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Biology of Typhula erythropus Fr. Koske, Richard E 1971

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THE BIOLOGY OF TYPHULA ERYTHROPUS FR. by RICHARD KOSKE .Sc., C a l i f o r n i a State Polytechnic College, I967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Botany We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1971 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of B Q T A N J ^ The University of British Columbia Vancouver 8. Canada Date L JVJU/ 1 ^ 1 i ABSTRACT The l i f e cycle of Typhula erythropus was studied i n the f i e l d and. i n culture. An attempt was made to correlate obser-vations from both and to explain the behavior of this organism i n nature. The effect of temperature, pH, various carbon:nitrogen r a t i o s , and. nutrient concentration on mycelial growth, scl e r o -tium formation, and basidiocarp production was examined. Vegetative growth, including sclerotlum formation, was favored by temperatures above 10 C, high strength media, and a pH of 4-5. Basidiocarp production was maximal at lower temperatures, on low strength media, at pH 6, and at a low C/N r a t i o . The conditions for sclerotium germination were determined, and sclerotium v i a b i l i t y was investigated. S c l e r o t i a produced at 4 C did not germinate u n t i l exposed to 15-20.C. These heat activated s c l e r o t i a and s c l e r o t i a grown to maturity at 15 C germinated rapidly when incubated at k C on water agar. A low germination rate resulted when s c l e r o t i a were incubated at 15 C. The growth zone of T. erythropus basidiocarps i s sub-a p i c a l . Expansion of the Typhula f r u c t i f i c a t i o n occurs i n the same manner as that i n the common mushroom, Agaricus bisporus. A complex relat i o n s h i p between the p a r t i c u l a r stage of development of the organism and c u l t u r a l conditions was evident. The i n t e r a c t i o n of environmental and n u t r i t i o n a l factors was especially obvious during sclerotium formation i n culture. T. erythropus appears to be f a c u l t a t i v e l y homothallic. In the absence of a compatible mating s t r a i n , a l l nine monokaryotic iso l a t e s became dika r y o t i c . However, when crossed i n a l l combinations, the monosporic i s o l a t e s exhibited t y p i c a l t e t r a -polar heterothallism. This ambivalence i n mating i s of rare occurrence i n the fungi. Species i d e n t i f i c a t i o n i n the genus Typhula i s based on such characters as basidiospore measurements, basidiocarp dimensions, coloration, substrate, number of basidiocarps per sclerotium, and s c l e r o t i a l anatomy. The s t a b i l i t y of these features under various conditions has never been tested. Four i s o l a t e s of T. erythropus were examined i n culture, and the constancy of certain taxonomically important c h a r a c t e r i s t i c s was noted. Basidiospore measurements, s c l e r o t i a l micro-morph-ology, and the coloration of basidiocarps a r i s i n g from s c l e r o t i a were stable features i n a l l i s o l a t e s . Other c h a r a c t e r i s t i c s were variable. i i i TABLE OF CONTENTS p a g e I n t r o d u c t i o n 1 L i t e r a t u r e Review 2 M a t e r i a l s and Methods 7 (1) I s o l a t e s Employed 7 (2) Culture Techniques and Growth Conditions 7 (3) Assessment of Growth and Reproduction 9 (4) Methods of I n o c u l a t i o n 9 (5) Monokaryons and Mating , 10 (6) S t a i n i n g Techniques 10 Results and Observations 11 I . L i f e Cycle i n Nature 11 I I . Basidiospore Germination 12 I I I . Growth of Monokaryotic Mycelium 12 IV. Matings of Monokaryons... 16 A. I n t r a s p e c i f i c P a i r i n g s 16 B. I n t e r s p e c i f i c P a i r i n g s 1? V. Growth of D i k a r y o t i c Mycelium 19 A. General C h a r a c t e r i s t i c s 19 B. Temperature E f f e c t s 21 C. pH E f f e c t s 21 D. Nitrogen U t i l i z a t i o n 22 E. Vitamin Requirements 2k VI. Sclerotium Formation.... 2k A. Morphology of Mature S c l e r o t i a 2k B. Development 25 C. Time of Formation 27 •Iv TABLE OF CONTENTS (Cont'd.) P a S e D. Temperature E f f e c t s 28 E. pH E f f e c t s 32 F. Carbon:Nitrogen Ratio E f f e c t s 34 G. N u t r i e n t Concentration E f f e c t s 34 H. Nitrogen U t i l i z a t i o n 37 I . Wheat Germ E f f e c t s (Medium C) 39 V I I . S clerotium Germination 44 A. General Observations 44 B. Temperature E f f e c t s . . . . . 45 (1) Temperature During Germination 45 (2) Temperature During Production 46 (3) Freezing of S c l e r o t i a 48 C. Other P h y s i c a l Factors...- 48 (1) Drying of S c l e r o t i a 48 (2) Washing of S c l e r o t i a 48 (3) Sclerotium Diameter 48 (4) I l l u m i n a t i o n of S c l e r o t i a 49 D. Medium Composition E f f e c t s 49 E. In S i t u Germination 50 V I I I . B a s i d i o c a r p Formation and Growth 50 A. General Observations 50 B. Morphology of the Mature B a s i d i o c a r p 5 1 C. B a s i d i o c a r p Development 52 D. Expansion of the B a s i d i o c a r p 53 E. Temperature E f f e c t s 56 F. pH E f f e c t s 59 V TABLE OF CONTENTS (Cont'd.) G. Photo Effects 59 H. Gravity Effects 61 I. Carbon:Nitrogen Ratio Effects 61 J. Nutrient Concentration Effects 62 K. Nitrogen U t i l i z a t i o n 6 4 L. Vitamin Requirements 64 Discussion .. 66 I. General Considerations 66 I I . Mating System and Monokaryons 66 I I I . Mycelial Growth, Dikaryotic 69 IV. Sclerotium Development.... 70 V. Sclerotium Germination 7^ VI. Basidiocarp Growth and Development 77 VII. Interaction of Nutritional/Environmental Factors 82 VIII. Taxonomic Aspects and Implications 85 Bibliography 88 Appendix A: Co l l e c t i o n Data 93 ( 1 ) Typhula erythropus 93 (2) Typhula s c l e r o t i o i d e s 93 Appendix B: Culture Media 9^ ( 1 ) Medium A 94 (2) Medium B 95 (3) Medium C 95 (4) Malt-Yeast-Peptone (MYP) 95 (5) Water Agar 95 v i LIST OF TABLES Table I: The E f f e c t of Incubation Temperature on Sclerotium Germination. Table I I : Duration of the Stages of Basidiocarp Development 58 v i i FIGURES AND ILLUSTRATIONS Page P l a t e 1: L i f e Cycle of T. erythropus i n Nature.... 13 P l a t e 2 : E f f e c t of Temperature on Basidiospore Germination 14 P l a t e 3 : P a i r i n g of Monokaryotic I s o l a t e s 18 P l a t e 4 : Growth of the Colony on Medium A 20 P l a t e 5 : Environmental E f f e c t s on M y c e l i a l Growth 2 3 P l a t e 6: R e l a t i o n s h i p of Sclerotium Development and M y c e l i a l Growth 2 9 P l a t e 7 : E f f e c t of Temperature on Sclerotium Development.. 31 P l a t e 8: E f f e c t of pH on Sclerotium Development 33 P l a t e 9 : E f f e c t of C/N on Sclerotium Formation 35 P l a t e 1 0 : Sclerotium Development 36 P l a t e 1 1 : E f f e c t of Medium Strength on Sclerotium Formation 38 P l a t e 1 2 : Nitogen Sources f o r Sclerotium Formation.... 40 P l a t e 1 3 : R e l a t i o n s h i p of Sclerotium Development and M y c e l i a l Growth on a Wheat Germ Medium 42 P l a t e 14: E f f e c t of Wheat Germ on Sclerotium Formation 4 3 P l a t e 1 5 : E f f e c t s of Incubation Temperature on the Rate of Sclerotium Germination 4 7 P l a t e 16: Growth of the B a s i d i o c a r p , 1 . . . . . 55 P l a t e 1 7 : Growth of the B a s i d i o c a r p , 2 57 P l a t e 18: E f f e c t of Temperature on B a s i d i o c a r p Development • 58 P l a t e 1 9 : E f f e c t of pH on B a s i d i o c a r p Production 60 P l a t e 2 0 : E f f e c t of C/N on Ba s i d i o c a r p Production 6 3 P l a t e 2 1 : Basidiocarp Development 65 V i l l FIGURES AND ILLUSTRATIONS (Cont'd.) Page Plate 22: Interrelationship of N u t r i t i o n a l and Environ-mental Factors and Fungal Development 83 ix ACKNOWLEDGMENT I wish to thank Dr. R. J. Bandoni for support and guid-ance during the period of my research and i n the preparation of this thesis. For t h e i r comments and suggestions during the writing of this manuscript I thank Dr. G.C. Hughes, Dr. I.E.P. Taylor, and the graduate students of the mycology lab. Special thanks are extended to Dr. B.N. Johri for his a s s i s t -ance i n preparing the photographic plates and for his helpful comments. Financial support was provided by a U.B.C. graduate fellowship, a research fellowship, and teaching assistantships from the department of botany. INTRODUCTION The genus Typhula was established by Fries (1821) to include c l a v a r i o i d species with a c y l i n d r i c a l f e r t i l e clavule and a d i s t i n c t s t e r i l e s t a l k . Fries placed t h i s genus i n the order Clavati of the Hymenomycetes. Karsten (1882) emended the genus to r e t a i n only those species with basidiocarps a r i s i n g from s c l e r o t i a . Remsberg (19^0) and Corner (1950) accepted Karsten !s d e f i n i t i o n and placed a l l sclerotium-forming c l a v a r i -oid fungi i n Typhula. In 1970, Corner proposed the family Clavariadelphaceae to include Typhula, Chaetotyphula, P i s t i l l a r i a , P i s t i l l i n a , Myxomycidium, Araecoryne, Ceratellopsis, and Clavar-iadelphus. The d i s t i n c t i o n between Typhula and P i s t i l l a r i a , a sclerotium-free Typhula (Corner, 1950), was questioned by Koske and Perrin ( I97I) who found s c l e r o t i a i n the l i f e cycle of P i s t i l l a r i a  setipes Grev. Corner (I97O) reported a sclerotium present i n the "Typhula-state" of P. p e t a s i t i d i s Imai. In addition, a sclerotium was found i n Pterula s c l e r o t i c o l a Berthier (Berthier, I967) , a species placed i n a d i f f e r e n t family of the c l a v a r i o i d fungi. Species i d e n t i f i c a t i o n i n Typhula i s very d i f f i c u l t . Such c h a r a c t e r i s t i c s as spore size, basidiocarp dimensions, coloration, substrate, and sclerotium anatomy are used as major c r i t e r i a i n distinguishing species (Corner, 1950, 1970). There i s evidence that sclerotium anatomy, at lea s t , may be a useless character for t h i s purpose (R^ed, I969 and see l i t e r a t u r e review). The s t a b i l i t y of these morphological features under various environmental conditions has not been tested. The aim of the present investigation was to determine the - 2 -effects of environmental and n u t r i t i o n a l factors on the developmental morphology of d i f f e r e n t phases i n the l i f e cycle of T. erythropus Fr. It was further hoped that a detailed study of a well defined species might provide insight as to which morphological features are stable and taxonomically useful i n t h i s genus. LITERATURE REVIEW Much of the l i t e r a t u r e dealing with Typhula concerns the symptoms and control of diseases caused by pathogenic species, but some c u l t u r a l studies have been reported. In I887, deBary described sclerotium formation i n T. v a r i a b i l i s Riess and T. phacorrhiza Fr. and basidiocarp development i n the former. Part of my work concerned the examination of these processes i n another species. Although T. erythropus i s the most common Typhula i n Europe (Corner, I95O), i t has been studied l i t t l e i n culture. Lehfeldt (I923) germinated spores of this species on malt extract agar. He followed the movement of nuclei during d i -karyotization, demonstrated heterothallism, and remarked on i t s psychrophily. Tasugi ( I929, 1935) examined T. incarnata Lasch ex Fr. (as T. graminum Karst.) and found that UV l i g h t was necessary for the development of f e r t i l e sporophores. The temperature range for growth was 5-23 C, with an optimum between 8-15 C. Maximum growth occurred at pH 7 . MacDonald (193^) reported no special l i g h t requirement for sporophore development i n T. s c l e r o t i o i d e s (Pers.) Fr. which he mi s i d e n t i f i e d as T. gyrans Fr. (Corner, 1950). Basidiocarps arose from colonies on potato dextrose agar (PDA) and from s c l e r o t i a incubated on moist s o i l . The fungus grew at tempera--3-tures from 0-25 C. The optimum temperatures for mycelial growth, sclerotium formation, and basidiocarp production were 15-17, 17-20, and 13-15 C, respectively. Details of sclerotium development and basidiocarp anatomy were also presented. Mac-Donald concluded that sporophore growth was a p i c a l and that basidiocarps were p o s i t i v e l y phototropic and negatively geo-t r o p i c . Nuclear migration i n T. t r i f o l i i Rostr., a pathogen of clover, was studied by Noble (1937). She also described the morphological c h a r a c t e r i s t i c s of the haploid and dikaryotic mycelia, clamp formation, and the development of basidiocarps. S c l e r o t i a germinated on moist s o i l a f t e r an undetermined dor-mancy period. Heterothallism was demonstrated, but only f i v e monosporic i s o l a t e s were made, and she could not determine i f the species was bipolar or tetrapolar. A major contibution to our knowledge of Typhula was made by Remsberg (1940) when she cultured and studied 1 4 North Ameri-can species. She determined the optimum temperature f o r mycelial growth of each species, noted general c u l t u r a l charac-t e r i s t i c s , and v e r i f i e d Tasugi's observations on the UV require-ment. She found that l i g h t of a wavelength of 265O-3250 & was necessary for the production of f e r t i l e f r u c t i f i c a t i o n s . A l l 1 4 species grew at 0 C, and the optimum temperatures for growth varied from 6-12 C. Stressing sclerotium morphology, she devised a key to the species i n her study. Complete descriptions were given for the 1 4 species, and an h i s t o r i c a l account of the genus was presented. The age of the host plant was shown to determine resistance to Typhula snow bl i g h t by Tomiyama (1952). He also noted the mutual antagonism between mycelia of T. incarnata (as T. itoana Imai) and T. i s h i k a r i e n s i s Imai. In 1955. he cultured both species and reported that spore germination of T. incarnata occurred at 0 C and that maximum growth i n this species was made at 7-15 C. Ekstrand (1955) determined the optimum temperature for mycelial growth of T. borealis Ekstrand (10 C) and T. hyper-borea Ekstrand (5 C). Both species are pathogens of winter cereals; W. C. MacDonald ( I96I) considers them synonyms of T. idahoensis Remsb. In i960, Potatosova published three papers on Typhula and typhulosis i n the USSR. In the f i r s t study (1960a) she surface s t e r i l i z e d s c l e r o t i a of T. incarnata (as T. itoana), T. variab-i l i s , T. idahoensis, and T. t r i f o l i i and buried them i n moist sand. The pots of sand were covered with glass and placed out-doors. F e r t i l e basidiocarps arose from s c l e r o t i a of T. v a r i a b i l i s and T. t r i f o l i i when the outside temperature dropped to 8-15 C. Sporophores of T. incarnata were produced at 1.4-13.5 C and of T. idahoensis at 1.4-4.6 C. Basidiocarps i n the l a t t e r two species remained s t e r i l e . Maximum germination resulted from s c l e r o t i a buried at a depth of 5 nun; below 10 mm no germination occurred. Her second paper (1960b) reported the range and optimum temperature for growth of T. incarnata (0-18 C; opt. 10 C) and T:. idahoensis (0-16 C; opt. 10 C). She found that the devel-opment of s c l e r o t i a was stimulated by dif f u s e l i g h t but inhib-i t e d by d i r e c t l i g h t . A description of the 14 species of Typhula found i n the USSR was the subject of her t h i r d paper (1960c). In i t she discussed methods of overwintering, periods of s c l e r -otium formation, s p e c i a l i z a t i o n , and v a r i a b i l i t y of some - 5 -morphological features. The ef f e c t of long wave UV ra d i a t i o n on sclerotium germ-ina t i o n and basidiocarp formation was investigated b r i e f l y by Leach ( I962) . S c l e r o t i a of several undetermined Typhulas were i r r a d i a t e d with UV at 12 hours/day for 3 -10 days. Sporo-phores arose from s c l e r o t i a incubated on moist sand. Jackson (I963) reported a snow scald of turf caused by H« incarnata. He v e r i f i e d that basidiocarps were autumnal. Basidiospores of this species germinated r e a d i l y on PDA, and s c l e r o t i a were produced a f t e r 7 -10 days i n culture. Lehmann (I965) examined some aspects of the pathogenic nature, growth, and r e s p i r a t i o n of T. incarnata. The f i r s t i n vestigation of the ultrastructure of Typhula s c l e r o t i a was performed by Scurti and Converso (I965). They described types of c e l l s i n the medulla: storage c e l l s with polysaccharides and fat s and metabolically active c e l l s . Hyphal c e l l s i n the s c l e r o t i a l r i n d (the outer layer of the cortex) of this undetermined species were devoid of contents. Lockhart ( I967) examined another undetermined Typhula species p a r a s i t i c on strawberry plants. He cultured i t on PDA at - 1 , 10, and 19 C. At 10 C vigorous mycelial growth ensued, f i l l i n g a 90 mm p e t r i plate i n 11 days. Increasing the concentration of GOg above 0 . 