was grown for 24 hours on MYS at room temperature. Cells were washed from the surface of the culture with s t e r i l e d i s t i l l e d water; the c e l l density was ascertained by haemacytometer and was subsequently adjusted to 10^ ce l l s per ml. Samples were transferred to st e r i l e 15 x 60 mm. plastic petri dishes, which were then exposed, with l i d removed, at a distance of 6 cm. from the source of radiation; the intensity at the level of the sam-ple was 5 x 10^ erg/cm2. Exposure time varied from zero to 2 minutes at 10 second intervals. Constant agitation of samples during irradiation was achieved by means of a mag-netic s t i r r e r . Following irradiation the samples were diluted to 10^, 10**", 10^, and 102 cells/ml., 0.1 ml. of each was pla-ted onto each of two HIS plates which were incubated, i n -verted, at room temperature for 48 hours. The colonies were then oounted and the survival rates calculated. In some cases the plates were incubated for an additional - 1 9 -2k hours and atypical colonies were transferred to fresh MYS plates i n an attempt to isolate ultraviolet-induced mutants• Attempts were made to dedlkaryotize S. .lohnsonll and UBC #5035 by the method described above; after irradiation colonies which appeared non-mycelial were maintained and, i f no signs of the dlkaryotic condition occurred, these isolates were crossed with each other and with some strains of Sporobolomyces odorus and S. salmonloolor. Dedikaryotization was also attempted by the use of chemical mutagens: 2-deoxy-D-glucose, sodium glycocholate, and cholic acid were incorporated into nutrient medium. After inoculation with the dikaryon to be tested, the plates were incubated at room temperature, 1 5°C, or 20°C. After two to four weeks the mycelium was macerated with s t e r i l e d i s t i l l e d water and single c e l l isolates were made on MYS as previously described. A l l colours mentioned i n this work are based on those i n Rayner (1970). 20-EESULTS Description of Sporobolomyces odorus dikaryon. The 3 day old culture of the UBC #948 x #949 dikaryon i n GYP produced no p e l l i c l e and only a very slight sediment. The ce l l s i n suspension were of two distinct types: aplculate ovate cells 4 - 7 x 7 - H p were often sterlgmate but were seldom observed with buds. Conversely cylindric to reniform cel l s 1.5 - 2.5 x 4 - 12y* were frequently seen with buds but never with attached ballistospores. Mycelium (diameter 1»5 - 3/*) was present; a few true clamps were observed but the majority of clamps did not fuse with hyphae. Many hyphal tips appeared abnormal, often the wall was broken with the cytoplasm extruding. The corresponding GYP sol i d culture consisted of cel l s of the same types and dimensions described above. The streak was pale Flesh-pink, glossy, and slimy i n texture with a small amount of mycelial fringe extending from isolated points along the margin. A heavy deposit of ballistospores formed a mirror-Image of the colony on the l i d of the petri plate. - 2 1 -After one month the cells In both so l i d and l i q u i d GYP were of the same types and dimensions as at 3 days, but with a great increase i n the amount of mycelium. In the l i q u i d culture a moderate sediment and thin* wrinkled, pale Salmon p e l l i c l e were present. The streak culture was a dull yellow-Flesh colour, the center raised and con-voluted while the margins were f l a t and smooth. Mycelial fringe extending beyond the margins of the streak was deeply creased and bore abundant chlamydospores. The Sporobolomyces odorus dikaryon i n 3% ME consisted mainly of budding cells of the same dimensions as i n GYP after 3 days. Also present were large o i l - f i l l e d c e l l s , globose, ovate, or lacrymoid, 4 - 8 x 5 - 10A* i n size with a wall 0.25 - 0.5^ thick. Sterigmata ranged up to 75/t i n length. Mycelium was scarce, 1.5 - 3/* i n diameter with a few young chlamydospores. As i n the GYP cultures, the hyphae tended to have thin, ruptured walls through which cytoplasm extruded. The solid MA culture was moist, glossy, pale Flesh after three days. After one month the streak was of the same texture, but gray-Salmon i n colour; the margins were ragged and mycelial fringe was present i n small patches. 22-The one month l i q u i d culture had no p e l l i c l e but many glossy Salmon-coloured i s l e t s . Sediment was moderate and consisted of mycelium with chlamydospores and some budding c e l l s . Dalmau plates were not made with Sporobolomyces odorus dikaryon, but type of growth on various media was recorded. Growth on PDA was very heavy and predominantly mycelial, but chlamydospores did not appear u n t i l the oulture was over 7 days old. The 7 day culture on CMA was almost totally mycelial, healthy, with many young chlamydospores. Hyphae were 2^ i n diameter; chlamydospores were typically terminal, each with a clamp at the base and a hyaline projection at the t i p . YMA plates of S. odorus dikaryon produced abundant chlamydospores on aeri a l hyphae, but the submerged mycelium was gnarled, thin-walled, with burst hyphal tips and no chlamydospores. In contrast, on CjM, the mycelium was mostly submerged, with large well-formed clamps and chlamydospores. The spores averaged 10/* i n diameter* contained o i l globules of 3 - bp i n size, and showed signs of sculpturing and pigment formation after only 7 days. Growth on the synthetic medium GA was l i k e that on CJM, but the development of chlamydospores was slower, taking 10 to 14 days for sculptur--23-ing and pigmentation to become apparent. Growth on MYS was heavy; mycelium was robust with healthy chlamydospores. MDA, however, produced short, gnarled, antler-like hyphae, and both hyphae and immature chlamydospores were often ruptured and extruding cytoplasm. Repeated single c e l l isolates from the dikaryon of Sporobolomyces odorus produced purely monokaryotic cultures. No mycelium of any kind was detected i n any of the isolates, even after two months on MYS at room temperature. To rule out survival of the original strains, mycelium was trans-ferred for several generations t i l l a completely mycelial culture was obtained. This was washed with s t e r i l e d i s t i l l e d water to recover conidia. These were then plated out to achieve single c e l l isolates, none of which were mycelial. Primary sporidia, recovered i n the same manner from germin-ating chlamydospores, produced identical results. Sporobolomyces odorus ( UBC #948 x UBC #949 ) pro-duced no measurable growth at 37°C, but did produce growth at 5°C. The optimum temperature for growth, both of mycelium and yeast c e l l s , was 20°C. on CJM. Figures l a and lb show the colony diameters of the S. odorus dikaryon after 7 -24-and 14 days respectively on this medium, at various temper-atures* -25-u © •P © at >» c o H o o 25 20 15 10 Sporobolomyces odorus dikaryon UBC #9^8 x #949. S t i p p l i n g myce l ia l f r i n g e . 