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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 P o l y t e c h n i c C o l l e g e ,  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 required  t h i s t h e s i s as conforming to the 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 by his representatives.  be granted by the Head of my Department or  It is understood that copying or publication  of this thesis for financial gain shall not be allowed without my written permission.  Department of  BQTANJ^  The University of British Columbia Vancouver 8. Canada  Date  L  JVJU/  1^1  i ABSTRACT The  life  c y c l e of Typhula  f i e l d and. i n c u l t u r e . v a t i o n s from both and in  erythropus  An attempt was  was  s t u d i e d i n the  made to c o r r e l a t e  to e x p l a i n the behavior  of t h i s organism  nature. The  e f f e c t of temperature, pH,  various  carbon:nitrogen  r a t i o s , and. n u t r i e n t c o n c e n t r a t i o n on m y c e l i a l growth, tium formation, and b a s i d i o c a r p p r o d u c t i o n was  favored  temperatures above 10 C, h i g h s t r e n g t h media, and a pH  4-5.  B a s i d i o c a r p p r o d u c t i o n was  on low  s t r e n g t h media, a t pH  The and  sclero-  examined.  V e g e t a t i v e growth, i n c l u d i n g s c l e r o t l u m formation, was by  obser-  maximal a t lower  6, and a t a low  C/N  temperatures, ratio.  c o n d i t i o n s f o r s c l e r o t i u m germination were determined,  s c l e r o t i u m v i a b i l i t y was  investigated.  S c l e r o t i a produced  a t 4 C d i d not germinate u n t i l exposed to 15-20.C. a c t i v a t e d s c l e r o t i a and  These heat  s c l e r o t i a grown to m a t u r i t y a t 15 C  germinated r a p i d l y when incubated a t k C on water agar. low germination 15  A  r a t e r e s u l t e d when s c l e r o t i a were incubated a t  C. The  apical.  growth zone of T. erythropus Expansion  same manner  of the Typhula  b a s i d i o c a r p s i s sub-  f r u c t i f i c a t i o n occurs i n the  as that i n the common mushroom, A g a r i c u s b i s p o r u s .  A complex r e l a t i o n s h i p between the p a r t i c u l a r stage development of the organism and The  of  i n t e r a c t i o n of environmental  e s p e c i a l l y obvious T. erythropus  c u l t u r a l c o n d i t i o n s was  of evident.  and n u t r i t i o n a l f a c t o r s  was  during sclerotium formation i n c u l t u r e . appears to be f a c u l t a t i v e l y h o m o t h a l l i c .  the absence of a compatible i s o l a t e s became d i k a r y o t i c .  In  mating s t r a i n , a l l nine monokaryotic However, when c r o s s e d i n a l l  combinations, the monosporic i s o l a t e s e x h i b i t e d t y p i c a l polar heterothallism.  tetra-  This ambivalence i n mating i s of r a r e  occurrence i n the f u n g i . 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 c h a r a c t e r s as b a s i d i o s p o r e measurements, b a s i d i o c a r p dimensions, c o l o r a t i o n , s u b s t r a t e , number of b a s i d i o c a r p s s c l e r o t i u m , and  s c l e r o t i a l anatomy.  f e a t u r e s under v a r i o u s  were examined i n c u l t u r e , and  constancy of c e r t a i n taxonomically noted.  ology,  and  s t a b i l i t y of these  c o n d i t i o n s has never been t e s t e d .  i s o l a t e s of T. erythropus  was  The  Basidiospore  Four the  important c h a r a c t e r i s t i c s  measurements, s c l e r o t i a l micro-morph-  the c o l o r a t i o n of b a s i d i o c a r p s a r i s i n g from  were s t a b l e f e a t u r e s i n a l l i s o l a t e s . were v a r i a b l e .  per  sclerotia  Other c h a r a c t e r i s t i c s  iii TABLE OF CONTENTS p a g e  Introduction  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)  C u l t u r e Techniques and Growth C o n d i t i o n s  7  (3)  Assessment o f Growth and R e p r o d u c t i o n  9  (4)  Methods of I n o c u l a t i o n  9  (5)  Monokaryons and M a t i n g  (6)  S t a i n i n g Techniques  ,  R e s u l t s and O b s e r v a t i o n s I. II. III. IV.  V.  VI.  10 10 11  L i f e Cycle i n Nature  11  Basidiospore Germination  12  Growth o f M o n o k a r y o t i c Mycelium  12  M a t i n g s of Monokaryons...  16  A.  Intraspecific Pairings  16  B.  Interspecific Pairings  1?  Growth o f 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.  V i t a m i n Requirements  2k  Sclerotium Formation....  2k  A.  Morphology o f Mature S c l e r o t i a  2k  B.  Development  25  C.  Time of F o r m a t i o n  27  •Iv TABLE OF CONTENTS (Cont'd.) P  D.  VII.  S e  28  E. pH E f f e c t s  32  F. Carbon:Nitrogen R a t i o E f f e c t s  34  G. N u t r i e n t C o n c e n t r a t i o n E f f e c t s  34  H. N i t r o g e n 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  Sclerotium Germination  44  A. G e n e r a l O b s e r v a t i o n s  44  B. Temperature E f f e c t s . . . . .  45  C.  VIII.  Temperature E f f e c t s  a  (1)  Temperature D u r i n g G e r m i n a t i o n  45  (2)  Temperature D u r i n g P r o d u c t i o n  46  (3)  Freezing of S c l e r o t i a  48  Other P h y s i c a l F a c t o r s . . . -  48  (1)  Drying of S c l e r o t i a  48  (2)  Washing o f S c l e r o t i a  48  (3)  S c l e r o t i u m 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  B a s i d i o c a r p F o r m a t i o n and Growth  50  A.  General Observations  50  B.  Morphology o f the Mature B a s i d i o c a r p  5  C.  B a s i d i o c a r p Development  52  D.  Expansion o f 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  1  V  TABLE OF CONTENTS (Cont'd.)  Photo E f f e c t s  H.  Gravity Effects  61  I.  Carbon:Nitrogen R a t i o E f f e c t s  61  J.  Nutrient Concentration  K.  Nitrogen U t i l i z a t i o n  64  L.  Vitamin Requirements  64  Discussion I. II. III. IV. V. VI. VII. VIII.  59  G.  Effects  ..  62  66  Considerations  66  Mating System and Monokaryons  66  M y c e l i a l Growth, D i k a r y o t i c  69  S c l e r o t i u m Development....  70  S c l e r o t i u m Germination  7^  B a s i d i o c a r p Growth and Development  77  I n t e r a c t i o n of Nutritional/Environmental Factors  82  Taxonomic Aspects and I m p l i c a t i o n s  85  General  88  Bibliography Appendix A:  C o 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:  C u l t u r e Media  9^  (1)  Medium A  94  (2)  Medium B  95  (3)  Medium C  95  (4)  Malt-Yeast-Peptone (MYP)  95  (5)  Water Agar  95  vi  LIST OF TABLES  Table I:  The E f f e c t of I n c u b a t i o n Temperature on S c l e r o t i u m Germination.  Table I I : D u r a t i o n of the Stages of B a s i d i o c a r p Development  58  vii FIGURES AND ILLUSTRATIONS Page  Plate  1:  L i f e C y c l e o f T. e r y t h r o p u s  Plate  2:  E f f e c t of Temperature on B a s i d i o s p o r e G e r m i n a t i o n  14  Plate  3:  P a i r i n g of Monokaryotic I s o l a t e s  18  Plate  4:  Growth o f the Colony on Medium A  20  Plate  5:  Environmental  23  Plate  6:  R e l a t i o n s h i p o f S c l e r o t i u m Development and  i n Nature....  E f f e c t s on M y c e l i a l Growth  13  29  M y c e l i a l Growth Plate  7:  E f f e c t o f Temperature on S c l e r o t i u m Development.. 31  Plate  8:  E f f e c t o f pH on S c l e r o t i u m Development  33  Plate  9:  E f f e c t o f C/N on S c l e r o t i u m F o r m a t i o n  35  Plate 10:  S c l e r o t i u m Development  36  Plate 11:  E f f e c t of Medium S t r e n g t h on S c l e r o t i u m Formation  38  Plate 12:  Nitogen  40  Plate 13:  R e l a t i o n s h i p o f S c l e r o t i u m Development and  Sources f o r S c l e r o t i u m F o r m a t i o n . . . .  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 S c l e r o t i u m F o r m a t i o n  43  Plate 15:  E f f e c t s of I n c u b a t i o n Temperature on the 47  Rate o f S c l e r o t i u m G e r m i n a t i o n P l a t e 16:  Growth o f the B a s i d i o c a r p , 1 . . . . .  55  Plate 17:  Growth o f 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 o f Temperature on B a s i d i o c a r p Development  •  58  Plate 19:  E f f e c t o f pH on B a s i d i o c a r p P r o d u c t i o n  60  Plate 20:  E f f e c t o f C/N on B a s i d i o c a r p P r o d u c t i o n  63  Plate 21:  B a s i d i o c a r p Development  65  V i l l  FIGURES AND ILLUSTRATIONS  (Cont'd.) Page  P l a t e 22:  I n t e r r e l a t i o n s h i p of N u t r i t i o n a l and E n v i r o n mental F a c t o r s and Fungal Development  83  ix  ACKNOWLEDGMENT I wish to thank Dr. R. J . Bandoni f o r support and g u i d ance d u r i n g the p e r i o d o f my r e s e a r c h and i n the p r e p a r a t i o n of t h i s t h e s i s .  For t h e i r comments and suggestions  the w r i t i n g o f t h i s manuscript  during  I thank Dr. G.C. Hughes, Dr.  I.E.P. T a y l o r , and the graduate students of the mycology l a b . S p e c i a l thanks a r e extended to Dr. B.N. J o h r i f o r h i s a s s i s t ance i n p r e p a r i n g the photographic comments.  p l a t e s and f o r h i s h e l p f u l  F i n a n c i a l support was provided by a U.B.C. graduate  f e l l o w s h i p , a r e s e a r c h f e l l o w s h i p , and t e a c h i n g a s s i s t a n t s h i p s from the department o f botany.  INTRODUCTION The  genus Typhula was e s t a b l i s h e d by F r i e s  (1821)  i n c l u d e c l a v a r i o i d s p e c i e s with a c y l i n d r i c a l f e r t i l e  to clavule  and a d i s t i n c t s t e r i l e s t a l k . F r i e s placed t h i s genus i n the order C l a v a t i of the Hymenomycetes. the genus to r e t a i n only those from s c l e r o t i a . Karsten s !  oid  (1882)  Karsten  emended  s p e c i e s with b a s i d i o c a r p s  Remsberg (19^0) and Corner  (1950)  accepted  d e f i n i t i o n and p l a c e d a l l s c l e r o t i u m - f o r m i n g  f u n g i i n Typhula.  