0 5 $ caused a decrease i n the growth rate. In a C 0 2 ~ f r e e atmosphere, mycelial growth was s i m i l a r l y retarded. The germination of s c l e r o t i a of T. idahoensis on s t e r i l e s o i l was studied by Protosenko (I967) and Huber and McKay (I968). Protosenko found that germination occurred i n October. He also reported that the fungus grew fas t e r on PDA at 6-8 C than at - 6 -18-20 C. Ruber and McKay observed that low temperatures ( 1 - 2 C) or b u r i a l i n s o i l had l i t t l e e f f e c t on s c l e r o t i u m v i a b i l i t y . At h i g h e r temperatures (24 C), however, marked r e d u c t i o n s i n germination a b i l i t y were noted. Of p a r t i c u l a r importance to the taxonomy of the genus was Reed's ( I 9 6 9 ) i n t e r s p e c i f i c c r o s s i n g of T. graminum and T. i n -c a r n a t a . Corner ( 1 9 7 0 ) p l a c e d these two s p e c i e s i n d i f f e r e n t subgenera on the b a s i s of t h e i r d i s t i n c t l y d i s s i m i l a r s c l e r o t i a l morphology. R^ed found that they were completely i n t e r f e r t i l e and r e l e g a t e d the former to synonomy with T. i n c a r n a t a . Corner ( I97O) examined specimens of T. i s h i k a r i e n s i s , a p a r a s i t i c s p e c i e s on winter c e r e a l s i n Japan and Scandanavia. He suggested t h a t i t might be the same as T. erythropus. However, T. erythropus has not been r e p o r t e d as a p a r a s i t e . A l s o , W. C. MacDonald ( I 9 6 I ) c o n s i d e r e d T. i s h i k a r i e n s i s to be a synonym of T. i d a h o e n s i s , a w e l l known p a r a s i t i c s p e c i e s . U n f o r t u n a t e l y , n e i t h e r i n v e s t i g a t o r attempted i n t e r s p e c i f i c matings of the s p e c i e s , and the problem remains unsolved. The most r e c e n t c u l t u r a l i n v e s t i g a t i o n of Typhula was that of D e j a r d i n and Ward ( I 9 7 I ) . They grew T. i n c a r n a t a , T. t r i -f o l i i , and T. i d a h o e n s i s i n agar c u l t u r e s and determined the optimum temperature and pH f o r growth of each s p e c i e s . Maximum growth o c c u r r e d a t pH 7 on a malt e x t r a c t - y e a s t e x t r a c t - g l u c o s e medium a t 5-10 C. Poor growth was observed a t -5 and 2 0 C. S c l e r o t i a were produced more abundantly above 1 0 C. Oxygen up-take by T. i d a h o e n s i s was optimal a t 20 C, n e a r l y 15° higher than the temperature f o r maximum growth. A v a r i e t y of sugars were u t i l i z e d as r e s p i r a t o r y s u b s t r a t e s . -7 -MATERIALS AND METHODS (1) Isolates Employed Four is o l a t e s of T. erythropus were examined i n culture and the detailed investigation was carried out with one of them (iso l a t e T-4). Cultures were obtained from basidiospores released by mature basidiocarps. C o l l e c t i o n data are given i n Appendix A. The culture of T. s c l e r o t i o i d e s , used f o r attempted matings with T. erythropus, was obtained from basidiospores shed from mature basidiocarps. C o l l e c t i o n data for this species are also given i n Appendix A. (2) Culture Techniques and Growth Conditions Since a defined medium has not been used f o r any studies of Typhula except f o r the r e s p i r a t i o n work of Dejardin and Ward (1971), comparisons based on e a r l i e r c u l t u r a l data are of ques-tionable value. For this reason, and to ensure reproducible r e s u l t s , T. erythropus was grown on a defined medium. The standard medium used was a modification of the synthetic basal medium of L i l l y and Barnett (1951)• Its composition i s given i n Appendix B. This basal medium was designated Medium A. Medium B was prepared by reducing the asparagine concentration i n Medium A from 2.0 g / l to 0.2 g / 1 . Medium C, used f o r s c l e r -otium production, contained flakes of wheat germ instead of asparagine i n Medium A. Details of i t s preparation are given i n Appendix B. For basidiocarp development studies, Medium A was prepared with 1.0 g / l of vitamin free casein hydrolysate i n place of asparagine. Water agar (Appendix B) was routinely used for sclerotium germination investigations. Stock cultures were maintained on Medium A at 15 C. For nitrogen u t i l i z a t i o n studies Medium A was prepared with various compounds i n place of asparagine to give the same con-centration of N. The asparagine used was a Difco product, and other amino acids were supplied by N u t r i t i o n a l Biochemicals. A l l other chemicals were of reagent grade. Vitamin requirement studies were carried out i n dichromate cleaned glassware, and conclusions were based on data from three s e r i a l transfers. The ingredients of a l l media were autoclaved together at 15 l b s . for 15 minutes. Twenty mis (±2) were dispensed into 90 mm glass or s t e r i l e p l a s t i c p e t r i plates. When the phosphate was autoclaved separately and added to the remainder of the cooled medium, no difference was observed i n the growth response of the fungus. The pH of a l l media was adjusted to 5.0 - 5 . ^ with KOH before autoclaving. In pH-effect studies, the pH of the media was adjusted with IN KOH or IN ^SO^ and checked with a Radiometer M28 pH meter before autoclaving. Addition of bromcresol green or bromcresol purple to the medium was useful i n detecting a pH change following s t e r i l i z a t i o n . Cultures were grown at 4 C (±1), 10 C ( ± 1 ) , 15 C (±2), and 20 C (±1). Those at 10 and 1 5 C received 12 hours/day i l l u m i n -ation at a distance of 6-12" from Westinghouse cool-white 20W fluorescent lamps. At 20 C, 12 hours/day i l l u m i n a t i o n was provided by overhead fluorescent lamps i n a walk-in incubator. Cultures at 4 C were grown i n a r e f r i g e r a t o r and exposed to dif f u s e overhead fluorescent l i g h t only when the door was open. A minimum of three plates was used f o r each experiment, and major experiments were repeated at least twice. For sclerotium -9-germination studies, 15-50 s c l e r o t i a were used, and experiments were repeated at l e a s t once. Investigations of the l i g h t requirement f o r f r u i t i n g were performed by wrapping p e t r i plates i n aluminum f o i l and allowing the fungus to develop i n darkness. Since light-grown s c l e r o t i a and mycelia were able to give r i s e to a t y p i c a l basidiocarps i n darkness, i t was necessary to make s e r i a l transfers from dark-grown cultures to eliminate possible carryover of f r u i t i n g -inducing substances. The dark transfers were accomplished i n a darkened room by the f a i n t l i g h t of an alcohol lamp placed four feet away. (3) Assessment of Growth and Reproduction Observations on sclerotium formation and germination and basidiocarp production were performed only on structures derived from dikaryotic colonies. Relative growth rates were determined by measuring the increase i n colony diameter. For temperature and pH studies, this method was adequate, but t o t a l growth on d i f f e r e n t n i t r o -gen sources could not be rel a t e d to l i n e a r extension. Rapid and very sparse growth was associated with a nutrient-poor medium. Sclerotium production and f r u i t i n g a c t i v i t y were rated by the average number of mature structures formed per p e t r i plate. Dry weights were obtained by f i l t r a t i o n and drying of the mycelial mat on pre-weighed f i l t e r papers at 50 C for 24 hours. (4) Methods of Inoculation It was found that the age, source, and manner of app l i c a t i o n of the inoculum was very important i n the growth response of cultures of T. erythropus. Small agar blocks 3 mm2 cut from colonies grown on Medium A at 15 C for 1-2 months were used as -10-the standard inoculum source. The mycelium growing from these blocks responded to c u l t u r a l conditions i n the same manner as an inoculum of basidiospores. Inocula from colonies grown on Medium A for less than one month or from other media did not produce colonies that behaved predictably. The growth response was usually d i f f e r e n t from that of a basidiospore suspension inoculum. Growth rate, basidiocarp development, and sclerotium formation were e r r a t i c when the standard inoculum was not used. In an e f f o r t to shorten the time required to obtain sporo-phores i n culture, fresh plates of Medium A were inoculated with a water suspension of Medium A-grown colonies macerated i n a Waring blendor. Basidiocarp development was retarded, and most basidiocarps did not mature when thi s inoculation method was used. (5 ) Monokaryons and Matings Monosporic cultures were obtained by the d i l u t i o n and p l a t i n g out of a water suspension of basidiospores. A micro-scopic examination insured that basidiospores did not s t i c k together when released. The recovered monosporic cultures were a l l derived from two f r u c t i f i c a t i o n s from i s o l a t e T-4. Mono-karyons were microscopically examined for clamp connections and observed f o r basidiocarps. Both were present i n dikaryotic cultures and absent from young monokaryotic cultures. Monokaryons were crossed i n a l l combinations on malt extract-yeast extract-peptone agar (MYP, Appendix B) at 10 C. Small blocks of mycelium (with agar) to be crossed were set side-by-side on fresh media, and observations were made on the mycelium growing from the zone of contact. (6) Staining Techniques For routine observation and photography, hyphae were - 1 1 -stained with KOH-phloxine (Martin, 1 9 5 2 ) . Nuclei i n developing and mature basidiospores were stained with a mixture of aceto-orcein and aceto-carmine ( 1 : 1 ) . Furtado's ( I97O) toluidine blue method was used to st a i n nuclei i n monokaryotic and d i -karyotic hyphae. RESULTS AND OBSERVATIONS I. L i f e Cycle i n Nature Observations were made on the l i f e history of T. erythropus i n nature and i n culture. The findings from the f i e l d studies are presented i n this section. S c l e r o t i a produce basidiocarps from l a t e September u n t i l mid-December i n the Vancouver area. Most of my co l l e c t i o n s of sporophores were made i n the moist l e a f l i t t e r on petioles of Acer macrophyllum Pursh. These petioles were detached from l e a f blades and had been on the ground approximately one year. The fungus was also found f r u i t i n g on old f r u i t s of Acer, petioles of Rubus parv i f l o r u s Nutt., petioles of Alnus rubra Bong., and stems of Urtica l y a l l i i Wats. Mature basidiocarps shed basidiospores that germinate and give r i s e to li m i t e d haploid mycelia on suitable substrates (Corner, 1 9 5 0)• The fleshy petioles of Acer leaves, dropped a few months p r i o r to basidiospore discharge, are the usual sub-strate i n Vancouver. Following dikaryotization of the mycelia, s c l e r o t i a are produced i n the cortex of the petioles on either side of the r i n g of perivascular f i b e r s . Immature s c l e r o t i a are present by mid-December, and, by mid-January, mature s c l e r -o t i a are abundant. Often petiole and blade are s t i l l attached when s c l e r o t i a f i r s t develop. Mature s c l e r o t i a remain dormant throughout the spring and -12-summer. Sporophores appear i n the autumn with the onset of cool, moist conditions. In my c o l l e c t i o n s up to 60% of the basidiocarps and s c l e r o t i a on a petiole were located i n the basal 1 cm of the p e t i o l e . This area of the petiole i s the most fleshy. A diagramatic representation of the l i f e cycle of T. erythropus i s shown i n figure 1 to i l l u s t r a t e the stages of development. I I . Basidiospore Germination Mature basidiospores of T. erythropus are haploid when shed and each contains one nucleus. In my studies, the spores germinated by the formation of one to four germ tubes. Basidio-spores shed onto a s t e r i l e glass s l i d e at k C were placed i n a drop of water on Medium A and incubated at 4, 10, 15, and 20 C. The appearance of a germ tube was taken as an i n d i c a t i o n of germination. Germination occurred most rapidly at 15 C, with 83$ of the spores germinating within 60 hours ( f i g . 2). At 10 C, a lag p e r i o i d slowed the i n i t i a l rate of germination, but t o t a l germination a f t e r 60 hours was comparable to that at 15 C. Germination at 4 C was retarded by a 2h hour lag period before 63$ germination was achieved at the termination of the experi-ment. At 20 C, less than J0% of the spores germinated. I I I . Growth of Monokaryotic Mycelium The mycelium of a young colony started from a single basidiospore was monokaryotic and lacked clamp connections. A l l nine single-spore i s o l a t e s grew much more slowly i n culture than did dikaryotic mycelia. Cultures were grown on Medium A and MYP, a natural, but undefined, medium. After 33 days growth on Medium A at 4, 10, 15, and 20 C, the average colony diameter -13-FACING PLATE 1 Figure 1: L i f e cycle of T. erythropus i n nature. sporulation germination / dormancy period £ Q basidiospores maturation Figure 1 -14-FACING PLATE 2 Figure 2: Effect of temperature on basidiospore germination on Medium A. Each point on the graph represents 100 basidiospores counted. HOURS Figure 2 -15-was 18, 22, 7, and 4 mm, respectively. Mycelial growth on MYP was s l i g h t l y improved, the above colony diameters being reached i n 21 days at the same four temperatures. At 10 C or above, the colonies were dark brown; at 4 C, the cultures were a greyish-white, comparable to dikaryotic cultures. The mycelium of a l l i s o l a t e s was submerged, and the colony surfaces were shiny. Haploid cultures grown at 4 C were indistinguishable microscopically from dikaryotic mycelium except that the former lacked clamp connections. Haploid cultures grown at 10 and 15 C showed frequent bulges i n the hyphae that were absent from dikaryotic mycelia. Three to four week old monosporic cultures grown at 4 and 10 C produced 0.2-0.6 mm sporophores i n the center of the c o l -ony. These sporophores resembled the cone-shaped stage I basidio-carps (see section VIII) of dikaryotic cultures, but bore basidia and basidiospores. The basidia were four-spored, and the spore dimensions were sim i l a r to those of spores formed on dikaryotic f r u c t i f i c a t i o n s . The presence of clamp connections on the hyphae of the basidiocarp and at the colony margin was especially i n t e r -esting. These clamp connections indicated that homodikaryoti-zation.had occurred. Colonies became dikaryotic at the time of basidiocarp f o r -mation, p r i o r to basidiospore production. In contrast, cultures started from a multispore inoculum were dikaryotic i n approxi-mately three days. The rate of mycelial extension on the agar surface did not increase following homodikaryotization. Homo-karyotic cultures also d i f f e r e d from heterodikaryotic cultures i n not forming s c l e r o t i a at any temperature. -16-IV. Matings of Monokaryons A. IntraspecifIc Pairings Several d i f f i c u l t i e s were encountered i n attempting to obtain monosporic cultures and cross them. Since maximum ex-tension of dikaryotic hyphae and maximum basidiospore germination occurred at 15 C, d i l u t i o n plates of basidiospores were incu-bated at 15 C. Ususally only dikaryotic cultures were recovered from these plates. When no monosporic cultures were v i s i b l e a f t e r 10 days, the plates were discarded. As was l a t e r d i s -covered, the growth of haploid mycelia was n e g l i g i b l e at this temperature. By incubating d i l u t i o n plates at 10 C, I was able to i s o -l a t e nine mososporic cultures. These were crossed i n a l l combinations, i n duplicate, on MYP at 10 C. It had previously been ascertained that the morphological c h a r a c t e r i s t i c s of the dikaryotic mycelium on MYP were the same as those on Medium A. MYP was used because the monosporic i s o l a t e s grew fas t e r on i t than on Medium A. Clamp formation and basidiocarp formation could not be taken as indications of successful, mating since these structures were present i n older monosporic cultures. However, basidio-carps produced by heterokaryotic mycelia were distinguishable from those of homodikaryotic mycelia by the i r larger s i z e . Both types of basidiocarps were a t y p i c a l l y cone-shaped at 10 C. The homokaryotic sporophore was very narrow and lacked the plumpness and length (to 1.5 mm) of heterodikaryotic basidio-carps. In addition, basidiospores shed from heterodikaryotic sporophores soon produced a clamped mycelium where they landed on the agar near the parent colony. Basidiospores from homo--17-dikaryotic f r u c t i f i c a t i o n s did not produce a clamped mycelium for several weeks. Because of inoculum-induced v a r i a t i o n (see below) sclerotium production on Medium A at 4 or 10 C was not i n d i c a t i v e of hetero-d i k a r y o t i z a t i o n . Transfer of a piece of the