10 15 20 room temp. 30 37 Temperature (degrees Cent igrade) . F i g . l a . Colony diameter of Sporobolomyces odorus dikaryon at various temperatures a f t e r 7 days on CjM. u © •p © o H O 25 r 20 15 10 n S.odorus dikaryon S t i p p l i n g myce l i a l f r i n g e . 5 10 15 20 room 30 37 temp. Temperature (degrees Cent igrade) . F i g . l b . Colony diameter of Sporobolomyces odorus dikaryon at various temperatures a f t e r 1 4 days on CJM. -26-Description of Sporobolomyces sp. JGH #3 After 3 days i n 3% ME, JGH #3 formed no p e l l i c l e and only a very lig h t sediment. The cel l s were ovate, 1 . 5 -3.5 x 4 . 0 - 7.5^; many budding cel l s were seen but no sterlgmate ce l l s or mycelium were observed. Most c e l l s contained large o i l reserves. On sol i d MA the cel l s were of the same type and dimensions; the streak was glossy Salmon, moist and with smooth margins. In cross section the colony was f l a t and only slightly raised. After one month i n ME, JGH #3 had s t i l l produced no p e l l i c l e and only lig h t sediment. Growth i n suspension was heavy and consisted of the same type and size of c e l l s as the 3 day culture i n the same medium. The streak culture was Flesh to Peach in colour, creamy and soft i n texture. The colony was f l a t and spreading, the margins fimbriate. Balllstospores were scarce i n both solid and l i q u i d media. After 7 days at room temperature on CMA, JGH #3 pro-duced Salmon colonies, semiglossy and moist with lobate margins. Beneath the coverslips on the Dalmau plate the growth was far less dense and pseudomycelium was abundant. Cells were of the same size and shape as i n ME, but ball!sto--27 spores were also abundant. These were asymmetrically reni-form and 1 . 5 - 4 x 5 - 7 / * i n size. Figures 2a and 2b show the colony diameter of JGH #3 on CJM at various temperatures. There was no significant difference between the amount of growth at 1 5°C, 2 0°C, room temperature, and 30°C.; therefore no optimum temper-ature can be assigned. Physiological tests were carried out with both JGH #3 and JGH #5. For both, fermentation was negative and arbutin s p l i t t i n g was positive. In vitamin-free medium JGH #5 showed strongly positive growth after one week; JGH #3 was only weakly positive after the same length of time. Both strains were able to assimilate potassium nitrate. The results of carbon source u t i l i z a t i o n are incorporated into Figure 3» - 2 8 -© •P CD § & O H O O 25 20 15 10 JGH #3 10 15 20 room 30 37 temp. Temperature (degrees Cent igrade) . F i g . 2 a . Colony diameter of Sporobolomyces sp. JGH #3 at various temperatures a f t e r 7 days on CjM. 2 5 ~ 20 u © © 15 § 5 i o 6 5 o ->H O n JGH #3 20 room 30 37 temp. 5 10 15 Temperature (degrees Cent igrade) . F i g . 2b. Colony diameter of Sporobolomyces sp. JGH #3 a t various temperatures a f t e r 14 days on CJM. -29-Figure 3. Carbon source u t i l i z a t i o n by Sporidlobolus sp. UBC #5035 and s t r a i n s of Sporobolomyces spp. Sporobolo- 3.odorus S . sa lnon ico lor myces sp. UBC Carbon source JGK #3 .JGH #5 #5035 UBC #983 .UBC #988 UBC #989 maltose + + mannitol + + + + + + c i t r i c a c i d + + + + + + galactose + + + - + -sucrose + + + + + + l ac tose - - - - - -xylose + + + - - -i n o s i t o l - - - - - -dextrose + + + + + + sorbose w+ - + - - -c e l l o b i o s e - + - - + + trehalose + + + + + melibiose - - - - -ra f f inose - - - - + + melezitose - - - - + + i n u l i n w+ + - — - — s tarch - - - - - — L-arabinose + + + - - -D-arabinose + + + + + + D-ribose - w+ - + + + rhamnose - - - — — _ ethanol + + + w+ + + g l y c e r o l + + + + + + e r y t h r i t o l - - - - - -r i b l t o l + + - + g a l a c t i t o l + + + - - — sorbi t o l + + + + + + s a l i c i n + + + - - -l a c t i c a c i d - - + + + s u c c i n i c a c i d + + + + + + « - m e t h y l - w+ w+ - - + + D-glucoside Symbols: + =growth; w+ =weak growth; - =no growth. - 3 0 -Descrlptlon of Sporldlobolus sp. UBC #5035 After 3 days In GYP, UBC #5035 produced a slight sedi-ment but no p e l l i c l e . The cells were of two types: a cylindric-a l type 1.5-2.5 X 7.5 - 9.5^ and an ovate type 3 - 5 x 5.5 -9/A • In both cases budding was observed but no b a l l i s t o -spores or sterigmate ce l l s were seen. Short segments of pseudomycelium were present, as were varying lengths of true mycelium (1.5 - 3M i n diameter) with clear clamp conn-ections. The c e l l walls of these hyphae were abnormal, extruding cytoplasm; likewise chlamydospores were often thin-walled and ruptured. No normal chlamydospores were observed. The corresponding agar culture contained identical c e l l types. The streak was a semi-glossy pale Flesh, creamy and soft, with a slightly wrinkled surface. Small amounts of mycelial fringe originated at points along the margin of the streak; the hyphae showed the same abnormalities described i n the l i q u i d culture. No chlamydospores or ballistospores were observed and there was no mirror image of spores produced on the l i d of the inverted petri dish. After one month the liq u i d GYP culture had not produced a p e l l i c l e . A heavy sediment was present; mycelium was s t i l l scarce and abnormal and chlamydospores were few and poorly developed. The solid culture after the same period had taken on a yellowish cast. The surface was wrinkled, glossy, and very tough i n texture. An extensive mycelial fringe extended below the surface of the medium and some white aerial mycelium occurred near the center of the colony. Normal chlamydospores were abundant. Yeast c e l l s were of the same types and dimensions as described at three days. On MA, UBC #5035 produced c e l l s of the same types and dimensions as on GYP. Most cylindrical c e l l s contained two uniform o i l globules ( 0 . 5 - 1 . 