Clavariadelphaceae Pistillina,  arising  clavari-  In 1970, Corner proposed the f a m i l y  to i n c l u d e Typhula, Chaetotyphula,  Pistillaria,  Myxomycidium, Araecoryne, C e r a t e l l o p s i s , and C l a v a r -  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 , f r e e Typhula (Corner,  1950),  was questioned  ( I 9 7 I ) who found s c l e r o t i a i n the l i f e s e t i p e s Grev. the  a sclerotium-  by Koske and P e r r i n  cycle of P i s t i l l a r i a  Corner (I97O) r e p o r t e d a s c l e r o t i u m present i n  "Typhula-state"  of P. p e t a s i t i d i s Imai.  In a d d i t i o n , a  s c l e r o t i u m was found i n P t e r u l a s c l e r o t i c o l a B e r t h i e r ( B e r t h i e r , I967),  a s p e c i e s p l a c e d i n a d i f f e r e n t f a m i l y 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  difficult.  Such c h a r a c t e r i s t i c s as spore s i z e , b a s i d i o c a r p dimensions, c o l o r a t i o n , s u b s t r a t e , and s c l e r o t i u m anatomy a r e used as major c r i t e r i a i n d i s t i n g u i s h i n g species i s evidence  1950, 1970).  There  t h a t s c l e r o t i u m anatomy, a t l e a s t , may be a u s e l e s s  c h a r a c t e r f o r t h i s purpose (R^ed, review).  (Corner,  I969  a  n d see l i t e r a t u r e  The s t a b i l i t y o f these morphological  f e a t u r e s under  v a r i o u s environmental c o n d i t i o n s has not been t e s t e d . The aim of the present  i n v e s t i g a t i o n was t o determine the  -2e f f e c t s of environmental and n u t r i t i o n a l f a c t o r s on the developmental morphology of d i f f e r e n t phases i n the l i f e of  T. erythropus F r .  I t was  cycle  f u r t h e r hoped that a d e t a i l e d  study of a w e l l d e f i n e d s p e c i e s might p r o v i d e i n s i g h t as to which m o r p h o l o g i c a l f e a t u r e s are s t a b l e and t a x o n o m i c a l l y u s e f u l i n t h i s genus. LITERATURE REVIEW Much of the l i t e r a t u r e d e a l i n g with Typhula concerns the symptoms and c o n t r o l of d i s e a s e s caused by pathogenic s p e c i e s , but some c u l t u r a l s t u d i e s have been r e p o r t e d .  In I 8 8 7 ,  deBary  d e s c r i b e d s c l e r o t i u m f o r m a t i o n i n T. v a r i a b i l i s R i e s s and p h a c o r r h i z a F r . and b a s i d i o c a r p development of  T.  i n the former.  Part  my work concerned the examination of these processes i n  another s p e c i e s . Although T. erythropus i s the most common Typhula i n Europe  (Corner, I 9 5 O ) , i t has been s t u d i e d l i t t l e  Lehfeldt  i n culture.  (I923) germinated spores of t h i s s p e c i e s on malt  e x t r a c t agar.  He f o l l o w e d the movement of n u c l e i d u r i n g d i -  k a r y o t i z a t i o n , demonstrated psychrophily.  Tasugi ( I 9 2 9 ,  h e t e r o t h a l l i s m , and remarked 1 9 3 5 ) examined T. i n c a r n a t a  Lasch ex F r . (as T. graminum K a r s t . ) and found that UV was  necessary f o r the development  temperature range f o r growth was between 8-15 C.  on i t s  of f e r t i l e  light  sporophores.  The  5-23 C, with an optimum  Maximum growth o c c u r r e d a t pH  7.  MacDonald ( 1 9 3 ^ ) r e p o r t e d no s p e c i a l l i g h t requirement f o r sporophore development  i n T. s c l e r o t i o i d e s  (Pers.) F r . which  m i s i d e n t i f i e d as T. gyrans F r . (Corner, 1 9 5 0 ) . arose from c o l o n i e s on potato dextrose agar s c l e r o t i a incubated on moist s o i l .  he  Basidiocarps  (PDA) and from  The fungus grew a t  tempera-  -3t u r e s from 0-25  C.  The  optimum temperatures f o r m y c e l i a l  growth, s c l e r o t i u m formation, 15-17, 17-20, and development and  13-15  and  basidiocarp production  C, r e s p e c t i v e l y .  were  D e t a i l s of s c l e r o t i u m  b a s i d i o c a r p anatomy were a l s o presented.  Donald concluded that sporophore growth was  a p i c a l and  b a s i d i o c a r p s were p o s i t i v e l y p h o t o t r o p i c and  Mac-  that  n e g a t i v e l y geo-  tropic. Nuclear m i g r a t i o n c l o v e r , was  s t u d i e d by Noble (1937).  morphological mycelia,  i n T. t r i f o l i i  Rostr., She  a pathogen of  also described  the  c h a r a c t e r i s t i c s of the h a p l o i d and d i k a r y o t i c  clamp formation,  and  the development of b a s i d i o c a r p s .  S c l e r o t i a germinated on moist s o i l a f t e r an undetermined dormancy p e r i o d .  H e t e r o t h a l l i s m was  demonstrated, but only  monosporic i s o l a t e s were made, and the s p e c i e s was  she  five  could not determine i f  b i p o l a r or t e t r a p o l a r .  A major c o n t i b u t i o n to our knowledge of Typhula was by Remsberg (1940) when she can s p e c i e s .  She  c u l t u r e d and  s t u d i e d 1 4 North Ameri-  determined the optimum temperature f o r  m y c e l i a l growth of each s p e c i e s , noted g e n e r a l t e r i s t i c s , and ment.  She  v e r i f i e d Tasugi's  1 4 s p e c i e s grew a t 0 C, and v a r i e d from 6-12  on the UV  characrequire-  C.  of f e r t i l e f r u c t i f i c a t i o n s .  was  All  the optimum temperatures f o r growth  S t r e s s i n g s c l e r o t i u m morphology, she  to the s p e c i e s i n her study.  g i v e n f o r the 1 4 s p e c i e s , and was  observations  cultural  found that l i g h t of a wavelength of 265O-3250 &  necessary f o r the p r o d u c t i o n  a key  made  devised  Complete d e s c r i p t i o n s were  an h i s t o r i c a l account of the genus  presented. The  age  of the host p l a n t was  shown to determine r e s i s t a n c e  to Typhula snow b l i g h t by Tomiyama (1952).  He a l s o noted  the  mutual antagonism between mycelia o f T. i n c a r n a t a Imai) and T. i s h i k a r i e n s i s Imai.  (as T. i t o a n a  In 1955. he c u l t u r e d both  s p e c i e s and r e p o r t e d that spore germination  o f T. i n c a r n a t a  occurred a t 0 C and that maximum growth i n t h i s s p e c i e s was made a t 7-15 C. Ekstrand  (1955) determined the optimum temperature f o r  m y c e l i a l growth of T. b o r e a l i s Ekstrand borea Ekstrand  (10 C) and T. hyper-  (5 C ) . Both s p e c i e s a r e pathogens o f winter  c e r e a l s ; W. C. MacDonald ( I 9 6 I )  c o n s i d e r s them synonyms o f  T. idahoensis Remsb. In i960, Potatosova t y p h u l o s i s i n the USSR.  p u b l i s h e d three papers on Typhula and In the f i r s t  study  (1960a) she s u r f a c e  s t e r i l i z e d s c l e r o t i a of T. i n c a r n a t a (as T. i t o a n a ) , T. v a r i a b ilis,  T. i d a h o e n s i s , and T. t r i f o l i i  sand.  The pots of sand were covered  doors. and  F e r t i l e b a s i d i o c a r p s arose  T. t r i f o l i i  and b u r i e d them i n moist with g l a s s and p l a c e d  out-  from s c l e r o t i a of T. v a r i a b i l i s  when the o u t s i d e temperature dropped to 8-15 C.  Sporophores o f T. i n c a r n a t a were produced a t 1.4-13.5 C and o f T. i d a h o e n s i s a t 1.4-4.6 C. s p e c i e s remained s t e r i l e .  B a s i d i o c a r p s i n the l a t t e r two  Maximum germination  r e s u l t e d from  s c l e r o t i a b u r i e d a t a depth o f 5 nun; below 10 mm no occurred.  germination  Her second paper (1960b) r e p o r t e d the range and  optimum temperature f o r growth of T. i n c a r n a t a (0-18 C; o p t . 10 C) and  T . idahoensis :  (0-16 C; o p t . 10 C ) . She found t h a t the d e v e l -  opment o f s c l e r o t i a was s t i m u l a t e d by d i f f u s e l i g h t but i n h i b i t e d by d i r e c t l i g h t .  A d e s c r i p t i o n of the 14 s p e c i e s of Typhula  found i n the USSR was the s u b j e c t of her t h i r d paper In i t she d i s c u s s e d methods of o v e r w i n t e r i n g , otium formation,  (1960c).  p e r i o d s of s c l e r -  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 e f f e c t of l o n g wave UV r a d i a t i o n on s c l e r o t i u m germi n a t i o n and b a s i d i o c a r p f o r m a t i o n was Leach ( I 9 6 2 ) .  investigated b r i e f l y  by  S c l e r o t i a of s e v e r a l undetermined Typhulas  were i r r a d i a t e d w i t h UV a t 12 hours/day f o r 3-10 days.  Sporo-  phores arose from s c l e r o t i a incubated on moist sand. Jackson (I963) r e p o r t e d a snow s c a l d of t u r f caused by H« i n c a r n a t a .  He v e r i f i e d  that b a s i d i o c a r p s were autumnal.  B a s i d i o s p o r e s of t h i s s p e c i e s germinated r e a d i l y on PDA, s c l e r o t i a were produced a f t e r 7 - 1 0 (I965) examined  days i n c u l t u r e .  and  Lehmann  some a s p e c t s of the pathogenic nature, growth,  and r e s p i r a t i o n of T. i n c a r n a t a . The f i r s t s c l e r o t i a was  i n v e s t i g a t i o n of the u l t r a s t r u c t u r e of Typhula performed by S c u r t i and Converso  (I965).  They  d e s c r i b e d types of c e l l s i n the medulla: storage c e l l s with p o l y s a c c h a r i d e s and f a t s and m e t a b o l i c a l l y a c t i v e c e l l s . c e l l s i n the s c l e r o t i a l r i n d  Hyphal  (the outer l a y e r of the c o r t e x ) of  t h i s undetermined s p e c i e s were d e v o i d of c o n t e n t s . Lockhart  (I967) examined another undetermined Typhula  s p e c i e s p a r a s i t i c on strawberry p l a n t s . PDA a t - 1 ,  10,  and 19 C.  ensued, f i l l i n g a 9 0 mm  At 10 C v i g o r o u s m y c e l i a l p e t r i p l a t e i n 11 days.  c o n c e n t r a t i o n of GOg above rate.  He c u l t u r e d i t on growth  I n c r e a s i n g the  0 . 0 5 $ caused a decrease i n the growth  In a C 0 2 ~ f r e e atmosphere,  m y c e l i a l growth was  similarly  retarded. The g e r m i n a t i o n of s c l e r o t i a of T. i d a h o e n s i s on s o i l was  sterile  s t u d i e d by Protosenko (I967) and Huber and McKay  Protosenko found that g e r m i n a t i o n o c c u r r e d i n October. r e p o r t e d t h a t the fungus grew f a s t e r on PDA a t 6-8  (I968).  He a l s o  C than a t  -618-20  C.  Ruber and  or b u r i a l  in soil  McKay o b s e r v e d  had  little  At h i g h e r temperatures germination a b i l i t y Of p a r t i c u l a r Reed's  carnata.  Corner  subgenera  on  and  relegated  parasitic He  suggested  T.  e r y t h r o p u s has  MacDonald T.  that  (I96I)  crossing  that  The that  and  o f T. graminum and  on w i n t e r c e r e a l s i t might  be  c o n s i d e r e d T.  