mycelium growing out from the contact zone to plates of Medium A f o r standardiza-t i o n was not fe a s i b l e f o r two reasons. In some cases no mycelium grew into the agar from two mated s t r a i n s . Secondly, during the i n i t i a l inoculum standardization testing of dikaryons, I observed that e r r a t i c r e s u l t s were obtained when the inoculum source was less than one month old, grown at a temperature other than 15 C, or grown on a medium other than Medium A. Inocula from such sources gave r i s e to colonies that f a i l e d to produce s c l e r o t i a under conditions usually favorable f o r sclerotium production. Production of basidiocarps from the agar surface was also influenced by the inoculum source. On the basis of the production of larger, heterodikaryotic basidiocarps, the compatibility reactions of the i r basidiospores, and the increased growth rate following heterodikaryotization, I grouped the nine i s o l a t e s into four mating types. Isolates #3, 9, a n d 10 were compatible only with i s o l a t e s #4, 5» and 6. Isolate #8 was compatible only with i s o l a t e s #1 and 2. These mating reactions f i t the tetrapolar pattern. The pair i n g reactions are shown i n figure 3. B. I n t e r s p e c i f i c Pairings Monosporic i s o l a t e s #1, 3, 4, 8, 9, and 10, representing a l l four mating strains of T. erythropus, were crossed with two monosporic cultures of T. s c l e r o t i o i d e s . Except for their coloration, f r u c t i f i c a t i o n s of T. s c l e r o t i o i d e s are very similar -18-FACING PLATE 3 Figure 3- Results of pairings of nine monosporic i s o l a t e s of T. erythropus. Heterodikaryon formed = +; heterodikaryon not formed = In keeping with standard mycological practice, the mating types are a r b i t r a r i l y designated AB, Ab, aB, and ab. AB 3 9 10 ab 4 5 6 Ab 1 2 aB 8 3 AB 9 10 — — — + + + + + + + + + — — — 4 ab 5 6 1 Ab 2 + aB 8 — F i g u r e 3 - 1 9 -to those of T. erythropus. No dlkaryons were formed i n any of the p a i r i n g s . A d e f i n i t e barrage e f f e c t (Burnett, I968) was apparent i n a l l crosses. V. Growth of the D i k a r y o t i c Mycelium A. General C h a r a c t e r i s t i c s The mycelium of a l l fo u r i s o l a t e s of T. erythropus grew sl o w l y , t a k i n g over 30 days to cover a 90 mm p e t r i p l a t e under optimal c o n d i t i o n s on a l l media t e s t e d . Most hyphal growth occurred on or below the agar surface; a e r i a l mycelium was l i m i t e d i n young co l o n i e s and was r e s t r i c t e d to a l a y e r of short, t h i c k - w a l l e d hyphal c e l l s i n the center of ol d e r c u l t u r e s ( f i g s . 1 4 , 1 7 ) . The c e l l s that make up t h i s c r u s t were o f t e n encrusted w i t h c r y s t a l s and were s i m i l a r i n appearance to the sheathing hyphae at the base of a b a s i d i o c a r p . Clamp connections were common but were not present a t a l l septa. Branches f r e q u e n t l y arose from the clamps. Hyphal c e l l s v a r i e d i n s i z e from 2 - 1 0 u x 4 0 - 1 5 0 j i . A f t e r one month on Medium A a t 1 5 C, the colony was a transparent greyish-white becoming l i g h t reddish-brown i n the center where the c r u s t was forming. The surface was uneven, i n t e r r u p t e d by numerous lumps of hyphae and bas i d i o c a r p s a r i s i n g d i r e c t l y from the mycelium. The r a t e of m y c e l i a l extension i n a p e t r i p l a t e followed the sigmoid curve ( f i g . 4 ) . A f t e r an i n i t i a l l a g p e r i o d of approximately seven days, the l o g phase commenced, wit h the colony diameter i n c r e a s i n g a t the r a t e of 2-3 mm per day. Before the p l a t e became f i l l e d the growth r a t e d e c l i n e d , p o s s i b l y i n response to an a u t o i n h i b i t o r produced during growth. Other i s o l a t e s grew a t r a t e s equivalent to that recorded f o r -20-FACING PLATE it-Figure 4: Growth of a colony of T. erythropus on Medium A at 15 C. - 2 1 -Isolate T-4 on a l l media tested. The reddish-brown crust that formed i n the center of the colony and expanded r a d i a l l y was influenced i n i t s development by temperature, pH, and carbon and nitrogen sources. Factors favoring rapid mycelial growth also favored maximum crust formation. When T. erythropus was grown on Medium A containing glucose at 1 . 0 , 3 . 0 , 5 . 0 , 1 0 . 0 , and 1 5 . 0 g / l , maximum growth and most extensive crust development occurred at the highest concentration. Least growth and no crust were found at 1 . 0 g / l glucose. The amount of asparagine i n the medium s i m i l a r l y influenced crust development. Increasing the concentration of asparagine i n Medium A from 0 . 1 to 3 . 0 g / l resulted i n thicker mycelial growth and increased crust formation ( f i g . 14). B. Temperature Effects The optimum temperature for mycelial growth, as measured by both dry weight and l i n e a r extension, was 15 C. Good growth also occurred at 10 C and f a i r growth at 4 C. A very low rate of growth was observed at 0 and 20 C. Figure 5 shows the t o t a l l i n e a r growth of 21 day old colonies incubated at four d i f f e r e n t temperatures and the dry weight of 11 day old colonies grown at three temperatures. At 20 C, there was a tendency for the slow growing colony to p i l e up, producing a hard amorphous lump covered with a bloom of white mycelium. A s l i g h t crust was produced at 20 C, abundantly at 10 and 15 C, and not at a l l at 4 C on Medium A. C. pH Effects The optimum pH for vegetative growth was determined by measuring l i n e a r extension of mycelium at 15 C on Medium A. The most vigorous spread of mycelium occurred near pH 5 ( f i g . 6). -22-but cultures grew well over a pH range of 4.0-6.0. Above pH 6.0 the growth rate declined. Linear extension could not be determined below pH 4 because the medium did not s o l i d i f y at lower pH values. The f i n a l pH of a l l culture media was determined a f t e r 36 days growth with the indicators bromcresol green, bromcresol purple, phenol red, and pH paper. The f i n a l pH of media with i n i t i a l pH values of 4.0, 5.0, 6.0, 7.0, 7.7, and 8.5 was 6.5. 6.8, 7.6, 7.8, 7.8, and 7.8, respectively. At 10 C, the optimum pH for l i n e a r extension of hyphae sh i f t e d to 4.0 ( f i g . 6). Cultures were grown fo r 25 days. Media with i n i t i a l pH values of 4.0, 5.0, 6.0, and 7.0 had a f i n a l pH of 7.2, 7.2, 7.5, and 6.8, respectively. D. Nitrogen U t i l i z a t i o n T. erythropus was able to u t i l i z e n i t r a t e , ammonium, amides, and amino acids as nitrogen sources f o r mycelial growth. A v i s u a l estimation of growth was made on the basis of colony diameter and density of the hyphal mat. On medium A, DL-aspar-agine and casein hydrolysate supported the most vigorous growth. DL-alanine, ammonium sulfate, and ammonium chloride gave good growth, but a rapid pH drop (to 3.8) on media with ammonium sa l t s quickly c u r t a i l e d growth. Nitrates as calcium and potas-sium s a l t s induced rapid l i n e a r extension, but growth was sparse. L-(-)-phenylalanine, L-proline, and L-tyrosine supported poor growth, and DL-methionine i n h i b i t e d growth. The optimum concentration of KNO^ for hyphal growth was investigated. Cultures were grown at 15 C with KNO^ added at 0.0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, and 10.0 g/1. At a l l con-centrations greater than 0.0 g / l the rate of l i n e a r extension was nearly equal, and no optimum could be determined. -23-FACING PLATE 5 Environmental Effects on Mycelial Growth Figure 5' E f f e c t of temperature on mycelial growth. Colony diameters were measured a f t e r 21 days, dry weights determined a f t e r 11 days. A l l cultures were grown on Medium A. Figure 6: E f f e c t of pH on mycelial growth at 10 and 15 C. Cultures at 10 C were grown for 25 days, those at 15 C f o r 36 days. A l l cultures were grown on Medium A. 50 -24-E. Vitamin Requirements A stock solution of the vitamins thiamine, b i o t i n , p y r i -doxine, and i n o s i t o l was routinely included i n the preparation of Medium A. When these vitamins were omitted, vegetative growth was very poor. Two s e r i a l transfers with 1 mm2 inoculum plugs were executed to exclude the p o s s i b i l i t y of carryover from the o r i g i n a l inoculum source. When thiamine (100 ug/1) was added to the Medium A lacking the vitamin stock solution, vigor-ous growth was restored. From these data i t appeared that thiamine was required for the growth of T. erythropus. VI. Sclerotium Formation A. Morphology of Mature S c l e r o t i a Corner (1950) described i n d e t a i l the micro-morphology of T. erythropus s c l e r o t i a . At that time he believed, as did Remsberg (1940), that certain species could be distinguished so l e l y on the basis of the i r s c l e r o t i a l morphology. The s t a b i l -i t y of this character was never established. In my studies, a l l four i s o l a t e s of T. erythropus retained t h e i r c h a r a c t e r i s t i c s c l e r o t i a l features under a variety of n u t r i t i o n a l conditions and incubation temperatures. Mature s c l e r o t i a from culture were d o r s i v e n t r a l l y flattened bodies 0.7-2.9 mm i n diameter. The f i n a l size of these subspherical s c l e r o t i a was dependent upon the medium and the incubation temperature. The reddish-brown to nearly black coloring was li m i t e d to the c u t i c l e . The unistratose cortex and prosenchymatous medulla were white. A cross section of a mature sclerotium i s shown i n figure 15. The dark pigment of the c u t i c l e was s l i g h t l y soluble i n - 2 5 -water and insoluble i n ethanol, ethyl ether, petroleum ether, chloroform, acetone, 12N HC1, IN KOH, and a saturated aqueous solution of FeCl^. The pH of mature s c l e r o t i a was near 7« This was determined by reaction with bromcresol green, bromcresol purple, and phenol red. S c l e r o t i a from culture retained only 36% of t h e i r fresh weight a f t e r drying at room temperature for one week. On drying, the s c l e r o t i a darkened and became s l i g h t l y wrinkled and hard. B. Development Four d i s t i n c t stages, i l l u s t r a t e d i n figure 11, were evident during the formation of s c l e r o t i a and t h e i r growth to maturity. For the purpose of th i s study, a mature sclerotium was defined as one able to produce a basidiocarp. The knot of hyphae destined to become a sclerotium f i r s t became v i s i b l e i n stage Ia. This Ia stage developed on or beneath the agar sur-face. The stage lb commenced when the s c l e r o t i a l primordium was a well-defined 0.8-1.2 mm diameter white sphere. At the top of the l b sclerotium a reddish-brown spot appeared that slowly and evenly spread over the sclerotium. Stage II began with the f i r s t appearance of this spot and continued u n t i l the entire sclerotium was darkened. One to several drops of clear l i q u i d frequently were exuded from s c l e r o t i a during stages l b and I I . Their occurrence was especially common on s c l e r o t i a produced at 4 C. When s c l e r o t i a were completely darkened, stage III and maturity were reached. Stage III s c l e r o t i a averaged 1.2 mm i n diameter when pro-duced at 1 5 C. If l e f t on the parent colony, these s c l e r o t i a continued to increase i n size up to 1.4 mm. -26-Th e four stages of development that were observed i n cultured s c l e r o t i a also appeared to be present during sclerotium formation i n the f i e l d . Developing s c l e r o t i a on petioles incubated i n moist chambers at 4 C were observed i n stages l b and I I I . It was not determined i f the darkening process of stage II occurred i n as orderly a manner as seen i n culture. Sclerotium formation i n a l l four i s o l a t e s grown i n culture followed the stages of development c i t e d f o r i s o l a t e T-4. The d e t a i l s of development of s c l e r o t i a from one or two parent hyphae were described by deBary (I887), MacDonald (193^), and Remsberg (1940) for several species of Typhula. Sclerotium formation i n T. erythropus was si m i l a r to that i n other species, but the medullary hyphae did not become thick-walled i n T. eryth-ropus . S c l e r o t i a l i n i t i a t i o n was of the "strand type" described by Townsend and Willets (1954). Increased branching, growth, and septation from a few in t e r c a l a r y hyphal c e l l s produced the nucleus of the s c l e r o t i a l primordium ( f i g . 12). These develop-ments were completed during stage Ia. Growth of the young sclerotium by c e l l d i v i s i o n and expansion b u i l t up the d i f f u s e knot of hyphae to a white sphere (stage l b ) . The outer layer of c e l l s , constituting the cortex, was not much d i f f e r e n t i a t e d from the medullary c e l l s u n t i l l a t e i n stage l b . The prosen-chymatous nature of the expanding, intertwining c e l l s of the medulla was constant throughout development ( f i g . 16). During stage II, c o r t i c a l c e l l s developed a c h a r a c t e r i s t i c jig-saw piece shape. It was not determined whether this d i s -t o r t i o n was a r e s u l t of stretching of the c e l l s by i n t e r n a l ex-pansion of the sclerotium, as deBary suggested, or by the d i f f e r e n t i a l growth of the c e l l s to accomodate the increased -27-sclerotium s i z e . Regardless, the r e s u l t i n g jig-saw-like pattern that was formed on the sclerotium surface was a con-stant feature of a l l mature s c l e r o t i a ( f i g . 1 3 ) . This surface pattern i s not r e s t r i c t e d s o l e l y to T. erythropus. T. viburni Remsb. and T. phacorrhiza have sim i l a r patterns on t h e i r s c l e r o t i a . However, the i n t e r n a l anatomy of these species i s much d i f f e r e n t from that of T. erythropus. While the c o r t i c a l c e l l s became deformed, a dark reddish-brown c u t i c l e was deposited on t h e i r outer walls. In cross section this c u t i c l e , with i t s short perpendicular projections down the r a d i a l walls of the cortex, resembled the c u t i c l e of higher plants and i t s r e l a t i o n s h i p to the epidermis. The dark c u t i c l e was separable from the hyphal walls. Often drops of l i q u i d exuded between the cortex and the c u t i c l e during stage II l i f t e d the c u t i c l e from the sclerotium surface. After the drop evaporated, the c u t i c l e returned to the sclerotium surface. This a c e l l u l a r c u t i c l e sometimes appeared as a membrane-like structure around the drops. When examined microscopically, i t carried the jig-saw pattern of the sclerotium surface. Patches of the c u t i c l e were re a d i l y scraped from the surface of mature s c l e r o t i a . The morphology of mature s c l e r o t i a was very constant under a l l c u l t u r a l conditions tested. A l l i s o l a t e s and a l l f i e l d c o l l e c t i o n s displayed the same s c l e r o t i a l morphology. G. Time of Formation The r e l a t i o n s h i p of sclerotium development to mycelial growth i n culture i s shown i n figure 7. Since sclerotium pro-duction was best studied on Medium B at 15 C, this medium was used to correlate the events. Stage Ia s c l e r o t i a were f i r s t -28-v i s i b l e on 12 day old colonies i n the log phase of mycelial growth. S c l e r o t i a had advanced to stage l b four days l a t e r , at the end of the log phase. The s c l e r o t i a began to darken si x days l a t e r as the growth rate of the colony declined. Mature s c l e r o t i a were present a f t e r an additional four days. The average time from i n i t i a t i o n to maturity was lk days. This was the shortest average time recorded. The i n t e r v a l was longer on other defined media and at d i f f e r e n t incubation temperatures. The above description applies only to the 6-10 s c l e r o t i a that mature f i r s t on a colony. The i n t e r v a l between each stage was r e l a t i v e l y constant, but the i n i t i a t i o n time for a l l s c l e r o t i a that formed on a plate was variable. S c l e r o t i a f i r s t were formed close to the point of inoculation i n the oldest hyphae of the colony. As the colony grew, young s c l e r o t i a appeared behind the margin. Mature s c l e r o t i a appeared f i r s t i n the center of the colony, forming a gradient of development to the immature s c l e r o t i a near the margin. New s c l e r o t i a were i n i t i a t e d and matured a f t e r the log phase of growth had passed. D. Temperature Effects The ef f e c t of temperature on sclerotium formation was related to the carbon:nitrogen r a t i o and the pH of the medium. At 15 C, few s c l e r o t i a were formed on Medium A. These s c l e r o t i a were not well-defined and were d i f f i c u l t to separate from the colony. Cultures grown at k and 10 C