5 / * i n diameter); such ce l l s were never observed budding. Neither mycelium nor ballistospores were seen i n so l i d or l i q u i d Malt Extract cultures of this age. A s t e r i l e microscope slide placed beneath the MA culture for two hours at room temperature revealed that b a l l i s t o -spores were being produced i n small numbers. The spores deposited on the slide were asymmetric and 2 - 4 x 5 - 8 / * i n size. The colony on MA was identical i n colour, texture, and general appearance to that on GYP agar. Likewise the ME 32 culture was i n a l l respects l i k e that i n l i q u i d GYP after the same length of time. After one month i n yf» ME. UBC #5035 showed a moderate sediment* no p e l l i c l e * but a few very tiny* filmy i s l e t s . Most of the ce l l s were i n suspension; no mycelium was observed. The corresponding agar culture showed very l i t t l e growth. The streak was Salmon* semi-glossy* with smooth surface and margins. The consistency was moist and creamy. An extensive mycelial fringe, totally below the surface of the agar* radiated from the streak. Chlamydospores were abundant, but hyphal growth was not as dense as on GYP. On CMA, UBC #5035 produced a very heavy growth of budding and ballistospore-forming ce l l s with a very dense mycelial fringe; chlamydospores were abundant. Hyphae were 1.5 - 2p. i n diameter, with chlamydospores 10 - 14/* . The hyphae were f i l l e d with o i l globules 0.5 - V I N diameter. Chlamydospores were usually intercalary or on short side branches; only a small number were terminal. Hyaline proj-ections were present, terminal when the spore was at the end of a branch, but extending from the side of intercalary spores. Clamp connections were consistently observed at the base of chlamydospores. On Dalmau plates the type - 3 3 -of growth described above occurred only aerobically: beneath the coverslips growth consisted of budding cells and pseudo-mycelium. The PDA Dalmau cultures showed the same types of growth as the CMA plates. On YMA, however, the aerobic growth consisted of budding and balllstospore-forming c e l l s , whereas beneath the coverslips there was, i n addition, chlamydospore-bearing mycelium. Dalmau plates of CjM showed no growth beneath the coverslips. Aerobic growth was l i g h t and consisted of budding and ballistospore-forming c e l l s (Plate 1C) with an extremely extensive mycelial fringe bearing abundant chlamydospores. Hyphae were 1.5 - 3/* i n diameter, averaging Zf*. , and containing large (2/0 o i l globules. Chlamydospores measured from 12 -15/n * averaging 13/* . Plate ID, E , F, and G show details of UBC #5035 on CjM. The corresponding cultures on GA showed no growth beneath the coverslips. Aerobic growth was much l i k e that on CjM, but there was a total absence of mycelium. The colony diameter of Sporldlobolus sp. UBC #5035 - 3 4 -at various temperature i s i l l u s t r a t e d i n Figures 4a and 4b. The optimum temperature for growth was between 20°C. and 25°C.; there was l i t t l e difference between growth at 20°C. and at room temperature (average 2 3 ° C ) . Although total growth was slightly greater at room temperature, mycelial production was sli g h t l y heavier at 20°C. UBC #5035 s p l i t arbutin but showed no a b i l i t y to ferment sugars. It was also unable to grow i n vitamin-free medium, but was able to u t i l i z e potassium nitrate as i t s source of nitrogen. The results of the carbon source experiments involving UBC #5035 are included i n Figure 3. - 3 5 -PLATE 1 A - Colonies on MYS at room temperature a f t e r 7 days. Arrangement duplicates that of carbon source inoculations. X 1 .5. At l e f t , top to bottom - Sporobolomyces odorus UBC #981; Sporobolomyces salmonlcolor UBC #988; S. salmonicolor UBC #989. At r i g h t , top to bottom - Sporobolomyces sp. JGH #3; Sporobolomyces sp. JGH #5; Sporldlobolus sp. UBC #5035. B - Sporidlobolus johnsonll yeast c e l l s , i n GXP-a f t e r 5 days at 20°C. X 700. C - Sporldlobolus sp. UBC #5035, yeast c e l l s , i n GXP a f t e r 5 days at room temperature. X 700. D to G - Sporldlobolus sp. UBC #5035, on CjM a f t e r 5 to 14 days at room temperature. D - Mycelium with clamps; haematoxylin s t a i n . X 2000, E - Yeast c e l l ; haematoxylin s t a i n . X 3000. P - Intercalary chlamydospores. X 2000. G - Intercalary chlamydospores. X 500. -36-© -p © a •H O 25 20 15 10 5 -Sporldlobolus sp. UBC #5035 S t i p p l i n g myce l ia l f r i n g e . 10 15 20 room 30 37 temp. Temperature (degrees Cent igrade) . F i g . 4 a . Colony diameter of Sporldlobolus sp. UBC #5035 at various temperatures a f t e r 7 days on CJM. 25 U 20 © © * 10 o H O O Sporldlobolus sp, UBC #5035 S t i p p l i n g myce l ia l f r i n g e . room 10 15 20 temp. 30 37 Temperature (degrees Centigrade) F i g . ^ b . Colony diameter of Sporldlobolus sp. UBC #5035 at various temperatures a f t e r 14 days on CJM. - 3 7 -Description of Sporldlobolus Johnsonll The ME culture a f t e r 3 days showed a convoluted Pink p e l l i c l e , but l i t t l e sediment. The c e l l s were c y l i n d r i o (dimensions 3 - 4 x 8 - lip. ) and often apiculate (Plate 1 c ) . Many were seen with buds attached and b a l l i s t o s p o r e -bearing c e l l s were also common. Ba l l i s t o s p o r e s were 3 - 7 x 7 - IJn i n s i z e . The corresponding s o l i d culture pro-duced a streak of a yellowish-Salmon, tough, convoluted appearance. The streak was glossy at f i r s t (3 days) be-coming d u l l a t one month. An extensive hyphal f r i n g e ex-tended beyond the streak. A f t e r one month the ME culture had produced a very thick d u l l , convoluted p e l l i c l e , but s t i l l l i t t l e s e d i -ment. The c e l l type was as above but mycelium was now also abundant. On the PDA Dalmau plates, S. Johnsonll produced a very heavy growth of budding and ballistospore-forming c e l l s upon the surface of the agar. The colony was deep Salmon with a convoluted surface. Beneath the surface of the agar heavy mycelial growth bore many chlamydospores. Growth beneath the coverslips was predominantly mycelial -38-and pseudomyoellal; chlamydospores were few. On t h i s medium the hyphae were 3 - V<- i n diameter, gnarled, and often ex-truded cytoplasm through ruptured hyphal walls. On CJK S. .lohnsonil showed no growth beneath the cover-s l i p s . A erobically, the colony was pale Salmon, dry and d u l l , and consisted of budding and ballistospore-forming c e l l s (Plate lb) with a small amount of mycelial f r i n g e plus chlamydospores. Hyphae were 1.5 - 3/^  i n diameter; chlamydospores were 12 - 17/* • On YMA S. "lohnsonil produced heaped Salmon colonies of budding c e l l s surrounded by submerged mycelial f r i n g e . The hyphae averaged 3/* and the clamps often curved l a t e r a l -l y about the hyphae. On CMA the colony was almost t o t a l l y mycelial, with yeast c e l l s very scarce; otherwise the growth was normal. Likewise,on GA the growth was much l i k e that on CMA,but on t h i s medium many chlamydospores arose i n a s e s s i l e fashion on clamp connections along the hyphae. These spores averaged 5^ smaller i n si z e than the normal terminal ones. On MYP S. .lohnsonil produced very f l a t spreading colonies with l i t t l e mycelial f r i n g e and only a few chlamydo-- 3 9 -spores. The culture on MDA was highly domed and very con-voluted, and consisted of budding and ezagerated sterigmate c e l l s ; mycelium was sparse. Figure $& and 5b show the colony diameter of Sporldlo-bolus .lohnsonll at various temperatures on CjH. The optimum temperature, both f o r t o t a l growth and f o r mycelial growth was 20°C. P h y s i o l o g i c a l studies were not c a r r i e d out on S. johnsonll. -40-25 u <D - P CD § O H O o 20 15 10 II Sporldlobolus johnsonl l 10 15 20 30 room temp. Temperature (degrees Cent igrade) . 