Ward  and  g r o w t h o c c u r r e d a t pH medium a t 5 - 1 0 Sclerotia  i n Japan  pH  Poor  Scandanavia.  e r y t h r o p u s . However, Also,  W.  C.  t o be a synonym o f  species.  investigation  Unfortunately,  matings  of  the  of Typhula  was  ( I 9 7 I ) . They grew T. i n c a r n a t a , T. c u l t u r e s and  f o r growth of each  determined species.  extract-yeast  above  substrates.  and  1 0 C.  o p t i m a l a t 2 0 C, n e a r l y A variety  tri-  the Maximum  extract-glucose  o b s e r v e d a t -5  more a b u n d a n t l y  f o r maximum g r o w t h .  as r e s p i r a t o r y  a  unsolved.  g r o w t h was  i d a h o e n s i s was  temperature  and  interspecific  7 on a m a l t  were p r o d u c e d  t a k e by T.  utilized  C.  ishikariensis,  ishikariensis  i d a h o e n s i s i n agar  optimum t e m p e r a t u r e  sclerotial  incarnata.  t h e same a s T.  remains  T. i n -  interfertile  not been r e p o r t e d as a p a r a s i t e .  the problem  T.  o f T.  was  in different  dissimilar  t o synonomy w i t h T.  most r e c e n t c u l t u r a l  and  species  distinctly  examined s p e c i m e n s  o f D e j a r d i n and  folii,  viability.  t h e y were c o m p l e t e l y  i n v e s t i g a t o r attempted  species,  ( 1 - 2 C)  taxonomy o f t h e genus  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  neither  the  to the  of t h e i r  the former  species  sclerotium  p l a c e d t h e s e two  found  (I97O)  on  temperatures  noted.  importance  (1970)  R^ed  Corner  were  the b a s i s  morphology.  effect  low  (24 C ) , however, marked r e d u c t i o n s i n  interspecific  (I969)  that  15°  20  C.  Oxygen  up-  higher  o f s u g a r s were  than  -7MATERIALS AND (1)  I s o l a t e s Employed Four i s o l a t e s of T. erythropus  and  METHODS  the d e t a i l e d i n v e s t i g a t i o n was  them ( i s o l a t e T - 4 ) .  were examined i n c u l t u r e c a r r i e d out with one  of  C u l t u r e s were obtained from b a s i d i o s p o r e s  r e l e a s e d by mature b a s i d i o c a r p s .  C o l l e c t i o n data are g i v e n i n  Appendix A. The  c u l t u r e 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  shed from mature b a s i d i o c a r p s .  obtained from b a s i d i o s p o r e s C o l l e c t i o n data f o r t h i s  species  are a l s o g i v e n i n Appendix A. (2)  C u l t u r e Techniques and Growth C o n d i t i o n s Since a d e f i n e d medium has not been used f o r any  Typhula  except  s t u d i e s of  f o r the r e s p i r a t i o n work of D e j a r d i n and Ward  (1971), comparisons based on e a r l i e r c u l t u r a l data are of questionable value. results, The  For t h i s reason,  T. erythropus  was  and  to ensure r e p r o d u c i b l e  grown on a d e f i n e d medium.  standard medium used was  a m o d i f i c a t i o n of the s y n t h e t i c  b a s a l medium of L i l l y and B a r n e t t  (1951)•  I t s composition  g i v e n i n Appendix B.  This b a s a l medium was  Medium B was  by r e d u c i n g the asparagine  prepared  i n Medium A from 2.0 otium p r o d u c t i o n , asparagine  prepared  g/1.  Medium A.  concentration  Medium C, used f o r s c l e r -  contained f l a k e s of wheat germ i n s t e a d of  i n Medium A.  i n Appendix B. was  g / l to 0.2  designated  D e t a i l s of i t s p r e p a r a t i o n are  given  For b a s i d i o c a r p development s t u d i e s , Medium A  with 1.0  g / l of v i t a m i n f r e e c a s e i n h y d r o l y s a t e  i n p l a c e of asparagine.  Water agar (Appendix B) was r o u t i n e l y  used f o r s c l e r o t i u m germination i n v e s t i g a t i o n s . Stock  is  c u l t u r e s were maintained  on Medium A a t 15  C.  For n i t r o g e n u t i l i z a t i o n s t u d i e s Medium A was prepared w i t h v a r i o u s compounds i n p l a c e of a s p a r a g i n e to g i v e the same conc e n t r a t i o n of N.  The a s p a r a g i n e used was a D i f c o product, and  o t h e r amino a c i d s were s u p p l i e d by N u t r i t i o n a l B i o c h e m i c a l s . A l l other chemicals were o f reagent grade. V i t a m i n requirement s t u d i e s were c a r r i e d out i n dichromate cleaned glassware, and c o n c l u s i o n s were based on data from three serial  transfers.  The i n g r e d i e n t s of a l l media were a u t o c l a v e d together a t 15 l b s . f o r 1 5 minutes.  Twenty mis (±2) were d i s p e n s e d i n t o  90 mm g l a s s or s t e r i l e p l a s t i c p e t r i p l a t e s . was a u t o c l a v e d s e p a r a t e l y and added  When the phosphate  to the remainder o f the  c o o l e d medium, no d i f f e r e n c e was observed i n the growth response of the fungus. The pH of a l l media was a d j u s t e d to 5 . 0 - 5 . ^ w i t h KOH b e f o r e autoclaving.  In p H - e f f e c t s t u d i e s , the pH of the media was  a d j u s t e d w i t h IN KOH or IN ^ S O ^  and checked w i t h a Radiometer  M28 pH meter b e f o r e a u t o c l a v i n g .  A d d i t i o n of bromcresol green  or bromcresol p u r p l e to the medium was u s e f u l i n d e t e c t i n g a pH change f o l l o w i n g  sterilization.  C u l t u r e s were grown a t 4 C (±1), 10 C ( ± 1 ) , 15 C (±2), and 20 C (±1). Those a t 10 and 1 5 C r e c e i v e d 12 hours/day a t i o n a t a d i s t a n c e o f 6-12" from Westinghouse f l u o r e s c e n t lamps.  illumin-  c o o l - w h i t e 20W  At 20 C, 12 hours/day i l l u m i n a t i o n was  p r o v i d e d by overhead f l u o r e s c e n t lamps i n a w a l k - i n i n c u b a t o r . C u l t u r e s a t 4 C were grown i n a r e f r i g e r a t o r and exposed to d i f f u s e overhead f l u o r e s c e n t l i g h t only when the door was open. A minimum of three p l a t e s was used f o r each experiment, and major experiments were repeated a t l e a s t t w i c e .  For sclerotium  -9g e r m i n a t i o n s t u d i e s , 15-50 s c l e r o t i a were used, and were repeated a t l e a s t  experiments  once.  I n v e s t i g a t i o n s of the l i g h t requirement f o r f r u i t i n g were performed the fungus  by wrapping  p e t r i p l a t e s i n aluminum f o i l and a l l o w i n g  to develop i n darkness.  Since l i g h t - g r o w n s c l e r o t i a  and m y c e l i a were a b l e to g i v e r i s e to a t y p i c a l b a s i d i o c a r p s i n darkness, i t was necessary to make s e r i a l t r a n s f e r s from darkgrown c u l t u r e s to e l i m i n a t e p o s s i b l e c a r r y o v e r of f r u i t i n g i n d u c i n g substances. a darkened  The dark t r a n s f e r s were accomplished i n  room by the f a i n t l i g h t of an a l c o h o l lamp p l a c e d  f o u r f e e t away. (3)  Assessment of Growth and Reproduction Observations on s c l e r o t i u m f o r m a t i o n and germination and  b a s i d i o c a r p p r o d u c t i o n were performed from d i k a r y o t i c  only on s t r u c t u r e s d e r i v e d  colonies.  R e l a t i v e growth r a t e s were determined by measuring the i n c r e a s e i n colony diameter. t h i s method was adequate,  F o r temperature  and pH s t u d i e s ,  but t o t a l growth on d i f f e r e n t  gen sources c o u l d not be r e l a t e d to l i n e a r e x t e n s i o n .  nitroRapid and  very sparse growth was a s s o c i a t e d w i t h a n u t r i e n t - p o o r medium. S c l e r o t i u m p r o d u c t i o n and f r u i t i n g a c t i v i t y were r a t e d by the average number of mature s t r u c t u r e s formed  per p e t r i  plate.  Dry weights were o b t a i n e d by f i l t r a t i o n and d r y i n g of the m y c e l i a l mat on pre-weighed (4)  filter  papers a t 50 C f o r 24 hours.  Methods of I n o c u l a t i o n I t was found that the age, source, and manner of a p p l i c a t i o n  of  the inoculum was very important i n the growth response of  c u l t u r e s of T. e r y t h r o p u s .  Small agar b l o c k s 3 mm  2  cut from  c o l o n i e s grown on Medium A a t 15 C f o r 1-2 months were used as  -10the standard inoculum source.  The mycelium growing from these  b l o c k s responded to c u l t u r a l c o n d i t i o n s i n the same manner as an inoculum of b a s i d i o s p o r e s .  I n o c u l a from c o l o n i e s grown on  Medium A f o r l e s s than one month or from other media d i d not produce c o l o n i e s that behaved p r e d i c t a b l y .  The growth response  was u s u a l l y d i f f e r e n t from that of a b a s i d i o s p o r e suspension inoculum.  Growth r a t e , b a s i d i o c a r p development,  and  sclerotium  f o r m a t i o n were e r r a t i c when the standard inoculum was not used. In an e f f o r t to s h o r t e n the time r e q u i r e d to o b t a i n sporophores i n c u l t u r e , f r e s h p l a t e s of Medium A were i n o c u l a t e d with a water suspension of Medium A-grown c o l o n i e s macerated i n a Waring b l e n d o r .  B a s i d i o c a r p development  was r e t a r d e d , and most  b a s i d i o c a r p s d i d not mature when t h i s i n o c u l a t i o n method was (5)  used.  Monokaryons and Matings Monosporic c u l t u r e s were o b t a i n e d by the d i l u t i o n and  p l a t i n g out of a water suspension of b a s i d i o s p o r e s .  A micro-  s c o p i c examination i n s u r e d that b a s i d i o s p o r e s d i d not s t i c k together when r e l e a s e d .  The r e c o v e r e d monosporic c u l t u r e s were  a l l d e r i v e d 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. karyons were m i c r o s c o p i c a l l y examined observed f o r b a s i d i o c a r p s .  Mono-  f o r clamp connections and  Both were p r e s e n t i n d i k a r y o t i c  c u l t u r e s and absent from young monokaryotic c u l t u r e s . Monokaryons were crossed i n a l l  combinations on malt  e x t r a c t - y e a s t extract-peptone agar (MYP, Small b l o c k s of mycelium  Appendix B) a t 10 C.  (with agar) to be c r o s s e d were s e t  s i d e - b y - s i d e on f r e s h media, and o b s e r v a t i o n s were made on the mycelium growing from the zone of c o n t a c t . (6)  S t a i n i n g Techniques For r o u t i n e o b s e r v a t i o n and photography, hyphae were  -11-  s t a i n e d with KOH-phloxine (Martin, 1 9 5 2 ) .  