on this medium produced up to 80 d i s t i n c t s c l e r o t i a per plate. Medium B supported the production of 15-30 well-defined s c l e r o t i a per plate at 15 C; at k and 10 C, the average number of s c l e r o t i a per plate was 5-15. It was discovered l a t e r that up to 200 s c l e r o t i a per plate could be obtained on Medium B at 15 C i f the inoculum was -29-FACING PLATE 6 Figure 7 : Sclerotium development and i t s rela t i o n s h i p to mycelial growth. Cultures were grown on Medium B at 15 C. -30-taken from a culture grown on Medium A prepared with KNO^ i n place of asparagine. A two to three week old culture, grown at 15 C on Medium A, produced numerous t y p i c a l s c l e r o t i a when incubated at 0, 4, or 10 C. Return of the culture with stage III s c l e r o t i a to 15 C often was accompanied by the incorporation of the s c l e r o t i a into the spreading crust. Medium C supported excellent sclerotium production at 15 C, with up to 90 s c l e r o t i a formed per plate. At lower temp-eratures fewer s c l e r o t i a were produced. In addition to a f f e c t i n g the number of s c l e r o t i a formed per plate, the incubation temperature also influenced the time of sclerotium i n i t i a t i o n and development. Mature s c l e r o t i a were formed e a r l i e s t on Medium B, of the defined media employed. The time between stages and the time to i n i t i a t i o n at 4, 10, and 15 C are presented i n figure 8. Data from the 4 and 10 C exper-iments are from observations of sclerotium formation on Medium A. The 15 C data are taken from cultures on Medium B. Scler-otium production on Medium A was greater than that on Medium B at these temperatures. When cultures were grown on Medium A, the time required to produce s c l e r o t i a at 4 C was similar to that observed i n nature. Stage II s c l e r o t i a appeared f i r s t i n cultures grown at 10 C. However, the darkening process was retarded at thi s temperature, and stage III s c l e r o t i a were formed e a r l i e s t at 15 C. The time of i n i t i a t i o n and the duration of maturation were considerably greater at 4 C than at 10 or 15 C. I l a t e r discovered that mature s c l e r o t i a could be produced i n 35 days instead of 67 days at k C when the asparagine i n Medium A was -31-FACING PLATE 7 Figure 8: The effect of temperature on the rate of sclerotium development. Cultures grown at 4 and 10 C on Medium A; cultures at 15 C on Medium B. 0 10 20 30 40 50 60 70 A G E , DAYS F i g u r e 8 -32-replaced by 1.0 g / l of casein hydrolysate. At 10 and 15 C, s c l e r o t i a were well separated from each other. At 4 C, they tended to coalesce, frequently forming compound s c l e r o t i a . The incubation temperature also affected the diameter of mature s c l e r o t i a . The average diameter of 3° s c l e r o t i a pro-duced at 15 C was 1.2 mm. At 4 C, s c l e r o t i a from Medium A were more than twice as large, averaging 2.6 mm. The diameter of s c l e r o t i a produced at 10 C was intermediate, averaging 1.5 mm. Isolates T-I82 and T-29 produced numerous s c l e r o t i a on Medium A at 4 and 10 C. Isolate T-18 1 formed less than 15 s c l e r o t i a per plate on this medium at 4 C. E. pH effects The e f f e c t of pH on sclerotium formation was examined at 10 and 15 C. The re s u l t s of these experiments are presented i n figure 9. Cultures were grown at 15 C on Medium A adjusted to pH values of 4.0, 5.0, 5.5, 6.0, 7.0, 7.7, and 8.5. After 26 days, the average number of s c l e r o t i a per plate was determined. The f i n a l pH of the respective media was 6.5, 6.5, 7.6, 7.6, 7.8, 7.8, and 7.8. S c l e r o t i a were produced at pH 4.0 and 5.0, but these were not well-defined from the colony surface. No s c l e r o t i a were produced at other pH l e v e l s . The experiment was terminated a f t e r 26 days because the spreading crust threatened to grow over the s c l e r o t i a . Cultures at 10 C were grown on Medium A adjusted to pH values of 4.0, 5.0, 6.0, and 7.0. After 32 days, the average number of s c l e r o t i a per plate was determined, and the f i n a l pH was measured. The f i n a l pH f o r these media was 7.2, 7.2, 7.5, and 6.8, respectively. Maximal sclerotium production occurred -33-PACING PLATE 8 Figure 9 : The effect of i n i t i a l pH on sclerotium formation. Cultures were grown at 10 and 15 C on Medium A. 40 •{ 3o^ MATURE SCLEROTIA 20 H PER PLATE 104 0 • • • • -15° Figure 9 -34-a t pH 4.0, w i t h 32 s c l e r o t i a per p l a t e ; at pH 5.0, 6.0, and 7.0 the average number of s c l e r o t i a per p l a t e was 16, 7, and 6. F. Carbon:Nitrogen (C/N) Ratio E f f e c t s The optimum C/N r a t i o f o r s c l e r o t i u m production was i n f l u -enced markedly by the temperature of i n c u b a t i o n . For the 15 C i n v e s t i g a t i o n , media wi t h C/N r a t i o s from 3.8:1 to 190:1 (g C/g N) were prepared by a d j u s t i n g the amount of asparagine i n Medium A. The pH of a l l media was adjusted to 5.0, and c u l -tures were grown f o r 60 days. The f i n a l pH of media w i t h C/N r a t i o s of 47:1 to 190:1 was 5 . 2 . Media wi t h r a t i o s of 19:1 and 9 . 5:1 had a f i n a l pH of 7.0 and 8.4, r e s p e c t i v e l y . Maximum s c l e r o t i u m production occurred at a C/N r a t i o of 9 5:1. with an average of 16 s c l e r o t i a formed per p l a t e ( f i g . 10). Media wi t h r a t i o s of 140:1, 6 3:1, 47:1, and 38:1 supported approximately one h a l f the production achieved on the 95:1 medium. No s c l e r o t i a were formed a t 15 C when the C/N r a t i o was lower than 1 9:1. In c o n t r a s t to the 15 C r e s u l t s , e x c e l l e n t s c l e r o t i u m pro-d u c t i o n occurred a t 4 and 10 C on a medium w i t h a C/N r a t i o of 9 . 5:1 (Medium A). Cultures were grown on media wi t h C/N r a t i o s of 9 . 5:1 and 9 5:1. At 4 and 10 C, the average number of s c l e r o t i a per p l a t e was 80 at 9 . 5:1 and 5 - 1 5 a t 95:1 (Medium B). G. N u t r i e n t Concentration E f f e c t s The c o n c e n t r a t i o n of glucose and asparagine i n Medium A was adjusted to make media of i to 5X r e g u l a r s t r e n g t h . A C/N r a t i o of 95:1 was maintained i n a l l media, and the pH was set a t 4.0, optimal f o r s c l e r o t i u m formation. Cultures were grown at 15 C f o r 31 days; the r e s u l t s are shown i n f i g u r e 18. The average number of s c l e r o t i a per p l a t e was p r o p o r t i o n a l to the s t r e n g t h of the medium. No stage I I I s c l e r o t i a were produced on -35-F A C I N G P L A T E 9 F i g u r e 10: T h e e f f e c t o f v a r i o u s C / N r a t i o s o n s c l e r o t i u m f o r m a t i o n a t 15 C . The m e d i a w e r e p r e p a r e d by-a d j u s t i n g t h e c o n c e n t r a t i o n o f a s p a r a g i n e w h i l e t h e g l u c o s e c o n c e n t r a t i o n was m a i n t a i n e d a t 10 g/1. Figure 10 - 3 6 -FACING PLATE-10 Sclerotium Development i n T. erythropus Figure 1 1 : Stages of sclerotium development. The darkness of the Ia and l b s c l e r o t i a i s a r e s u l t of the accumulation of bromcresol green. X2. Figure 12: Young stage Ia sclerotium. X300. Figure 1 3 : Surface of the mature sclerotium. X400. Figure 14: The e f f e c t of several C/N r a t i o s on sclerotium production at 1 5 C. The numbers on the plates indicate the g / l of asparagine i n Medium A. The values 0.2, 0.5, 1.0, 2.0, and 5.0 are equivalent to C/N r a t i o s of 9 5 = 1 , 3 8 : 1 , 1 9 : 1 , 9.5=1, and 3.8:1 respectively. S c l e r o t i a at 1.0 g / l are not well defined from the colony. Note crust development. Figure 15"- X.S. of a mature sclerotium. The dark edge i s the outer surface of the sclerotium. Note the homogeneous nature of the c e l l s of the medulla. The cortex, of one c e l l thickness, i s not evident at this magnification. X70. Figure 1 6 : C e l l s of the medulla of the sclerotium. Note their prosenchymatous nature. X1000. Figure 1 7 : Crusting hyphae from the surface of the colony. Note thickened, dark walls. X400. -37-the •§•' strength medium (5.0 g/1 glucose, 0.1 g/1 asparagine). On the plates of f u l l , 1.5X, 2X, and 5X strength media, the average number of s c l e r o t i a produced was 2, 4, 21, and 24, respectively. The r a d i a l spread of the colony p a r a l l e l e d the increase i n sclerotium production. Maximum mycelial growth occurred on the 5X strength medium. The c o r r e l a t i o n of colony diameter at 31 days to nutrient concentration i s also i l l u s t r a t e d i n figure 18. H. Nitrogen U t i l i z a t i o n S c l e r o t i a of T. erythropus were produced on media made with a variety of nitrogen sources. However, i t was d i f f i c u l t to determine which compounds were u t i l i z a b l e for this process since s c l e r o t i a could be formed on simple water agar inoculated with basidiospores. Nitrogen sources were added to medium A i n place of aspara-gine. The influence of temperature on the optimum C/N r a t i o f o r sclerotium production has already been noted. For this reason, media for use at 10 C were prepared with a C/N r a t i o of 9.5=1 and media for use at 15 C with a 95:1 r a t i o . The pH of a l l media was adjusted to 5.0, and cultures were grown for 35 days. The results are shown i n figure 19. At 15 C, aspara-gine supported the maximum production of s c l e r o t i a . Consistently less than 10 s c l e r o t i a per plate were formed on media made with DL-alanine, DL-methionine, casein hydrolysate, KNO3, and Ca(N0-^)2. The appearance of s c l e r o t i a on these poorly u t i l i z e d substrates was also much delayed. Mature s c l e r o t i a were not present on methionine media u n t i l the cultures were over 110 days old. No s c l e r o t i a were produced on L-tyrosine or NH^Cl. -38-FACING PLATE 11 Figure 18: The effect of medium strength on sclerotium formation and r a d i a l growth of a colony. Standard strength Medium A contains 10 g / l glucose and 2 .0 g / l asparagine. Cultures were grown at 15 C for 31 days. -39-Th e pH drop associated with ammonium u t i l i z a t i o n possibly was the reason for the f a i l u r e of this source to support sclerotium production. At 10 C, optimum sclerotium production was made on aspara-gine with 80 s c l e r o t i a formed per plate. Casein hydrolysate was the next most productive, with 4-5 per plate. Alanine and KNO-^  media supported less than 10 s c l e r o t i a per plate. L-proline and L-(-)-phenylalanine i n h i b i t e d sclerotium formation. I. Wheat Germ Effects (Medium C) Sclerotium production on Medium C was exceptionally v i g -orous i n 15 C grown cultures. Mature s c l e r o t i a were formed rapidly and i n large numbers. The re l a t i o n s h i p of mycelial growth to sclerotium development i s shown i n figure 20. The log phase of mycelial growth on Medium C commenced approximately four days a f t e r inoculation when cultures were grown at 15 C. Stage Ia s c l e r o t i a appeared i n 9 day old colonies. The other stages followed at nearly the same interv a l s as those recorded on Medium B at 15 C. The re l a t i o n s h i p of sclerotium formation to the growth rate of the colony also was the same as that on Medium B. Mature s c l e r o t i a were present i n 22 day old cultures. This was four days less than the time required on Medium B. The l ag period of mycelial growth also was four days less than on Medium B. At 4 and 10 C, sclerotium production on Medium C was i n f e r i o r i n numbers of s c l e r o t i a and maturation rate to that observed on Medium A at these temperatures. A l l is o l a t e s produced numerous s c l e r o t i a when cultured on Medium C at 15 C. To determine the optimum concentration of wheat germ i n Medium C f o r sclerotium production, p e t r i plates containing 1.0 to 25.0 g / l of wheat germ were prepared. Cultures were -40-FACING PLATE 12 Figure 1$: Nitrogen sources for sclerotium formation. The r a t i n g system refers to the number of stage III s c l e r o t i a per plate. 15u 10 ala + ala + asn + + asn +++ met + phe — tyr — pro — cas hy + cashy +++ CaN + KN + KN + amCI — sclerotia per plate + + + = >40 + + = 10-39 + = 1-9 - = 0 Figure 19 -41-incubated at 15 C. After 27 days, an average of 85 mature s c l e r o t i a were present i n cultures grown with 3.5-^.5 g/1 of wheat germ. In comparison, only 6-10 mature s c l e r o t i a were produced i n the same period of time on Medium A. As figure 21 shows, wheat germ concentrations from 2.5-9*0 g/1 supported the formation of large numbers of mature s c l e r o t i a , and these s c l e r o t i a were well-defined and e a s i l y l i f t e d from the colony. At concentrations below 2.5 g/1 the s c l e r o t i a remained a l i g h t reddish-brown and, though viable, were d i f f i c u l t to separate from the cartilaginous colony. On media with wheat germ con-centrations greater than 7.5 g/l» the dark mycelial crust soon developed and overgrew many mature s c l e r o t i a , rendering them inseparable from the colony. The active f r a c t i o n of the wheat germ was soluble i n cold water but not soluble i n ethanol, ethyl ether, or chloroform. A quantity of wheat germ flakes was extracted with 100 ml of water agitated by a magnetic s t i r r e r f or 20 minutes at 20 C. The water solution was f i l t e r e d and then used to make up 100 ml of Medium A lacking asparagine. Sclerotium production was compared with that on media prepared with the extract of 2.0, 4.0, and 6.0 g/1 of wheat germ. Cultures were grown at 15 C for 47 days. The results are shown i n figure 22. Maximum sclerotium production occurred when the extract of 6.0 g/1 was used. The water extract of wheat germ did not support the pro-duction of as many s c l e r o t i a as did wheat germ flakes. This water soluble factor(s) was found to be dialyzable and heat stable. No further attempt was made to characterize the active f r a c t i o n of wheat germ. -1*2-FACING PLATE 13 Figure 20: The relat i o n s h i p of sclerotium development to mycelial growth on Medium C at 15 C. -43-FACING PLATE 14 Effect of Wheat Germ on Sclerotium Formation Figure 2 1 : The eff e c t of wheat germ concentration on the production of s c l e r o t i a . Cultures were grown at 15 C f o r 27 days. Figure 2 2 : Sclerotium production on media prepared with three concentrations of a water extract of wheat germ. Cultures were grown for 4 7 days at 15 C. 90 60 30 i 0 5 10 15 wheat germ, g/\ F i g u r e 2 1 20 0) •+-• 03 d 0) d __ o _Q) O 50-40-30-20-10 0 2.0 4.0 6.0 wheat germ extracted, g/\ F i g u r e 2 2 VII. Sclerotium Germination A. General Observations In nature, and i n the laboratory, the s c l e r o t i a of T. erythropus germinate to produce the diminutive clavate basidio-carps c h a r a c t e r i s t i c of the species. S c l e r o t i a also germinate i n culture by producing dikaryotic hyphae when set on fresh media. Germination of the f i r s t type was the subject of this part of the investigation. Mature s c l e r o t i a from culture germinated well i n p e t r i plates of water agar. On Medium A mycelial growth from s c l e r -o t i a placed on the agar surface was much more vigorous than that on water agar, and no basidiocarps arose from s c l e r o t i a incubated on the nutrient medium. The temperature during production of s c l e r o t i a and the temperature during germination were the most important factors a f f e c t i n g sclerotium germination. The v i a b i l i t y of s c l e r o t i a was also influenced by the medium upon which they were produced. The young basidiocarp originates i n the medulla of the sclerotium. The hyphal d e t a i l s of germination i n T. erythropus are comparable to those reported for T. s c l e r o t i o i d e s by MacDonald ( 1 9 3 ^ ) • The sporophore primordium i n the medulla pushes outward, eventually causing the outer layer of c e l l s (the cortex i n the T. erythropus sclerotium) to rupture and allowing the young basidiocarp to grow out through a jagged hole. One to eight sporophores were produced from germinating T. erythropus s c l e r o t i a . Corner (1950) suggested that the l o c a t i o n of the basidio-carp may be determined during sclerotium formation. Observations on germinating s c l e r o t i a of T. erythropus confirmed this -45-o p i n i o n . The rounded upper side of a s c l e r o t i u m when attached to the parent colony f o r convenience was designated the d o r s a l surface, and the f l a t t e n e d lower side was the v e n t r a l s u r f a c e . S c l e r o t i a removed from c u l t u r e and i n v e r t e d on water agar f i r s t produced basidiocarps from the o r i g i n a l d o r s a l s i d e of the s c l e r o t i m . These sometimes grew downward i n t o the agar. When s c l e r o t i a were not i n v e r t e d before germination, sporophores arose from the d o r s a l s u r f a c e . This apparent predetermination e f f e c t was observed i n s c l e r o t i a produced at 4, 10, and 15 C. No dormancy per i o d p r i o r to germination was r e q u i r e d f o r s c l e r o t i a from c u l t u r e s grown at 10 and 15 C. However, s c l e r o t i a grown to maturity at 4 C r a r e l y germinated i f not given a 15-20 C treatment f o r 10-14 days before germination (see next s u b - s e c t i o n ) . B. Temperature E f f e c t s (1) Temperature During Germination V i a b l e s c l e r o t i a from c u l t u r e s grown on Medium C a t 15 C were placed on water agar and incubated at 4, 10, 15, and 20 C. Some s c l e r o t i a were a l s o given a f i v e day treatment at 4 C p r i o r to i n c u b a t i o n at 15 C on water agar. As shown i n Table I, 95-100$ of the s c l e r o t i a incubated at 4 and 10 C germinated. At 15 C, l e s s than 20% produced b a s i d i o c a r p s , but a f t e r the 4 C treatment the germination r a t e was 70$. At 0 and 20 C, no germination occurred. The r a t e of germination (time to appearance of young b a s i d i o c a r p s ) was a l s o a f f e c t e d by the temperature at germin-a t i o n . S c l e r o t i a incubated at 4 C germinated q u i c k l y , w i t h maximum germination reached i n 14-16 days ( f i g . 