37 S t i p p l i n g myce l ia l f r i n g e . F i g . 5 a . Colony diameter of Sporldlobolus johnsonl l at various temperatures a f t e r 7 days on CJM. M CD - P <P § 5 O rH O O 25 20 15 10 5 • Sporldlobolus Johnsonll S t i p p l i n g myce l ia l f r i n g e . n 10 15 20 room 30 37 temp. Temperature (degrees Cent igrade) . F i g . 5 o . Colony diameter of Sporidlobolus johnsonl l at various temperatures a f t e r 14 days on CjM. - 4 1 -MSporldlobolus r u l n e n l l " was grown only In J% ME and on 3% MA. The 3 day culture In ME produced l i t t l e sediment and no sign of a p e l l i c l e . The c e l l s were ovate to c y l i n d r i c a l , 3 - 4 . 5 x 7 - 12/4 and reproduced by budding. No b a l l i s t o -spores or mycelium were observed. On the s o l i d medium the c e l l s were of the same siz e and type but b a l l i s t o s p o r e s , mycelium, and chlamydospores were occasionally present. A f t e r one month the l i q u i d culture had produced a thick, convoluted, Salmon p e l l i c l e . Sediment was l i g h t . Mycelium and chlamydospores were abundant. The streak culture was Pink to Salmon, tough i n texture, with a hyphal f r i n g e o r i g i n a t i n g at points along the margin of the colony. The hyphae were 2 - 3/<i n diameter with t e r -minal or i n t e r c a l a r y chlamydospores of 12 - 1 8 ^ o f the t y p i c a l Sporldlobolus type. A spore drop onto a s t e r i l e microscope s l i d e revealed the b a l l i s t o s p o r e s to be asym-metrical and 4 - 6 x 6 - I3^u i n s i z e . - 4 2 -P h y s l o l o g l c a l tests Besides the r e s u l t s already reported f o r JGH #3 and #5 and UBC #5035* two uncompatible s t r a i n s of Sporobolo-myces salmonicolor (UBC #988 and #989) and one of S. odorus (UBC #981) were also put through various p h y s i o l o g i c a l t e s t s . The r e s u l t s of the carbon source u t i l i z a t i o n ex-periments are Included i n Figure 3« A l l three s t r a i n s were unable to ferment sugars, but were able to grow i n vitamin-fr e e medium. A l l could u t i l i z e potassium n i t r a t e , but only S. salmonicolor UBC #989 gave a po s i t i v e arbutln t e s t , and even that was l a t e n t . Mating Studies Previous mating experiments had been performed on r i c h media, such as MYS (Bandoni et a l , 1971). However superior r e s u l t s were obtained using CJM which favours mycelial growth. Repetition of several of Bandoni*s crosses (personal communication) l e d to the use of UBC #948 x #949 as a t y p i c a l S. odorus dikaryon. Crosses of the Sporobolomyces forms from Jamaica (JGH #1, 2, 3, 5. 6, and SF #251, 254, 255. 258, 261) were made - 4 3 -on CJM i n a l l possible combinations three separate times. A l l crosses were negative with the exceptions of JGH #5 x SF #261 and SF #251 x SF #261 which both produced mycelium and chlamydospores i d e n t i c a l to those of UBC #5035. The above 10 Jamaican i s o l a t e s were also crossed with represen-t a t i v e s t r a i n s of Sporobolomyoes odorus and S. salmonlcolor. but with no p o s i t i v e r e s u l t s . Single c e l l Isolates As stated e a r l i e r , s i n g l e c e l l i s o l a t e s of Sporobolo-myces odorus dikaryon (UBC #948 x #949) y i e l d e d cultures of purely yeast type. No mycelium of any kind was present a f t e r one month on MIS at room temperature and the budding and ball!stospore-forming c e l l s were of the t y p i c a l Sporo-bolomyoes odorus form. Single c e l l i s o l a t e s of Sporldlobolus Johnsonii. as previously reported (Nyland, 1948a , 1948b, 1949; L a f f i n and Cutter, 1959 a )» gave r i s e to clamped, ohlamydospore-bearing mycelium, i n addition to budding and b a l l i s t o -spore-forming c e l l s . WS_. r u l n e n l l w and UBC #5035 produced the same type of growth: the singl e c e l l i s o l a t e s gave r i s e to clamped mycelium bearing chlamydospores. - 4 4 -U l t r a v i o l e t Experiments The s u r v i v a l curves f o r Sporldlobolus .johnsonll. UBC #5035» and Sporobolomyces odorus (UBC #948) are represented i n Figure 6. The graph i s semilogarithmic with the per cent survivors p l o t t e d against exposure time i n seconds. A l l three organisms produced curves departing i n s i g n i f i c a n t l y from s t r a i g h t l i n e s , none showed the O p i o i d plateau* proposed by L a f f i n and Cutter U959*) f o r Sporldlobolus  .lohnsonll. In f a c t the organism producing the curve closest to sigmoid was Sporobolomyces odorus. Mutants of S. .lohnsonll derived by u l t r a v i o l e t i r -r a d i a t i o n showed a v a r i e t y of colour, c o l o n i a l and morpho-l o g i c a l abnormalities. Some i s o l a t e s were t o t a l l y mycelial (with or without clamp connections), but no t o t a l l y yeast-type cultures were recovered. None of the non-clamp or non-chlamydospore-forming cultures showed p o s i t i v e mating reactions when crossed with each other or with Sporobolo- myces odorus s t r a i n s . In a l l , 40 mutants were i s o l a t e d and tested i n t h i s study. In addition, 15 i s o l a t e s of u l t r a v i o l e t i r r a d i a t e d UBC #5035 were also observed but no non-inycelial s t r a i n s were recovered. - 4 5 -F i g . 6. U l t r a v i o l e t s u r v i v a l curves for Sporldlobolus  j ohnson i i . Sporobolomyces odorus. and UBC #5°35. -46-Chemlcal Mutagens Chemical mutagens produced va r i a t i o n s i n the mor-phology of hyphae, hut no c o l o n i a l or colour mutants were observed. Gnarled configurations, f a l s e (unfused) clamp connections and 'bow-tie' clamps (Miles and Raper, 1956) were common amongst cultures of Sporldlobolus johnsonii. UBC #5035, and Sporobolomyces odorus dikaryon (UBC #948 x #949) grown on GA and CJM plus 0.16$ 2-deoxy-D-glucose, (Plate 2A + B). On these media there was also an abundance of simple septa and abnormal, thin-walled chlamydospores, but 120 i s o l a t e s recovered by maceration of the aberrant mycelium f a i l e d to y i e l d any non-mycelial c u l t u r e s . GA and CjM plus sodium glycocholate or c h o l i c a c i d produced near normal morphology i n the above three s t r a i n s , with a s l i g h t l y higher occurrence of unfused clamp connec-ti o n s , and a reduced number of b a l l i s t o s p o r e s . No attempts at dedikaryotization were successful on t h i s or any medium. Chlamydospore germination After soaking i n d i s t i l l e d water, the chlamydospores of Sporldlobolus johnsonii germinated according to the -47-PLATE 2 A - Sporldlobolus johnsonll on CjM plus 2-deoxy-D-glucose. Abnormal clamps and chlamydospores. X 1500. B - Sporldlobolus sp. UBC #5035 on CjM plus 2-deoxy-D-glucose. Abnormal clamps, hyphae, and chlamydospores. Haematoxylin s t a i n . X 1700. -48-manner described