Nuclei i n developing  and mature b a s i d i o s p o r e s were s t a i n e d with a mixture of a c e t o o r c e i n and aceto-carmine  (1:1).  Furtado's  (I97O)  toluidine  b l u e method was used to s t a i n n u c l e i i n monokaryotic  and d i -  k a r y o t i c hyphae. RESULTS AND OBSERVATIONS I.  L i f e Cycle i n Nature Observations were made on the l i f e h i s t o r y of T. erythropus  i n nature and i n c u l t u r e . are  presented i n t h i s S c l e r o t i a produce  The f i n d i n g s from the f i e l d  studies  section. b a s i d i o c a r p s from l a t e September u n t i l  mid-December i n the Vancouver a r e a . of  sporophores  of  Acer macrophyllum Pursh.  Most of my  collections  were made i n the moist l e a f l i t t e r  on p e t i o l e s  These p e t i o l e s were detached  from  l e a f blades and had been on the ground approximately one year. The fungus was a l s o found f r u i t i n g on o l d f r u i t s of Acer, p e t i o l e s o f Rubus p a r v i f l o r u s Nutt., p e t i o l e s of Alnus r u b r a Bong., and stems of U r t i c a l y a l l i i Wats. Mature b a s i d i o c a r p s shed b a s i d i o s p o r e s that germinate and g i v e r i s e to l i m i t e d h a p l o i d m y c e l i a on s u i t a b l e s u b s t r a t e s (Corner, 1 9 5 0 ) •  The f l e s h y p e t i o l e s of Acer l e a v e s , dropped a  few months p r i o r to b a s i d i o s p o r e d i s c h a r g e , a r e the u s u a l subs t r a t e i n Vancouver.  F o l l o w i n g d i k a r y o t i z a t i o n of the m y c e l i a ,  s c l e r o t i a a r e produced  i n the c o r t e x of the p e t i o l e s on e i t h e r  s i d e of the r i n g of p e r i v a s c u l a r f i b e r s . are  Immature  sclerotia  present by mid-December, and, by mid-January, mature s c l e r -  o t i a a r e abundant. when s c l e r o t i a f i r s t  Often p e t i o l e and blade a r e s t i l l a t t a c h e d develop.  Mature s c l e r o t i a remain dormant throughout  the s p r i n g and  -12summer.  Sporophores appear i n the autumn with the onset of  c o o l , moist  conditions.  In my c o l l e c t i o n s up to 60% of the  b a s i d i o c a r p s and s c l e r o t i a on a p e t i o l e were l o c a t e d i n the b a s a l 1 cm of the p e t i o l e .  T h i s area of the p e t i o l e i s the  most f l e s h y . A d i a g r a m a t i c r e p r e s e n t a t i o n of the l i f e  c y c l e of T.  erythropus i s shown i n f i g u r e 1 to i l l u s t r a t e the stages of development. II.  Basidiospore  Germination  Mature b a s i d i o s p o r e s of T. erythropus a r e h a p l o i d when shed and each c o n t a i n s one n u c l e u s . germinated  In my s t u d i e s , the spores  by the f o r m a t i o n of one to f o u r germ tubes.  Basidio-  spores shed onto a s t e r i l e g l a s s s l i d e a t k C were p l a c e d i n a drop of water on Medium A and incubated a t 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  o c c u r r e d most r a p i d l y a t 15 C, with  83$ of the spores germinating w i t h i n 60 hours ( f i g . 2 ) . a l a g p e r i o i d slowed  At 10 C,  the i n i t i a l r a t e o f germination, but t o t a l  germination a f t e r 60 hours was comparable to that a t 15 C. Germination  a t 4 C was r e t a r d e d by a 2h hour l a g p e r i o d before  63$ germination was achieved a t the t e r m i n a t i o n of the e x p e r i ment. III.  At 20 C, l e s s than J0% of the spores Growth of Monokaryotic  germinated.  Mycelium  The mycelium of a young colony s t a r t e d from a s i n g l e b a s i d i o s p o r e was monokaryotic All  and l a c k e d clamp c o n n e c t i o n s .  nine s i n g l e - s p o r e i s o l a t e s grew much more s l o w l y i n c u l t u r e  than d i d d i k a r y o t i c m y c e l i a .  C u l t u r e s were grown on Medium A  and MYP, a n a t u r a l , but undefined, medium.  A f t e r 33 days growth  on Medium A a t 4, 10, 15, and 20 C, the average  colony  diameter  -13-  FACING PLATE 1 F i g u r e 1:  L i f e c y c l e of T. erythropus i n n a t u r e .  sporulation £ Q  germination  / dormancy period  maturation  Figure 1  basidiospores  -14-  FACING PLATE 2 F i g u r e 2:  E f f e c t of temperature on b a s i d i o s p o r e on Medium A.  germination  Each p o i n t on the graph r e p r e s e n t s  100 b a s i d i o s p o r e s counted.  HOURS Figure 2  -15-  was  18,  22, 7, and 4 mm,  respectively.  was  s l i g h t l y improved, the above colony diameters  i n 21 days a t the same f o u r  M y c e l i a l growth on being  MYP  reached  temperatures.  At 10 C or above, the c o l o n i e s were dark brown; a t 4 C, the c u l t u r e s were a g r e y i s h - w h i t e , comparable to d i k a r y o t i c cultures.  The mycelium of a l l i s o l a t e s was  colony s u r f a c e s were s h i n y .  submerged, and  the  H a p l o i d c u l t u r e s grown a t 4 C  were i n d i s t i n g u i s h a b l e m i c r o s c o p i c a l l y from d i k a r y o t i c mycelium except t h a t the former l a c k e d clamp connections. c u l t u r e s grown a t 10 and hyphae t h a t were absent  Haploid  15 C showed frequent bulges from d i k a r y o t i c  i n the  mycelia.  Three to f o u r week o l d monosporic c u l t u r e s grown a t 4 and 10 C produced 0.2-0.6 mm  sporophores  i n the center of the  col-  ony.  These sporophores  carps  (see s e c t i o n V I I I ) of d i k a r y o t i c c u l t u r e s , but bore b a s i d i a  and b a s i d i o s p o r e s .  resembled the cone-shaped stage I b a s i d i o -  The b a s i d i a were four-spored, and  the  spore  dimensions were s i m i l a r to those of spores formed on d i k a r y o t i c fructifications.  The presence  of clamp connections  of the b a s i d i o c a r p and a t the colony margin was esting.  These clamp connections  zation.had  indicated that  on the hyphae  especially  inter-  homodikaryoti-  occurred.  C o l o n i e s became d i k a r y o t i c a t the time of b a s i d i o c a r p f o r mation, p r i o r to b a s i d i o s p o r e p r o d u c t i o n .  In c o n t r a s t , c u l t u r e s  s t a r t e d from a m u l t i s p o r e inoculum were d i k a r y o t i c i n a p p r o x i mately three days.  The r a t e of m y c e l i a l e x t e n s i o n on the  s u r f a c e d i d not i n c r e a s e f o l l o w i n g h o m o d i k a r y o t i z a t i o n .  agar Homo-  k a r y o t i c c u l t u r e s a l s o d i f f e r e d from h e t e r o d i k a r y o t i c c u l t u r e s i n not forming s c l e r o t i a a t any  temperature.  -16IV. Matings o f Monokaryons A.  IntraspecifIc Pairings S e v e r a l d i f f i c u l t i e s were encountered i n attempting to  o b t a i n monosporic c u l t u r e s and c r o s s them.  Since maximum ex-  t e n s i o n of d i k a r y o t i c hyphae and maximum b a s i d i o s p o r e  germination  o c c u r r e d a t 15 C, d i l u t i o n p l a t e s of b a s i d i o s p o r e s were i n c u bated a t 15 C.  U s u s a l l y only d i k a r y o t i c c u l t u r e s were  from these p l a t e s .  When no monosporic c u l t u r e s were v i s i b l e  a f t e r 10 days, the p l a t e s were d i s c a r d e d . covered,  recovered  As was l a t e r  dis-  the growth of h a p l o i d mycelia was n e g l i g i b l e a t t h i s  temperature. By i n c u b a t i n g d i l u t i o n p l a t e s a t 10 C, I was a b l e to i s o l a t e nine mososporic c u l t u r e s .  These were crossed i n a l l  combinations, i n d u p l i c a t e , on MYP a t 10 C. been a s c e r t a i n e d t h a t the morphological  I t had p r e v i o u s l y  c h a r a c t e r i s t i c s of the  d i k a r y o t i c 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 f a s t e r on i t than on Medium A. Clamp formation and b a s i d i o c a r p f o r m a t i o n  c o u l d not be  taken as i n d i c a t i o n s of s u c c e s s f u l , mating s i n c e these s t r u c t u r e s were present  i n o l d e r monosporic c u l t u r e s .  However, b a s i d i o -  carps produced by h e t e r o k a r y o t i c mycelia were d i s t i n g u i s h a b l e from those of homodikaryotic  mycelia by t h e i r l a r g e r s i z e .  Both types of b a s i d i o c a r p s were a t y p i c a l l y cone-shaped a t 10 C. The  homokaryotic sporophore was very narrow and l a c k e d the  plumpness and l e n g t h carps.  (to 1.5 mm) of h e t e r o d i k a r y o t i c b a s i d i o -  In a d d i t i o n , b a s i d i o s p o r e s  shed from h e t e r o d i k a r y o t i c  sporophores soon produced a clamped mycelium where they on the agar near the parent  colony.  Basidiospores  landed  from homo-  -17d i k a r y o t i c f r u c t i f i c a t i o n s d i d not produce a clamped mycelium f o r s e v e r a l weeks. Because of inoculum-induced v a r i a t i o n (see below) s c l e r o t i u m production  on Medium A a t 4 o r 10 C was not i n d i c a t i v e o f hetero-  dikaryotization.  T r a n s f e r of a p i e c e of the mycelium growing  out from the contact  zone to p l a t e s of Medium A f o r s t a n d a r d i z a -  t i o n was not f e a s i b l e f o r two reasons.  In some cases no  mycelium grew i n t o the agar from two mated s t r a i n s . d u r i n g the i n i t i a l  Secondly,  inoculum s t a n d a r d i z a t i o n t e s t i n g of  I observed that e r r a t i c r e s u l t s were obtained  dikaryons,  when the inoculum  source was l e s s than one month o l d , grown a t a temperature other  than 15 C, or grown on a medium other  than Medium A.  I n o c u l a from such sources gave r i s e to c o l o n i e s that f a i l e d to produce s c l e r o t i a under c o n d i t i o n s u s u a l l y f a v o r a b l e f o r sclerotium production.  Production  of b a s i d i o c a r p s  from the  agar s u r f a c e was a l s o i n f l u e n c e d by the inoculum source. On t h e b a s i s of the p r o d u c t i o n basidiocarps, and  of l a r g e r , h e t e r o d i k a r y o t i c  the c o m p a t i b i l i t y r e a c t i o n s of t h e i r  basidiospores,  the i n c r e a s e d growth r a t e f o l l o w i n g h e t e r o d i k a r y o t i z a t i o n ,  I grouped the nine i s o l a t e s i n t o f o u r mating types.  