23). Up to 35 days were r e q u i r e d f o r maximum germination at 15 C f o l l o w i n g -46-th e 4 C pretreatment. This 35 day figure includes the f i v e days of pretreatment. Although s c l e r o t i a germinated at 15 C, basidiocarps devel-oping from s c l e r o t i a at this temperature often remained s t e r i l e . At 10 and 4 C, sporophores matured quickly and frequently grew to more than 20 mm i n length. Temperature effects on basidio-carp development are treated i n another section of this paper. Both percentage and rate of germination were also influenced by the medium on which the s c l e r o t i a were grown. This i s discussed i n another part of this section. S c l e r o t i a collected on petioles i n the f i e l d i n January and May did not germinate when incubated i n clear p l a s t i c moist chambers at 4, 1 0 , 1 5 , or 15 C a f t e r one week at 4 C. S c l e r o t i a from August c o l l e c t i o n s germinated readily at 4 C and 15 C following 4 C pretreatment. No germination occurred at 15 C without pretreatment. ( 2) Temperature During Production S c l e r o t i a grown at 10 and 15 C on suitable media were viable a f t e r reaching stage I I I . Up to 1 0 0 $ germination was obtained from these s c l e r o t i a when incubated at 4 C. However, s c l e r o t i a produced on Medium A at 4 C germinated only a f t e r a pretreatment at 1 5 - 2 0 C for 10-14 days. Following treatment, germination occurred at 4 C on water agar. The 1 5 - 2 0 C treat-ment was e f f e c t i v e on s c l e r o t i a s t i l l attached to the parent colony or separated from i t on water agar plates. The treat-ment was i n e f f e c t i v e when s c l e r o t i a were desiccated. A germ-ination rate of 8 5 $ was recorded from treated s c l e r o t i a grown at 4 C. The addition of casein hydrolysate or wheat germ did not - 4 7 -FACING PLATE 15 Figure 23: The e f f e c t of incubation temperatures on the rate of sclerotium germination. S c l e r o t i a were produced on Medium C at 15 C. Table I: The e f f e c t of Incubation Temperature on sclerotium germination. GERM. TEMP PRE-TREAT GERM. 10° 4° -7days 0-20 70  95-100 95-100 -48-a l l e v i a t e the necessity of the heat treatment. Since i t was possible that a l i g h t stimulus was involved i n the production of viable s c l e r o t i a , the 20 C treatment was performed i n darkness. Treated and untreated s c l e r o t i a i n the 4 C incubator were thus exposed to the same amount of l i g h t . S c l e r o t i a grown to stage II at 4 C and matured at 15 C on the parent colony were i d e n t i c a l i n t h e i r v i a b i l i t y to s c l e r o t i a produced at 15 C. ( 3 ) Freezing of S c l e r o t i a Viable s c l e r o t i a were frozen at - 5 C f o r two weeks on water agar plates. They were then placed on fresh plates and incubated at 15 C. A germination rate of 40$ was observed, 3 0 $ less than the 4 G pretreated control. C. Other Physical Factors ( 1) Drying of S c l e r o t i a Viable s c l e r o t i a were a i r dried at 20 C f o r 25 days and then placed on water agar at 4 G f o r germination. After 30 days, 5 5 $ of the s c l e r o t i a had produced basidiocarps. A 9 5 $ germination rate occurred i n the control. ( 2 ) Washing of S c l e r o t i a S c l e r o t i a grown on Medium C at 15 C were removed from the colony and washed i n fla s k s of s t e r i l e water at 20 C. To simulate the leaching e f f e c t of rai n , the fla s k s were set on a reciprocating shaker f o r 48 hours. The washed s c l e r o t i a were pretreated at 4 C for 5 days and incubated on water agar at 15 C. After 36 days, 7 1 $ germination had occurred, the same as the unwashed control. ( 3) Sclerotium Diameter It i s known that the larger s c l e r o t i a of Ciaviceps purpurea -49-(Fr.) Tul. are more viable than smaller ones (Cooke and Mitch-e l l , I 9 6 6 ) . S c l e r o t i a from several cultures of T. erythropus were measured and a record kept of the i r germination to deter-mine i f a sim i l a r c o r r e l a t i o n existed. Of the s c l e r o t i a with diameters greater than 0.7 mm, no coorelation between size and v i a b i l i t y was evident. S c l e r o t i a measuring less than 0.7 mm were seldom observed to germinate. In nature, and i n culture, the average sclerotium diameter was greater than 1.0 mm. (4) Illumination of S c l e r o t i a S c l e r o t i a grown to maturity i n the l i g h t were able to germinate when placed on water agar and incubated i n darkness. Although the sporophores that arose i n darkness usually were palmately branched and s t e r i l e , some t y p i c a l f e r t i l e basidio-carps were produced under these conditions. D. Medium Composition Effects S c l e r o t i a were formed i n culture on a variety of media at several d i f f e r e n t temperatures, but s c l e r o t i a from d i f f e r e n t sources were not equally viable. S c l e r o t i a grown on Medium B at 10 C germinated poorly (less than 5$) i n contrast to a high rate (95$) f o r 15 C grown s c l e r o t i a from Media A, B, and C. S c l e r o t i a from Medium C made with the water extract of wheat germ were equal i n v i a b i l i t y to those produced on Medium C. Varying the C/N r a t i o from 19:1 to.190:1 i n Medium A (refer to section VI) did not a l t e r the germination rate of s c l e r o t i a produced at 15 C. The s c l e r o t i a that formed on water agar were small (0.5-0.8 mm) and had a low germination rate. Whether t h e i r low v i a b i l i t y was an eff e c t of size or of nutrients was not determined. -50-E. In Situ Germination Stage III s c l e r o t i a that had developed on a colony on Medium A or B did not usually germinate i n place. If l i f t e d from the colony and relocated on the same plate, no germination resulted. Placement of a square of d i a l y s i s tubing between the sclerotium and the parent colony did not encourage germ-ina t i o n . Exceptions to this f a i l u r e to germinate i n s i t u were noted i n several month old colonies grown on Media A and C. F e r t i l e basidiocarps arose from s c l e r o t i a i n cultures on Medium C when incubated a 4 C a f t e r maturation at 15 C. Seven month old cultures on Medium A grown at k C produced basidiocarps from s c l e r o t i a ; the basidiocarps lacked heads and were s t e r i l e . S c l e r o t i a whose development was arrested between stage II and III often gave r i s e to f e r t i l e sporophores i n culture. This occurred most commonly on media with a low C/N r a t i o i n cultures incubated at 10 and 15 C. VIII. Basidiocarp Formation and Growth A. General Observations In nature, basidiocarps of T. erythropus are known to ar i s e only from s c l e r o t i a (Corner, 1950). No sclerotium-free specimens were found i n any of the c o l l e c t i o n s from B r i t i s h Columbia. However,, i n culture, basidiocarps were produced from s c l e r o t i a , d i r e c t l y from the colony surface, and from the edges of the inoculum plug. Often a medium that did not support f r u i t i n g from the agar surface induced some sporophore develop-ment from the inoculum block. When plugs were inverted p r i o r to inoculation, the appearance of basidiocarps was delayed, and the f i r s t f r u c t i f i c a t i o n arose from the lower side, nearest the agar. This orientation e f f e c t appeared to be a r e s u l t of -51-the much denser mycelial mat formed on the upper surface of a culture. Small sporophores arose from inoculum plugs on water agar plates and sometimes developed i n the agar away from the inoc-u l a t i o n point. On more favorable media basidiocarps arose d i r e c t l y from the agar as well as from the inoculum plug. Occasionally sporophores were produced from sclerotium-like lumps i n the colony. These i l l - d e f i n e d lumps were reported to be abortive s c l e r o t i a i n cultures of T. sc l e r o t i o i d e s (Mac-Donald, 193^). Since i t was possible to obtain basidiocarps without s c l e r o t i a , the effects of n u t r i t i o n a l and environmental factors on basidiocarp formation could be investigated free from the interference of t h i s p h y s i o l o g i c a l l y active structure. B. Morphology of the Mature Basidiocarp Corner (1950) has described the st r u c t u r a l d e t a i l s of mature T. erythropus basidiocarps. Except for the p o s s i b i l i t y of T. i s h i k a r i e n s i s , the many recombinations and synonyms assoc-iated with other Typhulas are not found i n T. erythropus. The c h a r a c t e r i s t i c coloring of the head and stalk, the general spore dimensions, and the structure of the sclerotium make this an e a s i l y i d e n t i f i a b l e species. Lehfeldt (I923) found this species convenient to use because of the certainty of i t s i d e n t i f i c a t i o n . I chose i t for the same reason. Basidiocarps were t y p i c a l l y unbranched, but i n culture branching was occasionally present. These branched f r u c t i f i c a -tions were always found on plates i n conditions unfavorable for development ( i . e . on water agar or i n darkness). No branched basidiocarps were seen i n f i e l d c o l l e c t i o n s . -52-C. Basidiocarp Development Sporophores followed the same pattern of development regardless of the i r source of i n i t i a t i o n ( i . e . from s c l e r o t i a , the agar surface, or inoculum plugs). For convenience, th e i r development has been divided into four stages, I , I I , I I I , and I V . At stage I the basidiocarp primordium f i r s t becomes v i s i b l e as a pure white cone, 0.1 x 0.2 mm. This body elongates with l i t t l e increase i n diameter (0.2 mm), assuming a gradually tapered candle-like shape up to 1.2 mm long. Stage I I commences when the base of the young basidiocarp begins to darken ( f i g . 31). This darkening i s caused by the sheathing c e l l s of the stalk becoming thick-walled and pigmented, much l i k e the crust that forms on the surface of older colonies. The tapered stage I I sporophore elongates for 3 6 - 4 8 hours when the upper 0.4 mm suddenly swells to delimit a well-defined clavate head ( f i g . 35). The appearance of the head indicates the onset of stage I I I . No hymenium i s present at thi s time. Sporophore elongation and head development continue. When the head i s approximately I mm long, i t becomes f e r t i l e , and stage I V commences ( f i g s . 30. 32,34). Sporulation continues for 10-15 days under favorable conditions of temperature and humidity. During this i n t e r v a l the stage I V basidiocarp continues to elongate (see l a t e r for details)'. Sporophores past maturity took on a watery appearance and collapsed. The head of a f a l l e n sporocarp gave r i s e to a dikaryotic mycelium or was converted to a sclerotium on the agar surface. The zone of encrusting hyphae that f i r s t appeared i n stage I I spread up the stalk a few mm behind the apex of the growing - 5 3 -b a s i d i o c a r p . Mature sporophores are t y p i f i e d by t h i s r e d d i s h -brown, horny s t a l k (Corner, 1 9 5 0 ) . Every b a s i d i o c a r p that arose from a s c l e r o t i u m possessed t h i s c h a r a c t e r i s t i c . However, not a l l b a s i d i o c a r p s that were produced from the mycelium had the reddish-brown s t a l k . The temperature of i n c u b a t i o n and the medium composition a f f e c t e d pigmentation. These e f f e c t s are d e t a i l e d i n a l a t e r sub-section. The l e n g t h of ba s i d i o c a r p s during d i f f e r e n t stages of development was r e l a t i v e l y constant. Stage I carpophores measured from 0 . 1 to 1 . 2 mm, stage II from 1 . 2 to 3 . 2 mm, stage III from 3 . 2 to 6 . 3 mm, and stage IV from 6 . 3 to 2 5 mm. The head of a 2 5 mm b a s i d i o c a r p averaged 4 mm i n l e n g t h . The d u r a t i o n of each stage and, thus, the time from i n i t i a -t i o n to maturity were v a r i a b l e , i n f l u e n c e d by temperature and medium composition. These e f f e c t s are presented i n d e t a i l i n another sub-section. Sporocarps a r i s i n g from the m y c e l i a l mat f i r s t appeared I 3 - I 9 days a f t e r i n o c u l a t i o n of Medium A and i n c u b a t i o n at 15 C. These f i r s t - f o r m e d b a s i d i o c a r p s were s i t u a t e d close to the i n o c -ulum plug. As the co l o n i e s grew, ba s i d i o c a r p s were produced f u r t h e r from the i n o c u l a t i o n point ( f i g . 3 3 ) • A 60 mm diameter colony included up to 60 f r u c t i f i c a t i o n s w i t h i n a 30 mm diameter inner c i r c l e . A l l i s o l a t e s produced sporophores from c u l t u r e s i n an i d e n t i c a l manner to that described f o r i s o l a t e T - 4 . D. Expansion of the B a s i d i o c a r p The growth of the stage I primordium i n t o a mature b a s i d i o -carp was brought about almost e n t i r e l y by the i n f l a t i o n of -54-pre-existing c e l l s . After early stage I, few new c e l l s formed at the apex of the sporophore. This was demonstrated by the ap p l i c a t i o n of marks to sporophores and by microscopic exam-inat i o n . Basidiocarps i n various stages of development from s c l e r o t i a and inoculum plugs were marked with spots of black ink or spores of Equisetum along t h e i r length. The distance between the spots was measured d a i l y with an", ocular micrometer, and the zone of expansion was determined. Unmarked basidiocarps developed at the same rate as those that were marked. The growth of a sporophore from 1 mm to 8.3 mm i s shown i n figure 24. Only the upper part of a basidiocarp expanded, leaving behind a non-expanding trunk. The t i p (0.2-0.4 mm) of a stage I or II sporophore elongated l i t t l e , contributing only to the head. When the head had been delimited as a 0.4 mm long body, i t s growth was independent of the elongation of the stalk. The growth zone of a stage II basidiocarp was confined to a 1-2 mm region approximately 0.4 mm behind the apex. In stage III and IV sporocarps, the growth zone was located just below the head. Relative to the base, the growth zone traveled up the s t a l k . The percentage of a basidiocarp elongating was i n -versely proportional to the t o t a l length of the f r u c t i f i c a t i o n . Figure 25 i l l u s t r a t e s t h i s r e l a t i o n s h i p . Approximately 70$ of the length of a young 2 mm sporophore was elongating. The hyphal c e l l s at the base (the remaining 3°$) n a d ceased i n f l a -t i o n . Less than 40$ of the length of a 7 mm basidiocarp was i n the process of elongation. The rate of extension of the sporophore increased as the structure developed. At 10 C, a stage IV basidiocarp elongated at an average rate of J.2 mm/24 hours; a stage I grew at just -55-FACING PLATE 16 Figure 2k: The growth of a b a s i d i o c a r p from 1 mm to 8.3 mm. Note that the lower part ceases elongation before the upper p a r t . The growth zone i s l o c a t e d j u s t below the head. 8--LENGTH mm I II III IV STAGE F i g u r e 2k -56-1.1 mm/24 hours. The elongation rates of the four stages are i l l u s t r a t e d i n figure 25. E. Temperature Effects Basidiocarps arose from inoculum plugs placed on Medium A a f t e r 2-3 days incubation at 4, 10, and 15 C. Since very vigorous sporophore production occurred when the plugs were placed on Medium A with 1.0 g / l of casein hydrolysate i n place of asparagine, this medium was used to study the effects of temperature. Basidiocarps were formed rapidly at 4 and 10 C; stage IV sporophores were observed on the inoculation plug 6-8 days a f t e r inoculation. At 15 C, development was retarded, requiring up to 10 days f o r basidiocarps to reach stage IV. The ef f e c t of temperature on the duration of each stage i s shown i n Table II and figure 27. These data represent average values. Not a l l stage I primordia completed th e i r development. The delay i n reaching stage IV at 15 C was seen i n the t r a n s i t i o n from stage II to I I I . Head formation frequently was delayed or t o t a l l y i n h i b i t e d at 15 C. The production of basidiocarps from the surface of the colony was li m i t e d to a much narrower temperature range than that f o r t h e i r development from inoculation plugs. On Medium A and Medium A with casein hydrolysate, sporophores were ra r e l y produced at 0, 4, or 10 C. Basidiocarps that did develop at these temperatures usually were very short and were composed of of a sub-globose head on a tiny s t a l k . These 1-2 mm high f e r t i l e sporophores lacked pigmentation. At 15 C, however, t y p i c a l basidiocarps developed f r e e l y from the mycelial mat of cultures grown on both media. Sixty or more sporophores commonly were produced i n a single p e t r i plate. These mature f r u c t i f i c a t i o n s -57-FACING PLATE 17 Growth of the Basidiocarp - 2 Figure 25: The relat i o n s h i p of the stage of development to the rate of extension of basidiocarps produced at 10 C. Figure 26: The cor r e l a t i o n of basidiocarp length to the length of the growth zone i n elongating basidiocarps. 