by Nyland (1948a, 1948b). The percentage of germination was small and large numbers of yeast c e l l s obscured observation. An attempt to repeat Nyland's (1948B) technique f o r germination on PDA at 35°C. was unsuccessful. Attempts to germinate the chlamydospores of UBC #5035 were t o t a l l y unsuccessful. None of the treatment used resu l t e d i n even one observable germination. Sporobolomyces odorus (UBC #948 I UBC #949) chlamydo-spores germinated on water agar, water agar plus potassium phosphate, and water agar plus ammonium sulphate, pro-vided they were previously well washed i n d i s t i l l e d water. Germination at 20°C. and at room temperature was by formation of a promycellum-lilce structure (Plate 3B) which gave r i s e to s p o r l d i a (Plate 3A, 4A, 4B). Occasionally under these conditions chlamydospores germinated by the production of mycelium (Plate 3C). In most cases observed, the promycelial structure was short and curved about the chlamydospore (Plate 3A, 3 B ) . Most were once septate, bearing one sporidium l a t e r a l l y from each of the two promycelial c e l l s . Some were aseptate with s p o r i d i a produced l a t e r a l l y (Plate 4 B ) . NO aseptate -49-PLATE 3 Germination of Sporobolomyoes odorus chlamydospores (UBC #948 x #949). X 5 0 0 . A - Formation of promyoelium-like structure and s p o r i d i a . On water agar, 6 to 14 days, at room temperature* B - Formation of promycelium-like structure. On water agar, a f t e r 8 days at room temperature. C - Formation of mycelium. On water agar, a f t e r 7 days at room temperature. S t i p p l i n g indicates the presence of cytoplasm. -50-promycelial structures were observed producing s p o r i d i a terminally. Generally the spo r i d i a were produced by budding on the promycelial structures? only a very few were observed to be sterigmate (Plate 4A) and the discharge of these s p o r i d i a was not observed. Upon germination the cytoplasm could be observed to move from the chlamydospore in t o the promycelial structure, and thence into the sp o r i d i a (Plate 3A, 3B, 3C; Plate 4 A , 4B, 4C). In some cases, secondary s p o r i d i a were produced upon the primary s p o r i d i a (Plate 4A), the cytoplasm moving from the primary to the secondary s p o r i d i a . This phenonemon may have been due to the f a c t that germination was observed only on non-nutritive media. On the three non-nutritive media used, chlamydospores of Sporobolomyces odorus (UBC #948 x UBC #949). when i n -cubated at 15°C, i n additi o n to the previously mentioned modes of germination, occasionally produced short mycelial segments bearing secondary chlamydospores (Plate 4C). No germination of Sporobolomyces odorus (UBC #948 x -51-UBC #9^9) w a s observed at any temperatures other than 15°C, 20°C, and at room temperature. On nutrient media (PDA, CJM, GA, GA+, and CMA) germination could not be ob-served owing to vast numbers of yeast c e l l s . Heating the preparation i n an attempt to destroy these vegetative c e l l s proved of l i m i t e d effectiveness, as bla s t o c o n i d i a were r a p i d l y formed on viable hyphal segments. Media containing ac t i v a t e d charcoal produced growth of yeast c e l l s , with only sparse mycelium and very few chlamydospores. Chlamydospores grown on any medium and subsequently subjected to f r e e z i n g f a i l e d to germinate. -52-PLATE 4 Germination of Sporobolomyces odorus chlamydospores (UBC # 9 4 8 x # 9 4 9 K A - Formation of promycelium-like structure and s p o r i d i a . On water agar, a f t e r 14 days at room temperature. X 500 B - Formation of s p o r i d l a , on water agar plus (NHj^SOij,, at room temperature. X 700 At l e f t - a f t e r 7 days. At r i g h t - a f t e r 8 days. C - Formation of chlamydospores. At l e f t - on water agar, a f t e r 8 days a t 15°C. X 700. At r i g h t - on water agar plus (NHj,)?S0fc, a f t e r 7 days at 15°C X 700. S t i p p l i n g indicates the presence of cytoplasm. A C -53-DISCUSSION As stated e a r l i e r , Phaff (personal communication) has expressed doubts that "Sporidiobolus r u l n e n l l " and Sporidiobolus Johnsonii d i f f e r s u f f i c i e n t l y to j u s t i f y t h e i r separation into two species. In his key to the species of the genus, Phaff ( i n Lodder, 1970) uses only the difference i n a b i l i t y to assimilate melezitose and ra f f i n o s e to d i s t i n g u i s h between S. Johnsonii and "S. r u l n e n l l " . Sporidiobolus sp. UBC #5035 has the d i s t i n c t i o n of a s s i m i l a t i n g neither, and of being unable to assimilate c e l l o b i o s e , f o r which the other two 'species 1 are p o s i -t i v e . The carbon source u t i l i z a t i o n r e s u l t s of t h i s study show that the three species d i f f e r i n t h e i r a b i l i t y to assimilate, i n a l l , twelve carbon compounds. Whereas these n u t r i t i o n a l a t t r i b u t e s are the only points of difference between S. johnsonii and"S. rulnenll"» UBC #5035 also d i f f e r s c o n s i s t e n t l y i n some morphological characters. The yeast c e l l s and b a l l i s t o s p o r e s of UBC #5035 i n v a r i a b l y are narrower i n proportion to t h e i r length and smaller i n o v e r a l l s i z e than those of the other species. An average UBC #5035 c e l l measures about 2 X 8/<i the aver-age S. Johnsonii or "S. r u l n e n l l " c e l l has dimensions of about 4 X 10^. -54-The mycelium and chlamydospores are morphologically the same i n a l l three species, with the exception of the f a c t that most UBC #5035 chlamydospores are i n t e r c a l a r y , whereas those of the other species are predominantly t e r -minal. In addition, when grown i n l i q u i d media, S. Johnson!! and MS. r u l n e n i l " both produce a thick, wrinkled p e l l i c l e . UBC #5035 forms no such p e l l i c l e , but rather a heavy growth i n suspension. The colour of S. Johnson!! and MS. r u l n e n i i * i s described as being Salmon to Pink, and occasion-a l l y Brick Red. UBC #5035 i s cons i s t e n t l y paler, with a s l i g h t yellowish cast, varying from Flesh to Salmon. The morphological and n u t r i t i o n a l differences de-scribed warrant the establishment of a separate species f o r UBC #5035. Snoridiobolus microsporus n. sp. Sporidia hyaline by transmitted l i g h t , Flesh to Salmon i n mass, c y l i n d r i c a l , (1.5) 2 (2.5) x (7.5) 8 (9.5)f<t or ovate, (3) 3.5 (5) x (5.5) 7 (9)/A. Sporidia germinate by ye a s t - l i k e budding or by formation of sterigmate b a l l i s t o -spores, 2 (4) x (5) 6 (8)^. Yeast c e l l s self-propagating by these methods. Dlkaryotic mycelium produced from single - 5 5 -monokaryotic yeast c e l l . 