Isolates  #3, 9, n d 10 were compatible only with i s o l a t e s #4, 5» and 6. a  I s o l a t e #8 was compatible only w i t h i s o l a t e s #1 and 2. mating r e a c t i o n s f i t the t e t r a p o l a r p a t t e r n .  These  The p a i r i n g  r e a c t i o n s a r e shown i n f i g u r e 3. B.  Interspecific Pairings Monosporic i s o l a t e s #1, 3, 4, 8, 9, and 10, r e p r e s e n t i n g  all  f o u r mating s t r a i n s of T. erythropus,  were crossed  two monosporic c u l t u r e s of T. s c l e r o t i o i d e s .  with  Except f o r t h e i r  c o l o r a t i o n , f r u c t i f i c a t i o n s o f T. s c l e r o t i o i d e s a r e very s i m i l a r  -18-  FACING PLATE 3 F i g u r e 3-  R e s u l t s of p a i r i n g s of nine monosporic i s o l a t e s of  T. e r y t h r o p u s .  Heterodikaryon  h e t e r o d i k a r y o n not formed = standard m y c o l o g i c a l p r a c t i c e , are a r b i t r a r i l y  formed = +;  In keeping the mating  with types  d e s i g n a t e d AB, Ab, aB, and ab.  AB ab 3 9 10 4 5 6 3 AB 9 10  —  —  —  +  +  +  +  +  +  +  + +  Ab aB 1 2 8 —  —  —  4 ab 5 6 Ab  1  +  2 aB 8  —  Figure 3  -19to those o f T. e r y t h r o p u s . of the p a i r i n g s .  No d l k a r y o n s were formed i n any  A d e f i n i t e barrage e f f e c t (Burnett,  was  apparent i n a l l c r o s s e s .  V.  Growth o f 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  I968)  The mycelium o f a l l f o u r i s o l a t e s o f T. e r y t h r o p u s grew s l o w l y , t a k i n g over 3 0 days t o c o v e r a 9 0 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 .  occurred  Most h y p h a l growth  on o r below t h e agar s u r f a c e ; a e r i a l mycelium was  l i m i t e d i n young c o l o n i e s and was r e s t r i c t e d t o a l a y e r o f short, t h i c k - w a l l e d hyphal c e l l s i n the center of o l d e r (figs. 14,17).  cultures  The c e l l s t h a t make up t h i s c r u s t were o f t e n  e n c r u s t e d w i t h c r y s t a l s and were s i m i l a r i n appearance t o t h e sheathing  hyphae a t t h e base o f a b a s i d i o c a r p .  Clamp c o n n e c t i o n s were common b u t were n o t p r e s e n t a t a l l septa.  Branches f r e q u e n t l y a r o s e from t h e 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, t h e c o l o n y was a transparent  greyish-white  becoming l i g h t r e d d i s h - b r o w n i n the  c e n t e r where t h e c r u s t was f o r m i n g .  The s u r f a c e was uneven,  i n t e r r u p t e d by numerous lumps o f hyphae and b a s 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 t h e mycelium. The  rate of m y c e l i a l extension  i n a petri plate  followed  the s i g m o i d 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 o f a p p r o x i m a t e l y seven days, t h e l o g phase commenced, w i t h t h e c o l o n y d i a m e t e r i n c r e a s i n g a t the r a t e o f 2 - 3 mm p e r day. B e f o r e the p l a t e became f i l l e d  t h e growth r a t e  declined,  p o s s i b l y i n response t o an a u t o i n h i b i t o r produced d u r i n g Other i s o l a t e s grew a t r a t e s e q u i v a l e n t  growth.  to that recorded f o r  -20-  FACING PLATE itFigure 4:  Growth of a colony of T. erythropus on Medium A a t 1 5 C.  -21-  I s o l a t e T-4 on a l l media t e s t e d . The reddish-brown c r u s t that formed i n the center of the colony and expanded r a d i a l l y was i n f l u e n c e d i n i t s development by temperature,  pH, and carbon and n i t r o g e n sources.  Factors  f a v o r i n g r a p i d m y c e l i a l growth a l s o f a v o r e d maximum c r u s t formation.  When T. erythropus was grown on Medium A c o n t a i n i n g  glucose a t 1 . 0 , 3 . 0 , 5 . 0 , 1 0 . 0 , and 1 5 . 0 g / l , maximum growth and most e x t e n s i v e c r u s t development occurred a t the h i g h e s t concentration. glucose.  Least growth and no c r u s t were found a t 1 . 0 g / l  The amount o f asparagine  i n f l u e n c e d c r u s t development. asparagine  i n the medium s i m i l a r l y  I n c r e a s i n g the c o n c e n t r a t i o n o f  i n Medium A from 0 . 1 to 3 . 0 g / l r e s u l t e d i n t h i c k e r  m y c e l i a l growth and i n c r e a s e d c r u s t formation B.  Temperature  ( f i g . 14).  Effects  The optimum temperature f o r m y c e l i a l growth, as measured by both d r y weight and l i n e a r extension, was 1 5 C. a l s o o c c u r r e d a t 1 0 C and f a i r growth a t 4 C. of growth was observed  a t 0 and 2 0 C.  Good growth  A very low r a t e  F i g u r e 5 shows the t o t a l  l i n e a r growth of 2 1 day o l d c o l o n i e s incubated a t f o u r d i f f e r e n t temperatures  and the d r y weight o f 11 day o l d c o l o n i e s grown  a t three temperatures.  At 2 0 C, there was a tendency f o r the  slow growing colony to p i l e up, producing a hard amorphous lump covered w i t h a bloom of white mycelium. produced a t 2 0 C, abundantly  A s l i g h t c r u s t was  a t 1 0 and 15 C, and not a t a l l a t  4 C on Medium A. C.  pH E f f e c t s The optimum pH f o r v e g e t a t i v e growth was determined by  measuring l i n e a r e x t e n s i o n of mycelium a t 1 5 C on Medium A. The most vigorous spread of mycelium o c c u r r e d near pH 5 ( f i g . 6 ) .  -22but c u l t u r e s grew w e l l over a pH range of 4.0-6.0. pH 6.0 the growth r a t e d e c l i n e d .  Above  L i n e a r e x t e n s i o n could not  be determined below pH 4 because the medium d i d not s o l i d i f y at  lower pH v a l u e s .  The f i n a l pH of a l l c u l t u r e media was  determined a f t e r 36 days growth with the i n d i c a t o r s 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, r e s p e c t i v e l y . At 10 C, the optimum pH f o r l i n e a r extension of hyphae  s h i f t e d to 4.0 ( f i g . 6 ) . C u l t u r e s were grown f o 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, r e s p e c t i v e l y . D.  Nitrogen  Utilization  T . erythropus  was a b l e to u t i l i z e n i t r a t e , ammonium, amides,  and amino a c i d s as n i t r o g e n sources f o r m y c e l i a l growth.  A  v i s u a l e s t i m a t i o n of growth was made on the b a s i s of colony diameter and d e n s i t y of the hyphal mat. agine and c a s e i n h y d r o l y s a t e supported DL-alanine,  On medium A, DL-aspar-  the most v i g o r o u s  growth.  ammonium s u l f a t e , and ammonium c h l o r i d e gave good  growth, but a r a p i d pH drop (to 3.8) on media with ammonium s a l t s q u i c k l y c u r t a i l e d growth. sium s a l t s induced  N i t r a t e s as calcium and potas-  r a p i d l i n e a r extension, but growth was sparse.  L - ( - ) - p h e n y l a l a n i n e , L - p r o l i n e , and L - t y r o s i n e supported  poor  growth, and DL-methionine i n h i b i t e d growth. The optimum c o n c e n t r a t i o n o f KNO^ f o r hyphal investigated. 0.0,  growth was  C u l t u r e s were grown a t 15 C with KNO^ added a t  0.5, 1.0, 2.0, 3.0, 4.0, 5.0, and 10.0 g/1.  At a l l con-  c e n t r a t i o n s g r e a t e r than 0.0 g / l the r a t e o f l i n e a r was  extension  n e a r l y equal, and no optimum could be determined.  -23-  FACING PLATE 5 Environmental E f f e c t s on M y c e l i a l Growth  F i g u r e 5'  E f f e c t of temperature on m y c e l i a l  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.  F i g u r e 6:  E f f e c t of pH on m y c e l i a l growth a t 10 and 15 C. C u l t u r e s a t 10 C were grown f o r 25 days, those a t 15 C f o r 36 days. Medium A.  A l l c u l t u r e s were grown on  50  -24E.  Vitamin  Requirements  A stock s o l u t i o n o f the vitamins doxine,  thiamine,  biotin,  pyri-  and i n o s i t o l was r o u t i n e l y i n c l u d e d i n the p r e p a r a t i o n  of Medium A.  When these vitamins were omitted,  growth was very poor.  vegetative  Two s e r i a l t r a n s f e r s with 1 mm  2  plugs were executed to exclude  inoculum  the p o s s i b i l i t y of c a r r y o v e r  from the o r i g i n a l inoculum source.  When thiamine  (100 ug/1) was  added to the Medium A l a c k i n g the v i t a m i n stock s o l u t i o n , v i g o r ous  growth was r e s t o r e d .  thiamine VI. A.  From these data i t appeared that  was r e q u i r e d f o r the growth of T. erythropus.  Sclerotium  Formation  Morphology o f Mature S c l e r o t i a Corner (1950) d e s c r i b e d i n d e t a i l the micro-morphology of  T. erythropus  sclerotia.  At that time he b e l i e v e d , as d i d  Remsberg (1940), that c e r t a i n s p e c i e s c o u l d be d i s t i n g u i s h e d s o l e l y on the b a s i s o f t h e i r s c l e r o t i a l morphology.  The s t a b i l -  i t y o f t h i s c h a r a c t e r was never e s t a b l i s h e d .  In my s t u d i e s , a l l  f o u r i s o l a t e s of T. erythropus  characteristic  retained their  s c l e r o t i a l f e a t u r e s under a v a r i e t y o f n u t r i t i o n a l and  i n c u b a t i o n temperatures.  Mature s c l e r o t i a from c u l t u r e  were d o r s i v e n t r a l l y f l a t t e n e d bodies The  conditions  0.7-2.9 mm i n diameter.  f i n a l s i z e of these s u b s p h e r i c a l s c l e r o t i a was dependent  upon the medium and the i n c u b a t i o n temperature. The  reddish-brown to n e a r l y b l a c k c o l o r i n g was l i m i t e d to  the c u t i c l e . were white.  The u n i s t r a t o s e c o r t e x and prosenchymatous medulla A c r o s s s e c t i o n o f a mature s c l e r o t i u m i s shown i n  f i g u r e 15. The dark pigment of the c u t i c l e was s l i g h t l y s o l u b l e i n  -25water and i n s o l u b l e i n e t h a n o l , e t h y l ether, petroleum ether, chloroform, acetone, 12N HC1,  IN KOH,  and a s a t u r a t e d aqueous  s o l u t i o n of F e C l ^ . The pH of mature s c l e r o t i a was  near 7«  This was  determined  by r e a c t i o n with bromcresol green, bromcresol p u r p l e , and phenol S c l e r o t i a from c u l t u r e r e t a i n e d only 36% of t h e i r f r e s h  red.  weight a f t e r d r y i n g a t room temperature  f o r one week.  