3.0 EXTENSION RATE M M / 2 4 N 2.0H 1.04 0-80-STAGE Figure 25 %OF 6 0 , LENGTH D U EXPANDING 40-I 20H 0 T " 7 BASIDIOCARP LENGTH mm Figure 26 8* 0 -58-FACING PLATE 18 F i g u r e 27: The e f f e c t of temperature on the r a t e of b a s i d i o c a r p development. The r a t e f o r sporo-phores produced a t k C was very s i m i l a r to that a t 10 C. T a b l e I I : D u r a t i o n o f t h e s t a g e s o f B a s i d i o -c a r p d e v e l o p m e n t DURATION OF STAGES, DAYS TEMP 0-I M l ll-lll lll-IV 0-IV 15° 2-3 1.5 3 2 8.5-9.5 10° 2-3 1.5-2 1.5-2 1-1.5 6-8 4° 2-3 2-3 1.5 1-2 65-8.5 STAGE IVH II-0 3 4 5 6 AGE, DAYS 8 T-9 Figure 27 -59-were indistinguishable from those a r i s i n g from s c l e r o t i a . Basidiocarps developing from the colony surface at 18 C lacked a hymenium. At 20 C, no sporophores were produced. The e f f e c t of temperature on basidiocarps produced from s c l e r o t i a was similar to that observed on sporophores a r i s i n g from inoculation plugs. At 4 and 10 C, large, t y p i c a l carpo-phores were produced from viable s c l e r o t i a . However, basidio-carps a r i s i n g from s c l e r o t i a at 15 C frequently grew to just 5-12 mm i n length and remained at stage I I . When these stage II sporophores were incubated at 4 or 10 C they resumed devel-opment and reached stage IV. No differences were noted between basidiocarps produced from s c l e r o t i a from culture or from the f i e l d . F. pH Effects The e f f e c t of pH on basidiocarp formation was determined by counting the number of mature sporophores produced i n c u l -tures on Medium A at 15 C. The f i n a l pH was also determined at this time. Cultures were grown f o r 31 days at pH 4.0, 5.0, 5.5, 6.0, 7.0, 7.7, and 8.5. The f i n a l pH of these media was 6.5, 6.5. 7.6, 7.6, 7.8, 7.8, and 7.8, respectively. Maximum f r u i t -ing occurred where the i n i t i a l pH was 5.5-6.0, with 15-18 basidiocarps formed per plate ( f i g . 28). At an i n i t i a l pH of 7.0. f r u i t i n g was less vigorous and was ne g l i g i b l e on media with and i n i t i a l pH of 4.0, 5.0, 7.7, or 8.5. G. Photo Effects Basidiocarps of T. erythropus are p o s i t i v e l y phototropic i n a l l stages of development. Apices of growing sporophores grow toward the l i g h t source. This growth response took place i n the zone of elongation, not at the apex. When the position - 6 0 -FACING PLATE 19 Figure 2 8 : The eff e c t of i n i t i a l pH on basidiocarp pro-duction on Medium A at 15 C. Cultures were grown for 31 days. CO CO CL 3 CD -H LO O CM LO O LO CO CL LU CC H < < O cu Q CC CO LU < CL CQ -61-of the l i g h t source was shifted, the growth of the sporophore toward the new source was evident i n less than nine hours ( f i g . 35). Blue and white l i g h t were the most e f f e c t i v e i n e l i c i t i n g the response. Light passing through red cellophane did not evoke a growth adjustment. Exposure to l i g h t appeared to be necessary for the devel-opment of mature basidiocarps. Dark-grown cultures started from light-grown mycelia frequently produced branched stage I I basidiocarps or did not produce any sporophores. S c l e r o t i a grown to maturity i n the l i g h t sometimes gave r i s e to f e r t i l e basidiocarps when germinated i n darkness, but most basidiocarps did not develop beyond stage I I . Stage I f r u c t i f i c a t i o n s were formed on the colony surface i n dark-grown cultures started from dark-grown mycelia. No f e r t i l e sporophores were produced i n these cultures. A control of dark-grown mycelium inoculated onto a plate and grown i n the l i g h t produced f e r t i l e sporophores. H. Gravity Effects Observations of basidiocarps produced i n darkness i n inverted p e t r i plates showed no signs of a posi t i v e or negative geotropic response. Sporocarps arose at various angles away from the agar surface. Since l i g h t has been implicated i n the geotropic response of some basidiomycetes (Taber, I966) , l i g h t -grown cultures were incubated at several angles. The orienta-t i o n of the basidiocarps was noted. No cultured sporophores of t h i s species were geotropic. I . Carbon:Nitrogen (C/N) Ratio Effects The asparagine concentration i n Medium A was adjusted to give C/N r a t i o s (g C/g N) of 3.8:1 to 190:1. The pH of a l l media was 5-5, and cultures were incubated at 15 C. After 26 - 6 2 -day s the number of stage IV basidiocarps was counted:, and the f i n a l pH of the media was determined. The results are i l l u s -trated i n figure 2 9 . Basidiocarp production was highest at a C/N r a t i o of 1 9 : 1 , with an average of 4 0 stage IV basidiocarps per plate. F r u i t i n g vigor was one half this value at C/N r a t i o s of 9.5:1 and 3 8 : 1 . At 6 2 : 1 only 1 5 sporophores were present, and at greater than 95:1 no f r u i t i n g occurred. The f i n a l pH of media with C/N of 1 9 : 1 to 1 9 0 : 1 was 5 . 5 . Media with r a t i o s of 9.5:1, 4 . 7 : 1 , and 3.8:1 had f i n a l pH values of 5.8, 7.0, and 8.0, respectively. There was a good co r r e l a t i o n between the degree of f r u i t i n g and basidiocarp length. The medium that yielded the maximum number of basidiocarps also produced the longest ones as well. Basidiocarps produced at a C/N r a t i o of 1 9 : 1 were long and slender, whereas those at 3.8:1 were short and stout. Other isol a t e s grown on C/N ra t i o s of 9 5 : 1 and 9-5:1 responded i n the same manner as i s o l a t e T-4. J . Nutrient Concentration Effects The concentration of glucose and asparagine i n Medium A was adjusted to make media of 1/10 to 2X regular strength. A C/N r a t i o of 9*5:1 was maintained i n a l l media. Cultures were grown at 1 5 C for 2 6 days. Maximum basidiocarp production from the colony surface occurred on the 1/10 strength medium ( 1 . 0 g / l glucose, 0.2 g / l asparagine). On this medium 60 mature sporophores were formed per plate. F r u i t i n g decreased as the nutrient concentration increased. The average number of mature basidiocarps on 1/2, f u l l , 1.5X, and 2X strength media was 40, 2 5 , 2 0 , and 2 per plate, respectively. Sporophores were formed e a r l i e s t on media with low nutrient concentrations. -63 -FACING PLATE 20 Figure 29= The effect of various C/N r a t i o s on basidio-carp production at 15 C. The concentration of asparagine was adjusted while the glucose con-centration was maintained at 10.0 g /1. C / N Figure 29 -64-In contrast to the decrease i n basidiocarp production as the strength of the medium increased, mycelial growth was most vigorous on the high strength media. K. Nitrogen U t i l i z a t i o n The d i f f i c u l t i e s already mentioned with regard to nitrogen n u t r i t i o n of sclerotium development apply to this section as well. Of the compounds tested, DL-asparagine and casein hydrol-ysate (1.0 g/1) supported maximum basidiocarp production. Few sporophores were formed on media made with DL-alanine, KNO^, or Ca(N0^)2« Although n i t r a t e s were poorly u t i l i z e d , they did support some basidiocarp production a f t e r three s e r i a l trans-f e r s . No basidiocarps were produced from cultures on media made with DL-methionine, L-(-)-phenylalanine, L-proline, or L-tyro-sine. Ammonium chloride and ammonium sulfate also supported no f r u i t i n g . The rapid drop i n pH, prohi b i t i v e to growth, prob-ably was responsible f o r the lack of basidiocarps. Cultures grown on 2% malt-extract agar (Difco), used i n preliminary work with T. erythropus, did not produce carpophores. Addition of 2.0 g/1 of ammonium sulfate or ammonium tartrate induced the formation of robust sporophores on the medium. It i s expected that ammonium sa l t s could be u t i l i z e d f o r basidiocarp formation i f the pH of the medium could be maintained at a sat i s f a c t o r y l e v e l . L. Vitamin Requirements No basidiocarps were produced from cultures grown on Medium A prepared without the vitamin stock solution. Addition of 100 ug/1 of thiamine to the medium i n place of the vitamin stock solution restored normal sporophore production. Two s e r i a l transfers v e r i f i e d that thiamine was the required vitamin. -65-FACING PLATE 21 B a s i d i o c a r p Development Figure JO: Stage IV b a s i d i o c a r p s on a p e t i o l e of Acer  macrophyllum. Xl.5 Figure 31: Stage I I b a s i d i o c a r p s developing from the inoculum plug on the c a s e i n hydrolysate medium. Culture was grown a t 15 C. Note the strong photo-t r o p i c response. X7. Figure 32: Stage IV b a s i d i o c a r p s developing from the inoculum plug. Note heavy spore drop below the heads of the f r u c t i f i c a t i o n s . Culture was grown at 10 C on Medium A w i t h c a s e i n hydrolysate. X7-I F i g u r e 33 : B a s i d i o c a r p s on Medium A w i t h casein hydrolysate. Note development from agar surface. A few stage I I I b a s i d i o c a r p s are present, but most are stage I I . Apparent branching i s an o p t i c a l i l l u s i o n caused by o v e r l a y i n g sporophores. X0.8. Figure J>k: Germinating s c l e r o t i a on water agar. Most sporo-phores are at e a r l y stage IV. Germination was a t 4 C. Approx. normal s i z e . Figure 35' Stage I I I b a s i d i o c a r p s . P l a t e had been r o t a t e d twice (90° each time) from the o r i g i n a l p o s i t i o n . The second r o t a t i o n occurred 9 hours before the photograph was made. Culture was grown on Med-ium A a t 15 C. - 6 6 -D I S C U S S I O N I. General Considerations The c u l t u r a l studies performed with T. erythropus provided data that may elucidate the l i f e cycle of this fungus i n nature. Except f o r the 15-20 C a c t i v a t i o n of s c l e r o t i a , T. erythropus i s able to complete i t s entire l i f e cycle at if -10 C. These temperatures are comparable to those prevalent during the growing season of the organism. In nature, s c l e r o t i a are formed i n the winter but remain dormant throughout the spring and summer. They germinate i n the f a l l when the habitat i s cool and moist. It appears l i k e l y that sclerotium a c t i v -ation occurrs i n the la t e spring when s c l e r o t i a are turgid. Desiccated s c l e r o t i a from culture could not be activated. Mature s c l e r o t i a collected on petioles i n May 19?0, did not germinate when incubated at 4, 10, or 15 C or a f t e r a pretreat-ment of 4 C f o r 5-1° days followed by 15 C incubation. S c l e r o t i a collected i n August, 1970, produced basidiocarps readily when incubated at 4 C but not at 15 C. Not a l l Typhulas are autumnal. Some species sporulate i n spring and autumn (Corner, 1950). Corner ascribed this single f r u i t i n g season to a f a i l u r e of the s c l e r o t i a to mature by spring, when they should otherwise germinate. This explana-tion does not agree with my f i e l d observations. Mature s c l e r o t i a of T. erythropus were present on petioles by February. T. sc l e r o t i o i d e s , another autumnal species, also was observed on Acer p e t i o l e s . S c l e r o t i a of this species were also mature before the end of winter. II . Mating System and Monokaryons T. erythropus appears to be f a c u l t a t i v e l y homothallic. -67-In the absence of a compatible mating s t r a i n , single-spore haploid cultures are able to become dikaryotized. However, before dikaryotization, single-spore mycelia are p o t e n t i a l l y h e t e r o t h a l l i c . In i t s h e t e r o t h a l l i c reactions, T. erythropus i s tetrapolar. Four mating strains were i d e n t i f i e d from the nine monosporic is o l a t e s derived from i s o l a t e T-4. The incom-p a t i b i l i t y system i n only one other Typhula i s known; T. i n -carnata i s also tetrapolar (Rj^ed, I 9 6 9 ) . Lehfeldt (I923), a student of Kniep, crossed single-spore mycelia of T. erythropus and observed fusion and clamp formation i n successful matings. He could not determine i f this species were bipolar or tetrapolar. Kniep (I928) reported the formation of haploid and " d i p l o i d " f r u c t i f i c a t i o n s by cultures of the same species. It i s not known i f these haploid f r u c t i f i c a t i o n s were actually homodikaryotic. In Sistotrema brinkmanni three "subspecies" are recognized, one homothallic, one with bipolar heterothallism, and one with tetrapolar heterothallism (Lemke, I 9 6 9 ) . The same s i t u a t i o n may exist i n T. erythropus, but further study i s needed. Noble (1937) was able to obtain only f i v e monosporic i s o -lates of T. t r i f o l l i • She demonstrated heterothallism but could not determine i f the species were bipolar or tetrapolar. Lemke (1969) noted that dikaryons could be homokaryotic or heterokaryotic and used the terms "homodikaryotic" and "hetero-di k a r y o t i c " to denote these s i t u a t i o n s . His terminology was used i n the present study. Homodikaryotic cultures of T. erythropus grew more slowly than heterodikaryons. This slow growth rate and the delay of s e l f - d i k a r y o t i z a t i o n f o r 3-4 weeks a f t e r inoculation would seem - 6 8 -to be favoring outbreeding. Dikaryotization of the monokaryotic colonies was f i r s t observed i n the young basidiocarps that arose i n the center of the colony. Later, clamp connections were formed on the hyphae at the margin of the colony. The homodikaryotization process might be s i m i l a r to that described i n Taphrina (Kramer, i 9 6 0 ) . In this genus the dikaryotic condition arises as a re s u l t of the mitotic d i v i s i o n of the single haploid nucleus during the germination of a blastospore or an ascospore. Typhula mycelium, l i k e the Taphrina blastospore, can produce haploid c e l l s f o r an indeterminate period. Under suitable con-dit i o n s a homodikaryon i s i n i t i a t e d , and the l i f e cycle can be completed. The slower growth rate of monokaryons i s common i n the higher basidiomycetes (Fincham and Day, I963; Raper, I966). I t should be emphasized that a l l nine single-spore i s o l a t e s were slow growing, and a l l developed basidiocarps and clamps within one week of each other. This would suggest that this f a c u l t a t i v e homothallism i s a well established genetic charac-ter i n T. erythropus and that homodikaryotization was not a re s u l t of a contamination with a compatible mating s t r a i n . T. t r i f o l i i produced haploid basidiocarps i n culture (Noble, 1937). These sporophores had the appearance of mini-ature dikaryotic sporophores. Four basidiospores were produced per basidium. The basidiospores were approximately one half the size of basidiospores from the dikaryon. Only three of Noble's f i v e haploid i s o l a t e s formed basidiocarps i n culture. These same three also formed haploid s c l e r o t i a i n culture. - 6 9 -J-« erythropus basidiospores from basidiocarps formed on single-spore cultures were equal i n size to normally produced basidiospores. Hanna (I928) reported that spores from haploid and dikaryotic f r u c t i f i c a t i o n s of Coprinus lagopus were of equal si z e . I l l . Mycelial Growth, Dikaryotic The general morphological features of mycelial growth of T. erythropus are si m i l a r to those reported f o r T. scle r o t i o i d e s by MacDonald (193*0 • The growth rate of 2-3 mm/day increase i n colony diameter i n cultures of T. erythropus was less than the rates recorded f o r other species. Dejardin and Ward ( 1 9 7 1 ) noted rates of 3.5 mm/day f o r T. incarnata, 3.8 mm/day f o r T. idahoensis, and 4.4 mm/day for T. t r i f o l i i . An undetermined Typhula species showed a rate of 8 mm/day (Lockhart, I 9 6 7 ) . T. erythropus was able to grow at temperatures from 0 to 20 C. Basidiospore germination occurred over the same range. The psychrophilic nature of many Typhula species i s well docu-mented (MacDonald, 1934; Tasugi, 1935; Remsberg, 1940; Terui, 1941; Ekstrand, 1 9 5 5 ; Tomiyama, 1 9 5 5 ; Potatosova, 1960a; Jack-son, I963; Lockhart, 196?; Corner, 1970; Dejardin and Ward, I 9 7 I ) . Doubtless, this a b i l i t y to grow at very low temperatures when other fungi are in h i b i t e d i s important to th e i r mode of existence. Psychrophily i s not uncommon i n the fungi. Other psychro-p h i l i c species, excluding Typhulas, amd thei r optimum tempera-tures f o r growth include S c l e r o t i n i a borealis, 0 C (Ward, I966), Phacidium infestans, 1 5 C (Perhson, 1948), Herpotrichia nigra, 1 5 C (Cochrane, I958) , and an unidentified basidiomycete, 12 -17 C (Ward, et a l , I 9 6 I ) . -70-Th e optimum pH for mycelial growth of T. erythropus was 4-6. Dejardin and Ward (I97I) reported maximum l i n e a r extension i n 5!