1.5 - 3/-t In diameter, with true clamp connections at every septum. Chlamydospores pro-duced lntercalarlly» some with l a t e r a l hyaline projections, occasionally terminally, then possessing a d i s t a l hyaline projection, each subtended by a clamp connection; hyaline and binucleate when young, becoming golden brown, uninu-cleate, and with a thick sculptured wall upon maturity, (10) 12 (14)^ In diameter. The d e s c r i p t i o n of S. mlcrosporus beginning on page 30 of t h i s work covers morphology on standard media, e f f e c t s of temperature on growth, carbon a s s i m i l a t i o n , etc. and w i l l not be repeated here. The Sporobolomyces form of Sporldlobolus mlcrosporus ( i e . JGH #3, JGH #5 etc.) appears to d i f f e r from previous-l y described species of Sporobolomyces, but i t i s beyond the scope of t h i s study to carry out a comparitive i n v e s t i -gation. Since some of the s t r a i n s do cross to form a Sporldlobolus-type organism, i t may be better to assign them to the genus Sporldlobolus as the imperfect state of S. mlcrosporus. The r e s u l t s of t h i s study, taking into account the - 5 6 -previous observations of Nyland (1948a, 1948b, 1949), L a f f i n and Cutter (1959*. 1959b), and Bandoni et a l (1971), indicate that the genus Sporldlobolus. as described, may represent a genetic teratology. The morphological s i m i -l a r i t i e s between Sporidiobolus species and the dikarya of Sporobolomyces species suggest that i n the former genus reduotion d i v i s i o n has been delayed past the normal b a s i d i a l stage. Thus Sporldlobolus alternates between a monokaryotic d i p l o i d and a d l k a r y o t i c haploid stage with no intervening monokaryotic haploid stage. Mating i s therefore impossible, and the organism e x i s t s i n a closed genetic system. In comparison, the Sporobolomyces odorus dikaryon possesses a normal smut l i f e c y c l e . Since a l l s i n g l e c e l l i s o l a t e s , even from purely mycelial cultures and from ger-minating r e s t i n g spores, produced no mycelium, a l l s p o r i d l a appear to be haploid. This would require that reduction d i v i s i o n take place i n the r e s t i n g spore. As conjugation of s p o r i d i a has previously been observed (Bandoni et a l . 1971), the l i f e cycle of t h i s organism i s probably com-plete, but a d d i t i o n a l work i s needed to confirm the s i t e of reduction d i v i s i o n . At present the perfect stage of Sporobolomyces odorus - 5 7 -cannot be described as a species of the genus Sporidlo-bolus . The current study shows the mode of germination of Sporobolomyces odorus r e s t i n g spores to be u s t i l a g i n -aceous. This does not agree with the observations of Ban-doni et a l (I97I) who reported t i l l e t i a c e o u s b a s l d i a under the conditions of t h e i r experiments. Since a l l studies agree that Sporldiobolus johnsonll produces t i l l e t i a c e o u s b a s i d i a , confirmation of the mode of germination of Sporo-bolomyces odorus r e s t i n g spores i s required before i t can be assigned to a genus. Even though morphologically a l -most i d e n t i c a l to Sporldlobolus johnsonll and MS. r u i n -e n l l n . the var i a t i o n s i n l i f e cycle - i e . the p o s i t i o n of reduction d i v i s i o n - might also warrant the ere c t i o n of a new genus f o r t h i s and r e l a t e d organisms. Throughout the present work the term chlamydospore has been used f o r the thlck-walled r e s t i n g spore of both Sporldlobolus and Sporobolomyces species; t h i s coincides with the usage of a l l previous authors on the subject. The term •chlamydospore 1, according to S n e l l and Dick (1957) r e f e r s to any thick-walled secondary spore. Alns-worth ( I 9 6 I ) and Fischer and Holton (1957) s t i p u l a t e that chlamydospores are asexual spores which germinate asex-u a l l y . The same authors define teliospores as t h i c k -walled r e s t i n g spores (of rusts and smuts) i n which -58-karyogamy and reduction d i v i s i o n occur, and which germinate, t y p i c a l l y , by the production of a promycelial structure. Therefore the r e s t i n g spores of the Sporobolomyces odorus dikaryon, and possibly those of the other synthesized Sporobolomyces dikarya, may properly be designated as t e l i o -spores. Sporldlobolus r e s t i n g spores, which undergo karyo-gamy but not reduction d i v i s i o n , do not f i t the precise d e f i n i t i o n of either 'chlamydospore* or • t e l i o s p o r e 1 . Due to the nuclear fusion and the mode of germination, thse spores should probably be considered to be teliospores which have l o s t the a b i l i t y to undergo meiosis. A great deal of study i s necessary i n the conoepts of genus and species i n the organisms studied i n t h i s i n v e s t -i g a t i o n , and i n the rela t e d f u n g i . The problem has been discussed by several authors ( F e l l , 1970; F e l l et a l , I 9 6 9 ; Fischer and Holton, 1957; Lodder, 1970; Sowell and Korf, i 9 6 0 ) . In many cases lack of morphological characters has neces-i t a t e d the use of p h y s i o l o g i c a l and n u t r i t i o n a l t e s t s . The r e s u l t i n g c l a s s i f i c a t i o n often s p l i t s s t r a i n s into species, f o r example i n Sporldlobolus. or maintains d i f f e r e n t organisms i n the same species. An example of the l a t t e r case, the discovery of perfect states i n Sporobolo-myces spp. has allowed the introduction into the c l a s s i f i --59-cation of such characters as mating reactions, d l k a r y o t i c morphology, and teliospore germination. Thus, Van der Walt (1970) has assigned one s t r a i n of Sporobolomyces salmoni- color to the perfect non-mycellal Aessosporon, while Ban-doni (unpublished) has crossed two s t r a i n s of the same im-perfect species to form a Sporldiobolus-type perfect s t a t e . In the same work, Bandoni also produced a Sporldlobolus-type dikaryon by crossing a s t r a i n of Sporobolomyces  salmonicolor with the type culture of Sporobolomyces  odorus. These r e s u l t s i ndicate that Sporobolomyces species are not homogeneous and that the c r i t e r i a used i n t h e i r c l a s s i f i c a t i o n require extensive r e v i s i o n . Standard descriptions i n the Sporobolomycetaceae and i n the genus Sporldlobolus are made according to morphology on r i c h media (malt extract, GYP, YMA). The r e s u l t s of t h i s study show that mating of Sporobolomyces haploids and the growth of mycelium of Sporldlobolus species and of Sporobolomyces dikarya are enhanced by the use of weak media. Due to t h i s f a c t , and to the morphological abnor-m a l i t i e s observed i n r i c h media, i t i s recommended that standard growth and descriptions of these organisms be made eit h e r on a weak medium (eg. CJM) or on s l i g h t l y r i c h e r mediums, such as MYS, which does not promote abnormalities. -60-Further attempts