On  d r y i n g , the s c l e r o t i a darkened and became s l i g h t l y w r i n k l e d 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 f i g u r e 11, were  e v i d e n t d u r i n g the f o r m a t i o n of s c l e r o t i a and t h e i r growth to maturity. was  For the purpose of t h i s study, a mature s c l e r o t i u m  d e f i n e d as one a b l e to produce a b a s i d i o c a r p .  The knot of  hyphae d e s t i n e d to become a s c l e r o t i u m f i r s t became v i s i b l e i n stage I a . face.  This Ia stage developed on or beneath the agar s u r -  The stage l b commenced when the s c l e r o t i a l  primordium  was  a w e l l - d e f i n e d 0.8-1.2 mm  diameter white sphere.  top  of the l b s c l e r o t i u m a reddish-brown spot appeared  slowly and evenly spread over the s c l e r o t i u m . w i t h the f i r s t appearance e n t i r e s c l e r o t i u m was  At the that  Stage II began  of t h i s spot and continued u n t i l  darkened.  One  the  to s e v e r a l drops of c l e a r  l i q u i d f r e q u e n t l y were exuded from s c l e r o t i a d u r i n g stages l b and I I .  T h e i r occurrence was  produced a t 4 C.  e s p e c i a l l y common on  sclerotia  When s c l e r o t i a were completely darkened,  stage I I I and m a t u r i t y were reached. Stage I I I s c l e r o t i a averaged 1.2 mm duced a t 1 5 C.  i n diameter when pro-  I f l e f t on the parent colony, these s c l e r o t i a  continued to i n c r e a s e i n s i z e up to 1.4  mm.  -26Th e f o u r stages of development that were observed i n c u l t u r e d s c l e r o t i a a l s o appeared f o r m a t i o n i n the f i e l d .  to be present d u r i n g s c l e r o t i u m  Developing s c l e r o t i a on p e t i o l e s  incubated i n moist chambers a t 4 C were observed i n stages l b and I I I .  I t was  not determined  i f the darkening process of  stage I I o c c u r r e d i n as o r d e r l y a manner as seen i n c u l t u r e . S c l e r o t i u m f o r m a t i o n i n a l l f o u r i s o l a t e s grown i n c u l t u r e f o l l o w e d 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 parent hyphae were d e s c r i b e d by deBary  (I887), MacDonald  and Remsberg (1940) f o r s e v e r a l s p e c i e s of Typhula. f o r m a t i o n i n T. erythropus was  two (193^),  Sclerotium  s i m i l a r to that i n other s p e c i e s ,  but the medullary hyphae d i d not become t h i c k - w a l l e d i n T. e r y t h ropus .  S c l e r o t i a l i n i t i a t i o n was  by Townsend and W i l l e t s  (1954).  of the " s t r a n d type" d e s c r i b e d Increased branching, growth,  and s e p t a t i o n from a few i n t e r c a l a r y hyphal c e l l s produced nucleus of the s c l e r o t i a l primordium ments were completed  ( f i g . 12).  d u r i n g stage I a .  the  These develop-  Growth of the young  s c l e r o t i u m 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 of c e l l s ,  (stage l b ) .  c o n s t i t u t i n g the cortex, was  The outer l a y e r  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 . chymatous nature of the expanding, medulla was  The  prosen-  i n t e r t w i n i n g c e l l s of the  constant throughout development ( f i g .  16).  During stage I I , 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 p i e c e shape. t o r t i o n was  I t was  not determined whether t h i s  dis-  a r e s u l t of s t r e t c h i n g of the c e l l s by i n t e r n a l  pansion of the s c l e r o t i u m , 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 i n c r e a s e d  ex-  -27sclerotium size. p a t t e r n that was  Regardless,  the r e s u l t i n g  formed on the s c l e r o t i u m s u r f a c e was  s t a n t f e a t u r e of a l l mature s c l e r o t i a p a t t e r n i s not r e s t r i c t e d Remsb. and  solely  T. p h a c o r r h i z a  sclerotia.  jig-saw-like  ( f i g . 13).  This  to T. erythropus.  have s i m i l a r  a consurface  T. v i b u r n i  p a t t e r n s on  their  However, the i n t e r n a l anatomy of these s p e c i e s i s  much d i f f e r e n t  from t h a t of T.  erythropus.  While the c o r t i c a l c e l l s became deformed, a dark r e d d i s h brown c u t i c l e was  deposited  on t h e i r outer w a l l s .  s e c t i o n t h i s c u t i c l e , with i t s short p e r p e n d i c u l a r  In  cross  projections  down the r a d i a l w a l l s of the cortex, resembled the c u t i c l e higher p l a n t s and dark c u t i c l e was  i t s relationship separable  to the epidermis.  from the hyphal w a l l s .  of l i q u i d exuded between the c o r t e x and II l i f t e d  This a c e l l u l a r  Often drops stage  A f t e r the  to the s c l e r o t i u m  surface.  c u t i c l e sometimes appeared as a membrane-like  s t r u c t u r e around the drops. carried  The  the c u t i c l e d u r i n g  the c u t i c l e from the s c l e r o t i u m s u r f a c e .  drop evaporated, the c u t i c l e r e t u r n e d  of  the jig-saw  When examined m i c r o s c o p i c a l l y , i t  p a t t e r n of the s c l e r o t i u m s u r f a c e .  of the c u t i c l e were r e a d i l y  scraped  Patches  from the s u r f a c e of mature  sclerotia. The morphology of mature s c l e r o t i a was all  cultural  collections G.  conditions tested.  very constant  under  A l l i s o l a t e s and a l l f i e l d  d i s p l a y e d the same s c l e r o t i a l morphology.  Time of Formation The  relationship  of s c l e r o t i u m development to m y c e l i a l  growth i n c u l t u r e i s shown i n f i g u r e d u c t i o n was  7.  Since s c l e r o t i u m  best s t u d i e d on Medium B a t 15 C,  used to c o r r e l a t e the events.  t h i s medium  prowas  Stage Ia s c l e r o t i a were f i r s t  -28v i s i b l e on 12 day o l d c o l o n i e s i n the l o g phase of m y c e l i a l growth. at  Sclerotia  had advanced  the end of the l o g phase.  to stage l b f o u r days l a t e r ,  The s c l e r o t i a began to darken  s i x days l a t e r as the growth r a t e of the colony d e c l i n e d . Mature s c l e r o t i a were present a f t e r an a d d i t i o n a l f o u r days. The average time from i n i t i a t i o n to m a t u r i t y was was  the s h o r t e s t average time r e c o r d e d .  on o t h e r d e f i n e d media and a t d i f f e r e n t  on a colony.  days.  This  The i n t e r v a l was  longer  i n c u b a t i o n temperatures.  The above d e s c r i p t i o n a p p l i e s only to the 6-10 mature f i r s t  lk  sclerotia  that  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 f o r a l l s c l e r o t i a that formed on a p l a t e was v a r i a b l e .  Sclerotia  first  were  formed c l o s e to the p o i n t of i n o c u l a t i o n i n the o l d e s t hyphae of the c o l o n y .  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  c e n t e r of the colony, forming a g r a d i e n t of development the  immature s c l e r o t i a near the margin.  i n i t i a t e d and matured a f t e r D.  Temperature  New  to  s c l e r o t i a were  the l o g phase of growth had passed.  Effects  The e f f e c t of temperature on s c l e r o t i u m f o r m a t i o n was r e l a t e d to the c a r b o n : n i t r o g e n 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  sclerotia  were not w e l l - d e f i n e d and were d i f f i c u l t to separate from the colony.  C u l t u r e s grown a t k and 10 C on t h i s medium produced  up to 80 d i s t i n c t s c l e r o t i a per p l a t e . p r o d u c t i o n of 15-30  Medium B supported the  w e l l - d e f i n e d s c l e r o t i a per p l a t e a t 15 C;  a t k and 10 C, the average number of s c l e r o t i a per p l a t e 5-15.  was  I t was d i s c o v e r e d l a t e r t h a t up to 200 s c l e r o t i a per  p l a t e c o u l d be o b t a i n e d on Medium B a t 15 C i f the inoculum was  -29-  FACING PLATE 6 Figure 7:  S c l e r o t i u m development and i t s r e l a t i o n s h i p to m y c e l i a l growth. Medium B a t 15 C.  C u l t u r e s were grown on  -30taken from a c u l t u r e grown on Medium A prepared w i t h KNO^ i n p l a c e of a s p a r a g i n e . A two to three week o l d c u l t u r e , grown a t 15 C on Medium A, produced numerous t y p i c a l 4, or 10 C.  Return o f the c u l t u r e with stage I I I s c l e r o t i a  to 15 C o f t e n was accompanied sclerotia  s c l e r o t i a when incubated a t 0,  by the i n c o r p o r a t i o n o f the  i n t o the s p r e a d i n g c r u s t .  Medium C supported e x c e l l e n t s c l e r o t i u m p r o d u c t i o n a t 15 C, with up to 90 s c l e r o t i a formed per p l a t e .  At lower temp-  e r a t u r e s fewer s c l e r o t i a were produced. In a d d i t i o n to a f f e c t i n g the number of s c l e r o t i a  formed  per p l a t e , the i n c u b a t i o n temperature a l s o i n f l u e n c e d the time of s c l e r o t i u m i n i t i a t i o n and development. were formed  Mature  sclerotia  e a r l i e s t on Medium B, of the d e f i n e d media employed.  The time between stages and the time to i n i t i a t i o n a t 4, 10, and 15 C a r e presented i n f i g u r e 8.  Data from the 4 and 10 C exper-  iments a r e from o b s e r v a t i o n s o f s c l e r o t i u m f o r m a t i o n on Medium A.  The 15 C data a r e taken from c u l t u r e s on Medium B.  Scler-  otium p r o d u c t i o n on Medium A was g r e a t e r than that on Medium B at the  these temperatures.  When c u l t u r e s were grown on Medium A,  time r e q u i r e d to produce  s c l e r o t i a a t 4 C was s i m i l a r to  t h a t observed i n n a t u r e . Stage I I s c l e r o t i a appeared f i r s t 10 C.  i n c u l t u r e s grown a t  However, the darkening process was r e t a r d e d a t t h i s  temperature, and stage I I I s c l e r o t i a were formed e a r l i e s t a t 15 C.  The time o f i n i t i a t i o n and the d u r a t i o n of maturation  were c o n s i d e r a b l y g r e a t e r a t 4 C than a t 10 o r 15 C. d i s c o v e r e d that mature s c l e r o t i a  I later  c o u l d be produced i n 35 days  i n s t e a d of 67 days a t k C when the asparagine i n Medium A was  -31-  FACING PLATE 7 Figure  8:  The e f f e c t of temperature on the r a t e of s c l e r o t i u m development.  