* t r i f o l i i , T. incarnata, and T. idahoensis at pH 5-7. Their work v e r i f i e d Tasugi's (1935) report on the optimum pH for T. incarnata (as T. graminum). My i s o l a t e s of T. erythropus were thiamine d e f i c i e n t ; a deficiency of this type i s common i n the higher basidiomycetes (Cochrane, 1958) , but has not been investigated i n other species of Typhula. The scant growth that did occur i n the apparent absence of thiamine probably can be attributed to impurities i n the other ingredients of the medium. Agar, asparagine, and glucose frequently are contaminated with b i o l o g i c a l l y s i g n i f -icant amounts of the vitamin (Cochrane, I 9 5 8 ) . Asparagine and casein hydrolysate as nitrogen sources supported maximum mycelial growth. They were also superior to other sources for sclerotium and basidiocarp production. Nitrates did not support the formation of a dense mycelial mat. The rapid drop i n pH to i n h i b i t o r y l e v e l s during ammonium u t i l -i z a t i o n by T. erythropus has been noted i n many other fungi (Apparao, 1956; Cochrane, 1958; Ward, 1964; Curren, I 9 6 8 ) . The i n i t i a l growth that was made on media with ammonium sa l t s was dense and comparable to that with asparagine. It appeared that ammonium would have supported the production of a dense mycelial mat had the pH not changed. IV. Sclerotium Development The development of s c l e r o t i a of T. erythropus was similar to that described f o r other Typhulas (deBary, 1887; MacDonald, 1934; Remsberg, 1940). S c l e r o t i a of T. s c l e r o t i o i d e s took 9-10 days to reach maturity a f t e r i n i t i a t i o n (MacDonald, 1 9 3 4 ) i n comparison to the 14-16 days required "by T. erythropus. The same four stages of development were noted during the f o r -mation of T. s c l e r o t i o i d e s s c l e r o t i a as were present i n their formation by T. erythropus. The exudation of l i q u i d from maturing s c l e r o t i a was f i r s t reported by deBary i n I887. He and l a t e r investigators (MacDonald, 1 9 3 ^ ; Remsberg, 1 9 4 0 ) be-l i e v e d that the l i q u i d was mainly water that was expelled during the compaction of the sclerotium. When the l i q u i d from T. erythropus s c l e r o t i a was evaporated on a glass s l i d e , a white c r y s t a l l i n e deposit remained. Remsberg observed the same phe-nomenon i n other Typhula species. The effects of temperature on sclerotium production were d i f f i c u l t to assess. A d e f i n i t e i n t e r a c t i o n between temperature of incubation and the C/N r a t i o of the medium was established. Sclerotium production was maximal at 1 $ C on a medium with a C/N of 9 5 : 1 ; at 4 and 10 C, the optimum C/N was 9 . 5 : 1 . Dejardin and Ward (I97I) mentioned that the greatest number of s c l e r o t i a were formed on a malt extract-yeast extract-glucose medium (note high C/N) at temperatures above 10 C. If the same temperature-C/N re l a t i o n s h i p exists f o r the species i n t h e i r study as f o r T. erythropus, t h e i r observation i s of l i t t l e value without more experimental data. The same may be said fo r MacDonald's (193*0 observation that maximal sclerotium pro-duction by T. s c l e r o t i o i d e s was at 1 3 - 1 5 C on PDA. The incubation temperature also influenced the time to sclerotium i n i t i a t i o n and to maturity. These processes were most rapid at 10 and 15 C, s c l e r o t i a appearing 8-12 days a f t e r -72-i n o c u l a t i o n of s u i t a b l e media. Remsberg (1940) reported s c l e r -otium formation o c c u r r i n g 5-14 days a f t e r i n o c u l a t i o n i n the 14 species she examined. MacDonald (193^) recorded a time of 21 days f o r s c l e r o t i a of T. s c l e r o t i o i d e s , and Dejardin and Ward (1971) 4 days f o r T. t r i f o l i i and T. idahoensis and 6 days f o r T. i n c a r n a t a . The l a t t e r species was reported by Jackson (I963) to r e q u i r e 7-10 days On PDA. The coalescence and f u s i o n of s c l e r o t i a at lower tempera-tures i n T. erythropus was described p r e v i o u s l y by Remsberg f o r other s p e c i e s . This e f f e c t was evident only when c u l t u r e s were grown on a r i c h medium or one w i t h a low C/N r a t i o . Corner (1950) proposed that the absence of compound s c l e r o t i a from f i e l d c o l l e c t i o n s was probably a r e s u l t of the l a c k of r i c h sub-s t r a t e s c o l o n i z e d by these f u n g i . The C/N r a t i o of f o r e s t l i t t e r i s 40:1 to 50:1 (Brock, I966). Townsend (1957) examined s c l e r o t i u m formation i n s e v e r a l species of fungi i m p e r f e c t i , some of which had p e r f e c t s t a t e s i n the hymenomycetes. She recognized three d i f f e r e n t stages of s c l e r o t i u m formation: i n i t i a t i o n , growth to f u l l s i z e , and maturation. Each stage d i f f e r e d i n i t s n u t r i t i o n a l r e q u i r e -ments. These stages correspond to stages I a , l b , and I I i n the present i n v e s t i g a t i o n . Stage I I I of the T. erythropus s c l e r o -tium i s equivalent to the "mature s c l e r o t i u m " i n Townsend fs work. N u t r i t i o n a l d i f f e r e n c e s were noted among the stages of s c l e r o t i u m formation i n T. erythropus• On Medium A prepared w i t h galactose, s c l e r o t i a d i d not develop beyond stage I a . Stage l b s c l e r o t i a o f t e n f a i l e d to become pigmented when pro-duced on a medium w i t h n i t r a t e as the s o l e n i t r o g e n source. - 7 3 -Except for the abortive s c l e r o t i a that developed on Medium A at 15 C, stage II s c l e r o t i a , once formed, continued on to stage I I I . Townsend ( 1 9 5 7 ) and others (Hawker, 1 9 5 0 ; Cochrane, 1 9 5 8 ) have reported that factors favoring vegetative growth also favor sclerotium production. This c o r r e l a t i o n was observed i n cultures of T. erythropus grown on media of various strengths. The richest medium supported maximal sclerotium production and the highest rate of l i n e a r extension of hyphae. Furthermore, the optimum temperature f o r mycelial growth and sclerotium formation was 15 C. This would indicate that sclerotium f o r -mation can be c l a s s i f i e d as vegetative growth i n contrast to reproductive growth. Cochrane ( 1 9 5 8 ) suggested that, "... sclerotium development, as a prelude to sexual reproduction, may be expected to be influenced by the same f a c t o r s . " How-ever, he presented no evidence i n support of t h i s . Such fac-tors as higher temperatures, high strength media, and high C/N r a t i o s at 15 C, which favored sclerotium production i n T. erythropus frequently were i n h i b i t o r y to basidiocarp f o r -mation. Wheeler and Waller (I965) found that the i n i t i a t i o n of Sclerotium r o l f s i i Sacc. s c l e r o t i a i n culture was delayed u n t i l the l a t e r a l extension of the mycelium had been checked. S c l e r o t i a did not appear u n t i l the plate was f i l l e d with myce-lium. This e f f e c t was not observed i n cultures of T. erythro-pus . Sclerotium i n i t i a t i o n , growth, and maturation were often completed before the plate was covered. The stimulatory e f f e c t of wheat germ on sclerotium f o r -mation was evident i n two ways. F i r s t , s c l e r o t i a were formed -74-e a r l i e r on Medium C than on any other media. It has been shown previously that factors favoring mycelial growth also favor sclerotium production. The short l ag period f o r the mycelial growth rate was r e f l e c t e d i n the more rapid appearance on mature s c l e r o t i a on Medium C. The second e f f e c t was to increase the number of s c l e r o t i a produced per plate. These b e n e f i c i a l effects of wheat germ could r e s u l t from the in c l u s i o n of new ingredients into the medium ( i . e . , vitamins, sugars, amino acids), the a l t e r a t i o n of the proportion of the other ingredients, or both. The factor or factors i n wheat germ were water soluble, heat stable, and dialyzable. V. Sclerotium Germination A l l previously reported attempts to germinate s c l e r o t i a of Typhulas were done by placing them on moist s t e r i l e sand or s o i l . The use of water agar f o r this purpose i n the present investigation was superior to previous methods for three reasons: 1) i t f a c i l i t a t e d observations, 2) results were ea s i l y reproducible, 3) n u t r i t i o n a l factors were minimized. No dormancy period or special treatment was necessary f o r sclerotium germination i n any species previously tested. Mature s c l e r o t i a of T. t r i f o l i i germinated r e a d i l y on the parent colony but required an undetermined resting period when sown on moist soil.(Noble, 1937). I t was not stated i n e a r l i e r publications i f s c l e r o t i a produced at temperatures s i m i l a r to those i n the f i e l d were as viable as those produced at the optimum temperature f o r mycelial growth. I found that s c l e r o t i a of T. erythropus produced at 4 C or from f i e l d c o l l e c t i o n s i n May did not germinate under conditions favorable f o r the germ-ina t i o n of s c l e r o t i a grown at 10 and 15 G. The 4 C grown -75-s c l e r o t i a required treatment at 15-20 C f o r germination to occur. A good cor r e l a t i o n between sclerotium size and v i a b i l i t y -has been shown i n Claviceps purpurea (Cooke and M i t c h e l l , I966). Large s c l e r o t i a of this species were s i g n i f i c a n t l y more viable than smaller s c l e r o t i a . This re l a t i o n s h i p was not noted i n germinating s c l e r o t i a of T. erythropus, except f o r very small s c l e r o t i a (0.7 mm) that never germinated. Corner (1950) interpreted the Typhula sclerotium as an adaptation to low temperature, but my results indicate that i t s function i s to carry the fungus through the warm, dry summer. During this time no fleshy, newly f a l l e n petioles are available to support mycelial growth. At temperatures above 20 C, mycelial growth was e s s e n t i a l l y n i l . Species of this genus, including T. erythropus, w i l l grow at very low tempera-tures. In regions with very severe winters the sclerotium may have the dual function of overwintering and oversummering. At le a s t two phases of sclerotium germination were i n f l u -enced by temperature. F i r s t , s c l e r o t i a produced at 4 C norm-a l l y required a treatment of 15-20 C f o r germination to occur. Ten to fourteen days at the elevated temperature were s u f f i c i -ent to ensure germination. The occasional germination of these s c l e r o t i a without heat a c t i v a t i o n may have been caused by t h e i r frequent exposure to room temperature during examina-ti o n . Only 6-10 month old s c l e r o t i a were observed to germinate without heat a c t i v a t i o n , and this was sporadic. A minimum time of heat treatment was not determined. It was noted that s c l e r o t i a produced at k, 10, and 15 C - 7 6 -had the areas of basidiocarp o r i g i n established within them during sclerotium formation. Corner ( 1 9 5 0 ) suggested that such a prelocation occurred. The effectiveness of the 1 5 - 2 0 C treatment on s c l e r o t i a grown at k C seems to be i n bringing the basidiocarp primordia to a l e v e l of d i f f e r e n t i a t i o n com-parable to that i n 15 C grown s c l e r o t i a . Cross sections of s c l e r o t i a produced at 10 and 15 C frequently showed areas of compacted hyphae and alignment i n the medulla i n d i c a t i v e of a sporophore primordium. No such area were noted i n s c l e r o t i a produced at 4 C. Thus, while the loc a t i o n of the basidio-carp o r i g i n may be phy s i o l o g i c a l l y determined during sclerotium formation at k C, morphological d i f f e r e n t i a t i o n apparently does not occur u n t i l the sclerotium i s exposed to temperatures of 10 C or higher. S c l e r o t i a had to be turgid f o r the heat a c t i v a t i o n to be e f f e c t i v e . S c l e r o t i a of Claviceps purpurea require a cold treatment f o r germination, and -this could be met only when the s c l e r o t i a were water soaked (Mitchell and Cooke, I968) . The second temperature e f f e c t concerned the germination of s c l e r o t i a produced at 15 C and of activated s c l e r o t i a grown at 4 C. The c e l l or c e l l s that d i f f e r e n t i a t e d i n the basidio-carp primordium p r i o r to germination seldom developed further when l e f t at 1 5 C. Rather, most rapid germination resulted when s c l e r o t i a were incubated at k C. At the reduced tempera-ture, basidiocarp formation was stimulated. This would explain the effectiveness of the 5-7 day treatment at k C on the i n -creased germination rate of 15 C grown s c l e r o t i a . When these s c l e r o t i a were returned to 15 C to germinate, the germination process had already been triggered. Continued sporophore devel--77-opment at 15 C was another matter, and i t i s discussed l a t e r . Potatosova (1960a) reported a temperature e f f e c t on s c l e r -otium germination s i m i l a r to the one discussed above. S c l e r o t i a of T. v a r i a b i l i s , T. t r i f o l i i , T. incarnata, and T. idahoensis were placed outdoors i n pots of s t e r i l e sand and covered with glass. F r u c t i f i c a t i o n s did not appear u n t i l the outside temp-eratures had dropped several degrees. Sporophores of T. variab-i l i s and T. t r i f o l i i were produced at 8-15.5 C, of T. incarnata at- 1.4-13.5 C, and of T. idahoensis at 1.4-4.6 G. T. erythropus s c l e r o t i a produced at 10 C germinated on water agar at 10 C, although more rapid germination occurred at 4 C. VI. Basidiocarp Growth and Development Remsberg (1940) and Tasugi ( I929, 1935) encountered considerable d i f f i c u l t y i n obtaining f e r t i l e sporophores from germinating s c l e r o t i a of 15 Typhula species. They found that UV radiation (265O-325O &), which was not transmitted through Pyrex glassware, was necessary f o r the development of f e r t i l e heads. In the absence of this quality UV l i g h t , long, s t e r i l e stalks were produced. However, not a l l Typhulas require this wavelength of UV to sporulate. T. s c l e r o t i o i d e s (MacDonald, 1934), T. t r i f o l i i (Noble, 1937; Potatosova, 1960a), and T. v a r i a b i l i s (Potatosova, 1960a) produced f e r t i l e sporophores when grown under glass. T. erythropus belongs to this group of species able to sporulate under glass. The basidiocarps of T. erythropus are strongly photo-tropic, as are those of T. s c l e r o t i o i d e s (MacDonald, I934) . Considering the microhabitat, the advantage to the fungus i n possessing this positive phototropic response i s obvious. In -78-the shaded l e a f l i t t e r a p o s i t i v e l y phototropic sporophore would grow toward a l i g h t source, avoiding contact with the moist debris p i l e d around the sclerotium. A negative geo-tropic response would reduce the effectiveness of spore d i s -charge by d r i v i n g the basidiocarp into the l i t t e r above the sclerotium. The sporophore of T. erythropus i s ensured of maximum e f f i c i e n y i n spore release by growing toward open areas i n the l i t t e r where basidiospores are most l i k e l y to be dispersed by the wind. MacDonald (193^) commented that the basidiocarps of T. s c l e r o t i o i d e s are negatively geotropic, but he presented no evidence. Blue and white l i g h t were the most e f f e c t i v e i n e l i c i t i n g the phototropic response. The growth adjustment of a basidio-carp toward a new l i g h t source occurred i n the zone of elonga-tion, below the head. It was not determined where the l i g h t stimulus was received. It was not conclusively