to dedlkaryotize Sporldlobolus species would best make use of chemicals such as 2-deoxy-D-glucose; u l t r a v i o l e t i r r a d i a t i o n produces too many genetic abnor-ma l i t i e s to be u s e f u l . The recovery of haploid c e l l s which could be crossed with each other and with Sporobolomyces haploids might provide a d e f i n i t i v e answer to the problem of the r e l a t i o n s h i p between the genus Sporldlobolus and the Sporobolomyces dikarya. The r e s u l t s of the current u l t r a v i o l e t i r r a d i a t i o n experiments do not agree with those of L a f f i n and Cutter (1959a). These authors presented a s u r v i v a l curve f o r the yeast c e l l s of Sporldlobolus .lohnsonil which exhibited an i n i t i a l plateau t y p i c a l of d i p l o i d organisms. The curve produced f o r t h i s species i n the present work was a s t r a i g h t l i n e i n d i c a t i n g a haploid organism, whereas that of Sporobolomyces odorus exhibited a s l i g h t i n i t i a l p l a -teau. Since the l i f e cycle of Sporldlobolus .lohnsonil suggests that the yeast c e l l s are indeed d i p l o i d , t h e i r ploidy should be confirmed by DNA a n a l y s i s . The numerous u l t r a v i o l e t mutants recovered i n attempts to i s o l a t e haploids of Sporldlobolus .lohnsonil and Sporldlo-bolus mlcrosporus exhibited a great range of morphological -61-v a r i a t i o n s at both the c o l o n i a l and the c e l l u l a r l e v e l . White and yellow colonies e s p e c i a l l y were common va r i a n t s . No attempts were made to Isolate biochemical mutants, but, considering the high number of v i s i b l e mutants, these were probably also induced. These r e s u l t s suggest that the c h a r a c t e r i s t i c s used f o r the c l a s s i f i c a t i o n of these organ-isms may be changed by a single induced mutation, and may therefore be suspect as taxonomic characters. Further study i n the genus Sporldlobolus i s c l e a r l y required, e s p e c i a l l y observation of r e s t i n g spore germination and d e f i n i t i o n of the s i t e of reduction d i v i s i o n . The i s o l a t i o n of haploid s t r a i n s by chemical and mechanical dedikaryotization would allow matings to be made with Sporobolomyces species, possibly e l u c i d a t i n g the rela t i o n s h i p s between the two genera. A c a r e f u l study of the species concept i n the Sporobolomycetaceae i s also required i n order to determine stable characters f o r the c l a s s i f i c a t i o n of these organisms. -62-SUMMARY 1 . D i k a r y a o f S p o r o b o l o m y c e s o d o r u s we re s y n t h e s i z e d , c o n f i r m i n g a n e a r l i e r r e p o r t ( B a n d o n i e t a l , I971). 2. S t r a i n s o f S p o r i d i o b o l u s s p p . a n d o f S p o r o b o l o m y c e s s p p . we re o b s e r v e d u n d e r s t a n d a r d c o n d i t i o n s ; o p t i m u m t e m p e r a t u r e s f o r g r o w t h , a n d a s s i m i l a t i o n o f c e r t a i n c a r b o n oompounds w e r e d e t e r m i n e d . 3 . A t t e m p t s we re made t o d e d l k a r y o t i z e c e r t a i n s t r a i n s o f S p o r l d l o b o l u s b y p h y s i c a l a n d c h e m i c a l m e a n s . 4. U l t r a v i o l e t s u r v i v a l c u r v e s w e r e p l o t t e d f o r two s p e c i e s o f S p o r l d l o b o l u s a n d o n e o f S p o r o b o l o m y c e s . 5. C h l a m y d o s p o r e s o f S p o r o b o l o m y o e s o d o r u s w e r e o b s e r v e d t o g e r m i n a t e b y t h e f o r m a t i o n o f u s t i l a g i n a c e o u s b a s l d l a . 6. A new s p e c i e s , S p o x l d l o b j o l u s j o l c r o s p o r u s . . i s d e s c r i b e d . -63-BIBLIOGRAPHY Alnsworth, G . C . I96I. D ic t ionary of the F u n g i . 5th E d i t i o n . Commonwealth Mycological I n s t i t u t e , Surrey. Bandoni, R . J . , K . J . Lobo, and S .A . Brezden. 197L Conjugation and chlamydospores i n Sporobolomyces odorus. Canadian Journal of Botany 49: 683-686. B u l l e r , A . H . R . 1933. Researches on Fung i . Volume 5: 171-206. Longmans, Green, and C o . , London. Da Costa, E .W.B. and R .M. K e r r u i s h . I962. Product ion of monokaryons i n Basidiomycete cu l tures by the a c t i o n of tox ic chemicals . Nature 195: 726-727. Derx, H . G . 1930. Etude sur l e s Sporobolomycetes. Annales Mycologic i 28: 1-23. Duran, R. and K . M . S a f e e u l l a . I968. Aspects of t e l i o spore germination i n some North American smut f u n g i . I . Mycologia 60: 231-243. Farkas, V . , A . Svoboda, and S. Bauer. I969. I n h i b i t o r y e f fec t of 2-deoxyglucose on the formation of the c e l l wa l l i n yeast p r o t o p l a s t s . Journal of Bac-t er io logy 98: 744-748. F e l l , J .W. 1970. Yeasts with Heterobasidiomycetous l i f e c y c l e s . Recent Trends i n Yeast Research. Spectrum 1: 49-66. F e l l , J . W . , A. S t a t z e l l , I . Hunter, and H . J . Phaf f . I969. Leucosporldium gen, n . , the heterobasidiomycetous stage of severa l yeasts of the genus Candida. Antonie van Leeuwenhoek 35s 433-462. F i s c h e r , B. and C. Brebeck. 1894. Zur Morphologie, B io log ie und Systematik der Kahmpilze, der Monl l i a candlda Hansen und der Soorerregers . Jena. F i s c h e r , G.W. and C S . E o l t o n . 1957. Biology and Contro l of the Smut Fung i . Ronald Press Co. New York. F r i e s , N. and K. Aschan. 1952. The p h y s i o l o g i c a l hetero-geneity of the d l k a r y o t i c mycelium of Polyporus  abie t lnus inves t igated with the a i d of m l c r u r g i c a l technique. Svensk Botanisk T i d s k r l f t 46: 4 2 9-^5. - 6 4 -Gui l l i ermond, A. 1 9 2 7 . Etude cytologique et taxonomique sur l e s levures du genre Sporobolomyces. E u l l e t i n de l a Societe Mycologique de France 4 3 : 2 4 5 - 2 4 8 . Henderson, S .A. and B . C . L u . I 9 6 8 . The use of haematoxylin f o r squash preparations of chromosomes. S t a i n Tech-nology 4 3 : 2 3 3 - 2 3 5 . Johnson, B . F . 1 9 6 8 a . L y s i s of yeast c e l l wal ls induced by 2-deoxyglucose at t h e i r s i t e s of glucan synthes i s . Journal of Bacter io logy 9 5 : 1 1 6 9 - 1 1 7 2 . . 1 9 6 8 b . D i s s o l u t i o n of yeast glucan induced by 2-deoxyglucose. Experimental C e l l Research 5 0 : 6 9 2 - 6 9 4 . Jones, E . S . 1 9 2 3 - Influence of temperature, moisture, and oxygen on spore germination of Ust l lago avenae. Journal of A g r i c u l t u r a l Research 24: 5 7 7 - 5 9 1 . K e r r u i s h , R . M . and E.W.B. Da Costa . I 9 6 3 . Monocaryotizat ion of cu l tures of Lenzi tes trabea (Pers . ) F r . and other wood-destroying Basidiomycetes by chemical agents. Annals of Botany N . S . 2 7 : 6 5 3 - 6 6 9 . Kluyver , A . J . and C . B . van N i e l . 