Cultures  grown a t 4  and 10 C on Medium A; c u l t u r e s a t 15 C on Medium B.  0  10  20  30 AGE,  40 DAYS  Figure 8  50  60  70  -32r e p l a c e d by 1.0 g / l o f c a s e i n h y d r o l y s a t e . At other.  10 and 15 C, s c l e r o t i a were w e l l separated from each At 4 C, they tended to c o a l e s c e , f r e q u e n t l y forming  compound  sclerotia.  The i n c u b a t i o n temperature a l s o a f f e c t e d 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 a t 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 l a r g e , a v e r a g i n g 2.6 mm.  The diameter  of s c l e r o t i a produced a t 10 C was i n t e r m e d i a t e , a v e r a g i n g 1.5  mm.  I s o l a t e s T-I82 and T-29 produced numerous s c l e r o t i a on Medium A a t 4 and 10 C.  I s o l a t e T-18  1  formed l e s s than 15  s c l e r o t i a per p l a t e on t h i s medium a t 4 C. E.  pH e f f e c t s The e f f e c t  10 and 15 C. f i g u r e 9.  of pH on s c l e r o t i u m f o r m a t i o n was examined a t  The r e s u l t s of these experiments a r e presented i n  C u l t u r e s were grown a t 15 C on Medium A a d j u s t e d to  pH v a l u e s o f 4.0, 5.0, 5.5, 6.0, 7.0, 7.7, and 8.5.  A f t e r 26  days, the average number of s c l e r o t i a per p l a t e was determined. The f i n a l pH of the r e s p e c t i v e media was 6.5, 6.5, 7.6, 7.6, 7.8, 7.8, and 7.8. but  Sclerotia  were produced a t pH 4.0 and 5.0,  these were not w e l l - d e f i n e d from the colony s u r f a c e .  s c l e r o t i a were produced a t other pH l e v e l s .  No  The experiment was  terminated a f t e r 26 days because the spreading c r u s t threatened to grow over the s c l e r o t i a . C u l t u r e s a t 10 C were grown on Medium A a d j u s t e d to pH v a l u e s of 4.0, 5.0, 6.0, and 7.0.  A f t e r 32 days, the average  number of s c l e r o t i a per p l a t e was determined, and the f i n a l was measured.  pH  The f i n a l pH f o r these media was 7.2, 7.2, 7.5,  and 6.8, r e s p e c t i v e l y .  Maximal s c l e r o t i u m p r o d u c t i o n o c c u r r e d  -33-  PACING PLATE 8 Figure 9:  The e f f e c t of i n i t i a l pH on s c l e r o t i u m f o r m a t i o n . C u l t u r e s were grown a t 10 and 15 C on Medium A.  40 • {  • • • • -15° 3o^ MATURE SCLEROTIA  20 H PER PLATE  104  0 Figure 9  -34a t pH 4.0,  w i t h 32 s c l e r o t i a per p l a t e ; a t pH 5.0,  7.0  the average number of s c l e r o t i a per p l a t e was  F.  C a r b o n : N i t r o g e n (C/N) The optimum C/N  r a t i o f o r s c l e r o t i u m p r o d u c t i o n was  (g C/g N) were prepared  9.5:1  r a t i o s from 3.8:1  pH of a l l media was  to 190:1  to  had a f i n a l pH of 7.0  asparagine  a d j u s t e d to 5.0, and  38:1  supported  on the 95:1 medium.  were formed a t 15 C when the C/N  r a t i o was  d u c t i o n o c c u r r e d a t 4 and 9.5:1  (Medium A ) .  of 9 . 5 : 1  and  per p l a t e was  95:1.  Media w i t h  lower  No  e x c e l l e n t s c l e r o t i u m pror a t i o of  C u l t u r e s were grown on media w i t h C/N  80 a t 9 . 5 : 1  10 C, the average number of  and 5 - 1 5  sclerotia  19:1.  than  10 C on a medium w i t h a C/N  At 4 and  an  approximately  one h a l f the p r o d u c t i o n a c h i e v e d  C results,  and  r a t i o of 95:1. w i t h  average of 16 s c l e r o t i a formed per p l a t e ( f i g . 10). and  C/N  r e s p e c t i v e l y . Maximum  s c l e r o t i u m p r o d u c t i o n o c c u r r e d a t a C/N  I n c o n t r a s t to the 15  cul-  Media w i t h r a t i o s of 19:1  and 8.4,  r a t i o s o f 140:1, 6 3 : 1 , 47:1,  C  190:1  The f i n a l pH of media w i t h  5.2.  was  influ-  F o r the 15  by a d j u s t i n g the amount of  t u r e s were grown f o r 60 d a y s . r a t i o s of 47:1  6.  Ratio Effects  i n v e s t i g a t i o n , media w i t h C/N  The  and  16, 7, and  enced markedly by the temperature of i n c u b a t i o n .  i n Medium A.  6.0,  a t 95:1  ratios sclerotia  (Medium B ) .  G. N u t r i e n t C o n c e n t r a t i o n E f f e c t s The was  c o n c e n t r a t i o n of g l u c o s e and a s p a r a g i n e  i n Medium A  a d j u s t e d to make media of i to 5X r e g u l a r s t r e n g t h .  r a t i o of 95:1 was a t 4.0, a t 15  maintained  i n a l l media, and  optimal f o r sclerotium formation.  the pH was  s t r e n g t h of the medium.  C/N set  C u l t u r e s were grown  C f o r 31 days; the r e s u l t s a r e shown i n f i g u r e 18.  average number of s c l e r o t i a per p l a t e was  A  The  p r o p o r t i o n a l to the  No stage I I I s c l e r o t i a were produced on  -35-  FACING Figure  10:  The  effect  of  formation  at  adjusting  the  the  glucose  PLATE  9  various 15  C.  C/N r a t i o s  on  The m e d i a were  concentration  concentration  of  was  sclerotium prepared  asparagine maintained  by-  while at  10  g/1.  F i g u r e 10  -36FACING PLATE-10 S c l e r o t i u m Development i n T. Figure 1 1 :  erythropus  Stages of s c l e r o t i u m development. of the Ia and  The  darkness  l b s c l e r o t i a i s a r e s u l t of  accumulation of bromcresol green.  X2.  F i g u r e 12:  Young stage Ia s c l e r o t i u m . X300.  Figure 1 3 :  Surface of the mature s c l e r o t i u m . X400.  F i g u r e 14:  The  e f f e c t of s e v e r a l C/N  p r o d u c t i o n a t 1 5 C.  the  r a t i o s on s c l e r o t i u m  The numbers on the p l a t e s  i n d i c a t e the g / l of asparagine  i n Medium A.  values  5 . 0 are  0.2,  0.5, 1 . 0 , 2.0,  and  The  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 a t 1.0 g / l are not w e l l  d e f i n e d from the colony. F i g u r e 15"-  X.S.  Note c r u s t development.  of a mature s c l e r o t i u m .  The dark edge i s  the outer s u r f a c e of the s c l e r o t i u m . homogeneous nature  of the c e l l s of the medulla.  The  c e l l t h i c k n e s s , i s not  cortex, of one  at this magnification. Figure 1 6 :  Note the  evident  X70.  C e l l s of the medulla of the s c l e r o t i u m .  Note  t h e i r prosenchymatous nature. X1000. Figure 1 7 :  C r u s t i n g hyphae from the s u r f a c e of the Note thickened,  dark w a l l s . X400.  colony.  -37the •§•' s t r e n g t h medium  (5.0 g/1 glucose, 0.1 g/1 a s p a r a g i n e ) .  On the p l a t e s of f u l l ,  1.5X, 2X, and 5X s t r e n g t h 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 i n c r e a s e  i n sclerotium production. the 5X s t r e n g t h medium. at  Maximum m y c e l i a l growth occurred on The c o r r e l a t i o n of colony  diameter  31 days to n u t r i e n t c o n c e n t r a t i o n i s a l s o i l l u s t r a t e d i n  f i g u r e 18. H.  Nitrogen  Utilization  Sclerotia  of T. erythropus were produced on media made  w i t h a v a r i e t y of n i t r o g e n sources.  However, i t was  difficult  to determine which compounds were u t i l i z a b l e f o r t h i s since s c l e r o t i a  process  could be formed on simple water agar i n o c u l a t e d  with basidiospores. N i t r o g e n sources were added to medium A i n p l a c e of asparagine.  The i n f l u e n c e of temperature on the optimum C/N  f o r s c l e r o t i u m p r o d u c t i o n has a l r e a d y been noted.  ratio  For this  reason, media f o r use a t 10 C were prepared w i t h a C/N  ratio  of  The pH  9.5=1  and media f o r use a t 15 C with a 95:1 r a t i o .  of a l l media was a d j u s t e d to 5.0, and c u l t u r e s were grown f o r 35 days.  The r e s u l t s a r e shown i n f i g u r e 19.  gine supported  At 15 C, aspara-  the maximum p r o d u c t i o n of s c l e r o t i a .  Consistently  l e s s than 10 s c l e r o t i a per p l a t e were formed on media made with DL-alanine, Ca(N0-^)2.  DL-methionine, c a s e i n h y d r o l y s a t e , KNO3, and The appearance of s c l e r o t i a on these p o o r l y u t i l i z e d  s u b s t r a t e s was a l s o much delayed. present on methionine days o l d .  Mature s c l e r o t i a were not  media u n t i l the c u l t u r e s were over  110  No s c l e r o t i a were produced on L - t y r o s i n e or NH^Cl.  -38-  FACING PLATE 11 F i g u r e 18:  The e f f e c t of medium s t r e n g t h on s c l e r o t i u m formation and r a d i a l growth of a  colony.  Standard s t r e n g t h Medium A c o n t a i n s glucose and 2 . 0 g / l asparagine. were grown a t 15 C f o r 31 days.  10 g / l  Cultures  -39Th e pH drop a s s o c i a t e d with ammonium u t i l i z a t i o n p o s s i b l y was the reason f o r the f a i l u r e of t h i s source to support sclerotium  production.  At 10 C, optimum s c l e r o t i u m p r o d u c t i o n gine w i t h 80 s c l e r o t i a formed per p l a t e . was the next most p r o d u c t i v e ,  was made on a s p a r a -  Casein  hydrolysate  w i t h 4-5 per p l a t e .  Alanine and  KNO-^ media supported l e s s than 10 s c l e r o t i a per p l a t e . and  L-(-)-phenylalanine  I.  Wheat Germ E f f e c t s (Medium C) Sclerotium  i n h i b i t e d sclerotium  production  L-proline  formation.  on Medium C was e x c e p t i o n a l l y  vig-  orous i n 15 C grown c u l t u r e s .  Mature s c l e r o t i a were formed  r a p i d l y and i n l a r g e numbers.  The r e l a t i o n s h i p of m y c e l i a l  growth to s c l e r o t i u m development i s shown i n f i g u r e 20. l o g phase of m y c e l i a l growth on Medium C commenced  The  approximately  f o u r days a f t e r i n o c u l a t i o n when c u l t u r e s were grown a t 15 C. Stage Ia s c l e r o t i a appeared i n 9 day o l d c o l o n i e s .  The other  stages f o l l o w e d a t n e a r l y the same i n t e r v a l s as those on Medium B a t 15 C.  The r e l a t i o n s h i p of s c l e r o t i u m  to the growth r a t e of the colony Medium B.  recorded formation  a l s o was the same as that on  Mature s c l e r o t i a were present  i n 22 day o l d c u l t u r e s .  This was f o u r days l e s s than the time r e q u i r e d on Medium B. The  l a g p e r i o d of m y c e l i a l growth a l s o was f o u r days l e s s than  on Medium B.  