demonstrated that l i g h t was required f o r basidiocarp i n i t i a t i o n . Light was necessary f o r the formation of the head and hymenium on basidiocarps a r i s i n g from the mycelium. However, s c l e r o t i a grown i n conditions,of l i g h t were sometimes able to produce f e r t i l e basidiocarps when germinated i n darkness. Thus i t seems that the l i g h t stimulus fo r head development might be stored i n the sclerotium u n t i l an emergent basidiocarp had reached stage I I . It was previously noted that sclerotium germination and basidiocarp development were independent events. The development of the T. erythropus basidiocarp was a r b i t r a r i l y divided into four stages of primordium, headless sporophore, head formation, and sporulation. A s i m i l a r -79-approach using four stages has been followed i n studies of carpophore development i n Agaricus bisporus (Lange) Imbach (Bonner et a l , 1956; Gruen, I963). Komagata and Okunishi (I969) recognized seven stages i n development of carpophores of Coprinus kimurae. In I887, deBary recognized that the growth zone i n sporo-phores of T. v a r i a b i l i s was limi t e d to the apex. Moreover, he believed that c e l l d i v i s i o n continued i n the growing t i p u n t i l the basidiocarp had attained i t s f u l l length. He also noted that no further augmentation occurred at the base of the sporophore. MacDonald (±93^) commented that growth was a p i c a l i n the sporophore of T. s c l e r o t i o i d e s . Corner (1950) stated, i n reference to Typhula sporophores, that, "... api c a l growth may be arrested very early and i n f l a -t i o n may be so prolonged that the fruit-bodies appear to emerge as though from a button-stage, as i n t y p i c a l agarics, but devel-opment i s never r e a l l y i n d i r e c t with a period of api c a l growth followed by a separate period of expansion." This would i n d i -cate that the period of c e l l i n f l a t i o n overlaps part of the period of c e l l d i v i s i o n . Further, i n f l a t i o n continues a f t e r c e l l d i v i s i o n has ceased. What Corner said i s true f o r T. erythropus, but he misjudged the nature and the duration of the overlap of d i v i s i o n and i n f l a t i o n . The stage I primordium i s b u i l t up by c e l l formation and s l i g h t i n f l a t i o n . When the basidiocarp elongates rapidly during stages II-IV, i t i s as a r e s u l t of i n f l a t i o n . However, new c e l l s are formed at a low rate i n the head region. These new c e l l s produce basidia on the slowly growing head. Basidio-- 8 0 -carp elongation and basidium formation are concurrent events that cease at approximately the same time. There appears to be no s i g n i f i c a n t difference between the development of the T. erythropus sporophore and that of an agaric sporophore. Bonner et a l ( 1 9 5 6 ) marked carpophores of Agaricus bisporus with dots of carmine and noted the region of basidiocarp expansion and the movement of this zone up the stipe of a developing basidiocarp. They found, as had Bul l e r ( 1 9 2 4 ) and Borriss (193*+)» that maximum elongation occurs i n the upper part of the stipe, below the cap. This region i s responsible f o r the great increase i n the height of a carpo-phore. After the 2 cm button stage, no new c e l l s are formed i n the stipe, and c e l l i n f l a t i o n accounts for basidiocarp expan-sion. The expansion of the agaric pileus i s independent of stipe elongation. In stage I T. erythropus basidiocarps, the region 0 . 2 -0.5 mm below the t i p i s the growth zone. As this zone moves upward, i t increases to a 1-2 mm long region traveling away from the previously expanded lower part of the stalk. Head formation and expansion are independent of stalk growth. In both agaric and Typhula f r u c t i f i c a t i o n s , the length of the growth zone (zone of expansion) i s inversely proportional to the height of the basidiocarp. A smaller percentage of the length of a t a l l , older sporophore i s expanding than that of a short, younger sporophore. No new c e l l formation occurs at the base of either type of basidiocarp. The time from i n i t i a t i o n to maturity of basidiocarps i s not known for most species of Typhula. Corner ( 1 9 5 0 ) recorded a time of 1 5 - 2 0 days f o r T. v a r i a b i l i s , and MacDonald ( 1 9 3 4 ) - 8 1 -reported 1-4 days fo r growth to f u l l size of T. s c l e r o t l o i d e s . This time i n t e r v a l i n T. erythropus i s dependent upon incubation temperature. At 4 and 10 G, a sporulating basidiocarp i s present 5-6 days a f t e r the appearance of stage I. Growth to f u l l size requires another 3-5 days. At 15 C, sporulation i s frequently i n h i b i t e d ; sporophores do not exceed stage II i n t h e i r development. Basidiocarp production was not so subject to the i n t e r -action between incubation temperature and C/N r a t i o s that influenced sclerotium formation. At temperatures below 15 C, sporophores seldom were produced from the colony surface. Similar responses to incubation temperature were observed i n a l l sporophores, regardless of t h e i r o r i g i n ( i . e . , s c l e r o t i a , colony surface, or inoculum plug). The optimum pH f o r basidiocarp formation from the colony surface was 6.0. This contrasts with the lower pH optima f o r mycelial growth and sclerotium formation. The int e r n a l pH of mature s c l e r o t i a i s near 7.0. The optimum C/N r a t i o f o r basidiocarp production was 19:1. Madelin reported maximum sporocarp formation from cultures of Coprinus lagopus grown on a medium with a C/N r a t i o of 15=1 when glucose and alanine were the main constituents (Madelin, 1956). The relevance of a low C/N r a t i o f o r f r u i t i n g from the colony i n T. erythropus to f r u i t i n g from s c l e r o t i a on maple petioles i n the f i e l d i s not e n t i r e l y clear. Brock (I966) gives the C/N r a t i o of "microorganisms" as approximately 10:1. Since the Typhula sclerotium i s r i c h i n stored glycogen and fat s (Scurti and Converso, I965K the C/N of a sclerotium i s - 8 2 -probably higher than 1 0 : 1 . This would agree with data from culture. S c l e r o t i a contained s u f f i c i e n t reserve materials to support the production of up to eight basidiocarps without an exogenous nutrient supply. Maximum sporophore formation from the medium occurred on a 1/10 strength medium. This response i s i n accord with the usual d i s t i n c t i o n s noted between vegetative and reproductive growth (Cochrane, I958) . Reproduction i n T. erythropus i s favored by a weak medium, and vegetative growth, including sclerotium formation, i s favored by a r i c h medium. VII. Interaction of Nutritional/Environmental Factors As results from experiments with T. erythropus and other fungi have shown, a single ingredient of the medium does not operate independently of the other ingredients, t h e i r propor-tions, environmental conditions, or the phase of growth of the organism. The effects of temperature, pH, the molecular con-f i g u r a t i o n and concentration of carbon and nitrogen sources, the C/N r a t i o , vitamins, s a l t s , and fungal response are complexly i n t e r r e l a t e d . In figure 36 I have attempted to depict these relationships graphically. Manipulation of one factor changes the optimum l e v e l of the other 6 factors. The p a r t i c u l a r fungal structure c i t e d i n the chart may be the mycelium, the sclerotium, or the basidiocarp i n any stage of i t s development. Temperature appears to be the most important single factor. Although a d e f i n i t e c o r r e l a t i o n between temperature and the optimum C/N r a t i o f o r sclerotium production was noted, this r e l a t i o n s h i p i s probably of l i t t l e importance i n the f i e l d . Cultures on high or low C/N r a t i o media produced only s c l e r o t i a - 8 3 -FACING PLATE 22 The Interrelationship of N u t r i t i o n a l and Environmental Factors and Fungal Development Figure J>6: A change i n any of the 7 factors w i l l i n f l u -ence the uptake of certain substances, the optimum concentration of certain substances, or the morphological response of the fungus. Each factor i n the hexagon aff e c t s and i s affected by the others, and a l l may operate as l i m i t i n g factors i n the development of a p a r t i c u l a r fungal structure. particular fungal structure Figure 3 6 -84-when incubated at temperatures prevalent during sclerotium formation i n nature. The uptake of carbon and nitrogen compounds by certain fungi has been found to be affected by temperature (Burnett, I 9 6 8 ) . It i s possible that the e f f e c t i v e C/N r a t i o available to the hyphae of T. erythropus when grown on Medium A at 15 C i s d i f f e r e n t than that i n cultures incubated at 4 or 10 C. The f a i l u r e of t y p i c a l s c l e r o t i a to form on Medium A at 15 C could have been the r e s u l t of several factors. The pH was seen to be very important i n sclerotium formation at 15 C. Cultures grown on media with a pH of 4 or 5 did form some s c l e r o t i a . The uptake of organic and inorganic nitrogen com-pounds by certain fungi i s dependent upon pH and the concentra-tion of the compounds (Fries, 1956; Jones, I 9 6 3 ; Nicholas, I 9 6 6 ) . Amino acid synthesis i n some fungi has been reported to be a l i m i t i n g factor at higher temperatures (Deverall, 1966). Certain compounds are not as readily u t i l i z e d as others. Thus, a C/N r a t i o of 40:1-50:1 ci t e d for l i t t e r (Brock, 1966) cannot t e l l us very much of the s u i t a b i l i t y of a substrate f o r a p a r t i c u l a r fungus. Much of the C or N may be i n forms unavail-able to the organism. Inorganic sa l t s and vitamins have been reported to be l i m i t i n g factors under certain conditions of growth (Cochrane, 1958; Casselton, I 9 6 6 ) . The i n t e r a c t i o n of environmental and n u t r i t i o n a l factors must be examined i n a two-way relat i o n s h i p with the p a r t i c u l a r stage i n the l i f e cycle of an organism. Hawker (1957) and Cochrane (1958) reported that the n u t r i t i o n a l requirements and optimum physical conditions often d i f f e r i n - 8 5 -i n d i f f e r e n t stages of development of the same species. VIII. Taxonomic Aspects and Implications The small size and s i m p l i c i t y of the Typhula f r u c t i f i c a t i o n makes morphological c h a r a c t e r i s t i c s of taxonomic value at a premium. Corner ( 1 9 5 0 , 1 9 7 0 ) uses head size and shape, badidiospore dimensions, stalk length and width, coloration, substrate, number of sporophores per sclerotium, and sclerotium structure as major taxonomic c r i t e r i a i n distinguishing species. The v a l i d i t y of s c l e r o t i a l characters for taxonomic perposes was negated by Rj^ed (I969) when he successfully crossed T. i n -carnata with T. graminum. Corner ( 1 9 5 0 , 1 9 7 0 ) had placed these two species i n d i f f e r e n t sub-genera on s c l e r o t i a l differences. The f a i l u r e of successful mating between the monokaryotic is o l a t e s of T. erythropus and T. s c l e r o t i o i d e s helped to define the l i m i t s of both species. Corner ( 1 9 5 0 ) stressed that, "But for the red-brown color of the stem and sclerotium i t jjT. eryth-ropus j would hardly be distinguishable from T. s c l e r o t i o i d e s . " Although the f r u c t i f i c a t i o n s are similar, the s c l e r o t i a l struc-ture of the two species i s quite d i f f e r e n t . The s c l e r o t i a l hyphae of T. s c l e r o t i o i d e s are thick-walled and those of T. erythropus are thin-walled. Also, the surface pattern and thickness of the cortex d i f f e r . Cultural data from the present study showed that the length of the head and stalk was not constant but increased with age. While a sporulating basidiocarp grew from 6.3 to 2 5 mm, the head length increased from 1.2 to 4.0 mm. Color-ation and stalk proportions were r e l a t i v e l y constant i n basidio-carps produced from s c l e r o t i a . However, when sclerotium-free sporophores were formed on a medium with a low C/N r a t i o , the -86-stalks were thick and short and were a p l a i n brown color. The number of basidiocarps per sclerotium was also variable. Although Corner (1950) gives the number f o r T. erythropus s c l e r o t i a as one or rarely two to three, up to 8 sporophores were produced from cultured s c l e r o t i a . S c l e r o t i a that gave r i s e to this number of basidiocarps were no larger than those from f i e l d c o l l e c t i o n s . Basidiospore size from cultured sporophores of T. eryth-ropus varied from 6-8 JJL X 2-3.6 )i. Ekstrand (1955) has placed considerable importance on the length/width r a t i o of basidio-spores of two species he described, and, i n f a c t , the species were distinguished by this single character. W. C. MacDonald ( I96I) did not agree and relegated both to synonomy with T. idahoensis, a species described by Remsberg. Species i n this genus can be divided into a large-spored group and a small-spored group; T. erythropus i s i n the l a t t e r . Although J . A. MacDonald (193*+) thought that, "spore size may turn out to be the only stable character i n the group," the v a r i a t i o n i n certain species i s so great that this prediction i s u n l i k e l y . A single basidiocarp of T. phacorrhiza produced spores measur-ing from 11-20^ x k.7-?.5p (Corner, 1950). The length/width r a t i o varied from 1.6 to 2.9. The nature of the substrate i n i d e n t i f y i n g saprophytic species might be more the r e s u l t of l i m i t e d c o l l e c t i o n data than an i n d i c a t i o n of substrate s p e c i a l i z a t i o n (Corner, 1950). Thus, nearly a l l currently used taxonomic c r i t e r i a i n this genus are quite variable. U n t i l crosses are made between many species, we cannot know the l i m i t s of morphological var-- 8 7 -i a t i o n within a single species. 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The u t i l i z a t i o n of nitrogen compounds, especially ammonia, by a low temperature basidiomycete. Can. J . Bot., 42:1071-1086. (67) . I966. Preliminary studies on the physiology of S c l e r o t i n i a borealis, a highly psychrophilic fungus. Can. J . Bot., 44:237-246. (68) Wheeler, B. E. J . and J . M. Waller. I965. The production of s c l e r o t i a by Sclerotium r o l f s i i , I I . the r e l a t i o n s h i p between mycelial growth and i n i t i a t i o n of s c l e r o t i a . Trans. B r i t . Mycol. S o c , 48:303-314. - 9 3 -APPENDIX A Colle c t i o n Data ( 1 ) Typhula erythropus Fr i e s (a) i s o l a t e T-4; UBC culture c o l l e c t i o n # 5OI9 Location: U.B.C. campus, Vancouver, B.C. Date: 30 September, I969 Habitat: on petioles of Acer macrophyllum (b) i s o l a t e s T-lo^ and T-18 2; UBC culture # 5 0 2 0 , 5 0 2 1 Location: U.B.C. campus, Vancouver, B.C. Date: 1 5 October, I97O Habitat: on petioles of Acer macrophyllum (c) i s o l a t e T-29; UBC culture # 5 0 2 2 Location: U.B.C. campus, Vancouver, B.C. Date: 12 November, 1 9 7 0 Habitat: on petioles of Acer macrophyllum ( 2 ) Typhula s c l e r o t i o i d e s (Pers.) Fries (a) i s o l a t e T-19; UBC culture # 5 0 2 3 Location: U.B.C. campus, Vancouver, B.C. Date: 1 3 October, I97O Habitat: on petioles of Acer macrophyllum Dried specimens of a l l c o l l e c t i o n s have been deposited i n the U.B.C. mycological herbarium. -94-A P P E N D I X B Culture Media Medium A substance amount glucose 10.0 g asparagine 2.0 g KH2P0i). 1.0 g MgS0^-?H20 0 . 5 g vi t a m i n stock s o l n . 1.0 ml micro-elements stock s o l n . 2.0 ml agar (K&S brand, high g e l strength) 12.0 g, d i s t i l l e d water 1.0 1 vi t a m i n stock s o l u t i o n thiamine 50 mg pyrid o x i n e 0 , 5 mg i n o s i t o l 2 5 mg b i o t i n 25 ug Diss o l v e i n 5°0 ml of 20$ ethanol, store i n r e f r i g e r a t o r . m i c r o e s s e n t i a l elements stock s o l u t i o n Fe(N0 3 ) 3'9H 20 181 mg ZnSO^*7H20 110 mg MnS0^.'4H20 51 mg Diss o l v e i n 150 ml d i s t i l l e d water. Add IN H2S0i|, u n t i l s o l u t i o n becomes c o l o r l e s s . Then add 110 mg CuS04'5H20 and 100 ml d i s t . water. Store i n r e f r i g e r a t o r . -95-Medium B Same as Medium A except asparagine concentration i s 0.2 g/1 instead of 2.0 g/1. Medium C Make up Medium A lacking asparagine. Pour 20 ml of hot s t e r i l e medium into 90 mm p e t r i plates containing 0.12-0.17 g of autoclaved wheat germ flakes (Rockhill "brand, Wild Rose M i l l s , Vancouver, B. C ) . Malt extract-yeast extract-peptone agar (MYP) substance amount malt extract (Difco) 7.5 g yeast extract (Difco) 0.5 g peptone 1.0 g agar (K&S brand) 12.0 g d i s t i l l e d water 1.0 1 Water Agar 1.2$ K&S brand high gel strength agar i n d i s t i l l e d water. 

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