1 9 2 5 . Uber s p i e g e l b i l d e r erzeugende Hefenarten und die neue Hefengattung Sporobolomyces. C e n t r a l b l a t t fur B a k t e r i o l o g l e , Parasitenkunde und Infect ionskrankhei ten , Ab. I I , 6 3 : 1 - 2 0 . . 1 9 2 7 . Sporobolomyces - e in Basldiomyzet? Annales Mycologic i 2 5 : 3 8 9 - 3 9 4 . L a f f i n , R . J . and V . M . Cut ter , J r . 1 9 5 9 a . Invest igat ions on the l i f e cyc le of Sporldlobolus j o h n s o n i i . I I r r a d i a t i o n and c y t o l o g i c a l s tud ie s . Journal E l i s h a M i t c h e l l S c i e n t i f i c Society 7 5 : 8 9 - 9 6 . . 1 9 5 9 b . Invest igat ions on the l i f e cyc le of S p o r l d l o -bolus . lohnsonil . I I . Mutants and micromanipulat ion. Journal E l i s h a M i t c h e l l S c i e n t i f i c Society 7 5 : 9 7 - 1 0 0 . Lodder, J . (ed.) 1 9 7 0 . The Yeasts . A Taxonomic Study. North Holland P u b l . Co. Amsterdam.' Lodder, J . and N.J .W. Kreger-van R i J . 1 9 5 2 . The Yeasts . A Taxonomic Study. North Holland P u b l . Co. Amsterdam. - 6 5 -Lohwag, H. I926. Sporobolomyces - Kein Easldiomyzet. Annales Mycologici 24: 194-202. Lowther, C.V. 1948. Low temperature as a f a c t o r i n the germination of dwarf bunt chlamydospores. Phyto-pathologla 38: 309-310. M cClaren, M. 1970. Chemical dedikaryotization of Coprlnus  mycellooephalus (Agaricales). Canadian Journal of Botany 47: 787-790. Martin, G.W. 1952. Revision of the North Central Tremellales. Studies i n Natural History. Iowa University 19: 1-122. Meiners, J.P. and J.T. Waldher. 1959. Factors a f f e c t i n g spore germination of twelve species of T l l l e t l a from cereals and grasses. Phytopathologia 49: 724-728. Miles, P.G. and J.R. Raper. 1956. Recovery of the component st r a i n s from d l k a r y o t i c mycelia. Mycologia 48: 484-494. Moore, D. and G.R. Stewart. 1 9 7 L Dedikaryotization of Coprlnus lagopus following growth with 2-deoxy-D-glucose. B r i t i s h Mycological Society Transactions 56: 311-313. Nakase, T. and K. Komagata. I968. Taxonomic s i g n i f i c a n c e of base composition of yeast DNA. Journal of Gen-e r a l and Applied Microbiology 14: 345-357. Nielson, J . I 9 6 6 . Changes In germination type i n some Ustllago spp. caused by an u n i d e n t i f i e d v o l a t i l e substance. Canadian Journal of Botany 44: 163-170. Nyland, G. 1948a. Preliminary observations on the morphology and cytology of an undescrlbed heterobasidiomycete from Washington state, Mycologia 40: 478-481. . 1948b. Studies on some unusual Heterobasldio-mycetes from Washington state. Ph.D. t h e s i s . State College of Washington; Pullman, Washington. . 19^9. Studies on some unusual Heterobasldiomycetes from Washington state. Mycologia 41: 686-701. Papazian, H.P. 1955. Sectoring variants i n Schlzophyllum. American Journal of Botany 42: 394-400. -66-Rayner, R.W. 1970. A Mycological Colour Chart . Commonwealth Mycological I n s t i t u t e , Kew, Surrey. S h i f r i n e , M . , H . J . Phaff , and A . L . Demain. 1954. Determin-a t i o n of carbon a s s i m i l a t i o n patterns of yeasts by r e p l i c a p l a t i n g . Journal of Bacter io logy 68: 28-35. S l o d k i , M . E . , L . J . Wickerham, and R . J . Bandoni. I966. E x t r a -c e l l u l a r heteropolysaccharides from Cryptococcus and Tremel la: a poss ib le taxonomic r e l a t i o n s h i p . Canadian Journal of Microbio logy . 12: 489-494. S n e l l , W.H. and E . A . D ick . 1957. A Glossary of Mycology. 1st E d i t i o n . Harvard U n i v e r s i t y Press; Cambridge, Massachusetts. Sowell , G. and R . P . K o r f . I960. An emendation of the genus I t e r s o n l l l a based on studies of morphology and pathogenic i ty . Mycologla 53: 934-945. Storck, R . t C . J . Alexopoulos, and H . J . Phaff . I969. Nucleo-t ide composition of DNA of some species of Crypto- coccus. Rhodotorula, and Sporobolomyces. Journal of Bacter io logy 93; 1069-1072. Van der Walt, J . P . 1970. The perfect and imperfect states of Sporobolomyces sa lmonlcolor . Antonie van Leeuwen-hoek, Journal of Microbiology and Serology 36: 49-55. Van der Walt, J . P . and M . J . P l t o u t . I969. P lo idy d i f ferences i n Sporobolomyces salmonlcolor and Candida a l b i c a n s . Antonie van Leeuwenhoek 35: 227-231. -6? APPENDIX Media Unless otherwise stated, a l l media were s t e r i l i z e d by autoclavlng at 15 l h . pressure f o r 15 minutes. C 1 M - Conjugation medium soytone 2 g. dextrose 2 g. water 1 1. agar 15 g. MYS - Malt Yeast Soytone medium malt extract 7 g» yeast extract 0.5 g. soytone 1 g. water 1 1. agar 15 g. ME - Malt Extract malt extract 3 g. water 100 ml. For MA (malt agar) add 1.5 g. agar. GYP - Glucose Yeast Peptone Medium glucose 20 g. peptone 10 g. yeast extract 5 g» water 1 1. For GYP Agar add 1.5 g. agar. PDA - Potato Dextrose Agar Difco PDA made according to package directions, -69-YMA - Yeast Morphology Agar Difco YMA made according to package d i r e c t i o n s . Arbutin medium arbutln 0.5 g. yeast extract 0.2 g. water 100 ml. agar 1.5 S. Ingredients were mixed, heated In a water bath to melt the agar, dispensed into tubes (5 ml. each), and autoclaved. The tubes were slanted t i l l c o o l . Vitamin-free Medium dextrose 1 g. ammonium sulphate 0.5 S. Vogel's Salts Sol'n 2 ml. water 100 ml. MYP - Malt Yeast Peptone Medium malt extract 15 g. yeast extract 0.5 g» soytone 2.5 S» water 1 1. agar 15 &• GA - Glucose Asparagine Medium glucose 2 S* asparagine 2 ' s. MgSOif 0.5 g. water 1 1. agar 15 g. Vogel's Salts sol'n 20 ml. GA+ - GA plus KH2POi4, - 0.5 g . / l . CMA - Corn Meal Agar Difco CMA made according to package d i r e c t i o n s . -70 Vogel's Salts Solution - Stock Solution Sodium citrate KH2P0h anhydrous MgS02j,.7H20 CaCL9.2H20 Vogel's Trace Elements water chloroform Add in order l i s t e d , s t i r r i n g continuously. Dilute 50 fold before use. 12.3 g. 25 g. 10 g. 1 g. 0.5 g. 0.5 ml. * 75 ml. 1.2 ml. * Vogel's Trace Elements Solution c i t r i c acid 5 g. ZnS0j . .7H 20 5 g. Fe ( N S 4 ) 2 t S 0 4 ) 2 . 6 H 2 0 1 g. CuS0i|..5H20 0.25 g. MnSO^.lH^O 0.05 g. H3BO3 anhydrous 0.05 g. Na2MbOj^.2H20 0.05 g. water 95 ail. chloroform 1 ml. MIN - Minimal Medium glucose KHgPOjj, MgS04.7H20 CaCNOoJg thiamine trace elements water agar * Trace Elements FeSO^ ZnSoj^ MnSojj, water 2 g. 1 g» 0.5 g. 0.5 g. 100 ug. 1 ml. * 1 1. 15 g. 0.02 g. 0.02 g* 0.02 g. 100 ml. -71 Haematoxylin Nuclear Stain - Henderson and Lu, I968. F i x a t i v e -F i x material d i r e c t l y on s l i d e f o r 5 minutes. Staining s o l u t i o n A -2% (w/v) haematoxylin i n 50% propionic a c i d . Staining s o l u t i o n B -0.5% (w/v) i r o n alum i n 50% propionic a c i d . Method -Af t e r f i x a t i o n , add one drop of Solution A and of Solution B d i r e c t l y to the material on the s l i d e . Cover and observe. n-butyl alcohol a c e t i c a c i d chromic a c i d 9 parts 6 parts 2 parts 

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