At 4 and 10 C, s c l e r o t i u m p r o d u c t i o n  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 r a t e to that observed on Medium A a t these temperatures.  A l l isolates  produced numerous s c l e r o t i a when c u l t u r e d on Medium C a t 15 C. To determine the optimum c o n c e n t r a t i o n Medium C f o r s c l e r o t i u m p r o d u c t i o n ,  of wheat germ i n  petri plates  1.0 to 25.0 g / l of wheat germ were prepared.  containing  C u l t u r e s were  -40-  FACING PLATE 12 F i g u r e 1$:  N i t r o g e n sources f o r s c l e r o t i u m  formation.  The r a t i n g system r e f e r s to the number of stage I I I s c l e r o t i a per p l a t e .  15  10  u  ala asn met tyr cas hy CaN KN amCI  + ++ + —  + +  ala asn phe pro cashy KN  + —  sclerotia per plate + + + = >40 + + = 10-39 + = 1-9 - = 0 F i g u r e 19  + +++ —  —  +++ +  -41incubated  a t 15 C.  A f t e r 27 days, an average of 85 mature  s c l e r o t i a were present wheat germ.  i n c u l t u r e s grown with 3.5-^.5 g/1 of  In comparison, only 6-10 mature s c l e r o t i a were  produced i n the same p e r i o d of time on Medium A. 21 shows, wheat germ c o n c e n t r a t i o n s the formation  As f i g u r e  from 2.5-9*0 g/1 supported  of l a r g e numbers of mature s c l e r o t i a ,  and these  s c l e r o t i a were w e l l - d e f i n e d and e a s i l y l i f t e d from the colony. At c o n c e n t r a t i o n s  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 v i a b l e , were d i f f i c u l t from the c a r t i l a g i n o u s colony. centrations greater  On media w i t h wheat germ con-  than 7.5 g/l» the dark m y c e l i a l c r u s t soon  developed and overgrew many mature s c l e r o t i a , inseparable The  rendering  them  from the colony.  active fraction  of the wheat germ was s o l u b l e i n c o l d  water but not s o l u b l e i n ethanol, A quantity  to separate  e t h y l ether,  or  chloroform.  of wheat germ f l a k e s was e x t r a c t e d with 100 ml  of  water a g i t a t e d by a magnetic s t i r r e r f o r 20 minutes a t 20 C. The  water s o l u t i o n was f i l t e r e d and then used to make up 100 ml  of Medium A l a c k i n g a s p a r a g i n e .  Sclerotium  production  was  compared w i t h that on media prepared with the e x t r a c t of 2.0, 4.0, and 6.0 g/1 of wheat germ. f o r 47 days.  C u l t u r e s were grown a t 15 C  The r e s u l t s a r e shown i n f i g u r e 22.  sclerotium production  occurred  Maximum  when the e x t r a c t of 6.0 g/1  was used. The water e x t r a c t of wheat germ d i d not support the prod u c t i o n of as many s c l e r o t i a as d i d wheat germ f l a k e s .  This  water s o l u b l e f a c t o r ( s ) was found to be d i a l y z a b l e and heat stable.  No f u r t h e r attempt was made to c h a r a c t e r i z e the a c t i v e  fraction  of wheat germ.  -1*2-  FACING PLATE 13 F i g u r e 20:  The r e l a t i o n s h i p of s c l e r o t i u m development to m y c e l i a l growth on Medium C a t 15 C.  -43FACING PLATE 14 E f f e c t of Wheat Germ on S c l e r o t i u m Formation Figure 2 1 :  The e f f e c t of wheat germ c o n c e n t r a t i o n on the p r o d u c t i o n of s c l e r o t i a .  C u l t u r e s were grown  at 1 5 C f o r 2 7 days.  Figure 2 2 :  S c l e r o t i u m p r o d u c t i o n on media prepared w i t h three c o n c e n t r a t i o n s of a water e x t r a c t of wheat germ. at  1 5 C.  C u l t u r e s were grown f o r 4 7 days  90  60  30 i  5  0  10 15 wheat germ, g/\ Figure 2 1  500)  •+-•  03  d 0) d  __  o _Q) O  40302010 0  2.0 4.0 6.0 wheat germ extracted, g/\ Figure 2 2  20  VII. A.  S c l e r o t i u m Germination General  Observations  In nature, and i n the l a b o r a t o r y , the s c l e r o t i a of T. erythropus germinate  to produce  the d i m i n u t i v e c l a v a t e b a s i d i o -  carps c h a r a c t e r i s t i c of the s p e c i e s .  Sclerotia also  germinate  i n c u l t u r e by producing d i k a r y o t i c hyphae when s e t on f r e s h media.  Germination of the f i r s t  type was  the s u b j e c t of t h i s  p a r t of the i n v e s t i g a t i o n . Mature s c l e r o t i a from c u l t u r e germinated w e l l i n p e t r i p l a t e s of water agar.  On Medium A m y c e l i a l growth from  o t i a p l a c e d on the agar s u r f a c e was  scler-  much more vigorous than  that on water agar, and no b a s i d i o c a r p s arose from  sclerotia  incubated on the n u t r i e n t medium. The temperature temperature  d u r i n g p r o d u c t i o n of s c l e r o t i a and  d u r i n g germination were the most important  a f f e c t i n g s c l e r o t i u m germination. was  the factors  The v i a b i l i t y of s c l e r o t i a  a l s o i n f l u e n c e d by the medium upon which they were  produced.  The young b a s i d i o c a r p o r i g i n a t e s 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 r e p o r t e d f o r T. s c l e r o t i o i d e s MacDonald ( 1 9 3 ^ ) •  The sporophore  primordium  by  i n the medulla  pushes outward, e v e n t u a l l y causing the outer l a y e r of c e l l s (the c o r t e x i n the T. erythropus s c l e r o t i u m ) to r u p t u r e and a l l o w i n g the young b a s i d i o c a r p to grow out through a jagged hole.  One  to e i g h t sporophores  T. erythropus Corner carp may  were produced  from  germinating  sclerotia.  ( 1 9 5 0 ) suggested  be determined  that the l o c a t i o n of the b a s i d i o -  during sclerotium formation.  on germinating s c l e r o t i a of T. erythropus confirmed  Observations this  -45-  opinion.  The rounded upper s i d e of a s c l e r o t i u m when a t t a c h e d  to the p a r e n t c o l o n y f o r convenience was d e s i g n a t e d the d o r s a l s u r f a c e , and the f l a t t e n e d l o w e r s i d e 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 b a s i d i o c a r p s 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 a g a r .  When s c l e r o t i a were not i n v e r t e d b e f o r e g e r m i n a t i o n , sporophores a r o s e from the d o r s a l s u r f a c e .  T h i s apparent p r e d e t e r m i n a t i o n  e f f e c t was observed i n s c l e r o t i a produced a t 4, 10, and 15  C.  No dormancy p e r i o d p r i o r t o g e r m i n a t i o n 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 a t 10 and 15 C.  However, s c l e r o t i a  grown t o m a t u r i t y a t 4 C r a r e l y germinated i f not g i v e n a 15-20  C t r e a t m e n t f o r 10-14  days b e f o r e g e r m i n a t i o n (see n e x t  sub-section). B. (1)  Temperature E f f e c t s Temperature D u r i n g G e r m i n a t i o n 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 p l a c e d on water agar and i n c u b a t e d a t 4, 10, 15, and 20  C.  Some s c l e r o t i a were a l s o g i v e n a f i v e day t r e a t m e n t a t 4 C p r i o r to i n c u b a t i o n a t 15 C on water a g a r .  As shown i n Table I ,  95-100$ of the s c l e r o t i a i n c u b a t e d a t 4 and 10 C germinated.  At  15 C, l e s s t h a n 20% produced b a s i d i o c a r p s , but a f t e r the 4 C t r e a t m e n t the g e r m i n a t i o n r a t e was 70$.  A t 0 and 20 C,  no  germination occurred. The r a t e of g e r m i n a t i o n (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 a t germination.  S c l e r o t i a i n c u b a t e d a t 4 C germinated q u i c k l y , w i t h  maximum g e r m i n a t i o n r e a c h e d 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 g e r m i n a t i o n a t 15 C f o l l o w i n g  -46t h e 4 C pretreatment.  This 3 5 day f i g u r e i n c l u d e s the f i v e  days of pretreatment. Although s c l e r o t i a germinated  a t 15 C, b a s i d i o c a r p s d e v e l -  oping from s c l e r o t i a a t t h i s temperature o f t e n remained At 10 and 4 C, sporophores to more than 2 0 mm  sterile.  matured q u i c k l y and f r e q u e n t l y grew  i n length.  Temperature e f f e c t s on b a s i d i o -  carp development a r e t r e a t e d i n another s e c t i o n of t h i s Both percentage and r a t e of germination were a l s o by the medium on which the s c l e r o t i a were grown. d i s c u s s e d i n another p a r t of t h i s  This i s  i n January  when incubated i n c l e a r p l a s t i c moist  chambers a t 4, 1 0 , 1 5 , or 1 5 C a f t e r one week a t 4 C. from August c o l l e c t i o n s germinated f o l l o w i n g 4 C pretreatment. without (2)  influenced  section.  S c l e r o t i a c o l l e c t e d on p e t i o l e s i n the f i e l d and May d i d not germinate  paper.  Sclerotia  r e a d i l y a t 4 C and 15 C  No germination o c c u r r e d a t 15 C  pretreatment.  Temperature During P r o d u c t i o n S c l e r o t i a grown a t 10 and 15 C on s u i t a b l e media were  v i a b l e a f t e r r e a c h i n g 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 a t 4 C. s c l e r o t i a produced  on Medium A a t 4 C germinated  pretreatment a t 1 5 - 2 0 C f o r 10-14 days.  However,  only a f t e r a  F o l l o w i n g treatment,  germination o c c u r r e d a t 4 C on water agar.  The 1 5 - 2 0 C t r e a t -  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 a t t a c h e d to the parent colony or separated from i t on water agar p l a t e s .  The t r e a t -  ment was i n e f f e c t i v e when s c l e r o t i a were d e s i c c a t e d .  A germ-  i n a t i o n r a t e of 8 5 $ was recorded from t r e a t e d s c l e r o t i a grown a t 4 C. The a d d i t i o n of c a s e i n h y d r o l y s a t e or wheat germ d i d not  -47-  FACING PLATE 15 Figure 23:  The e f f e c t of i n c u b a t i o n temperatures on the r a t e of s c l e r o t i u m g e r m i n a t i o n . produced on Medium C a t 15 C.  S c l e r o t i a were  Table I :  The e f f e c t of I n c u b a t i o n Temperature on s c l e r o t i u m g e r m i n a t i o n .  GERM. TEMP  10°  PRETREAT  GERM.  0-20 4° -7days 70 95-100 95-100  -48a l l e v i a t e the n e c e s s i t y of the heat treatment. Since i t was p o s s i b l e that a l i g h t stimulus was  involved  i n the p r o d u c t i o n of v i a b l e s c l e r o t i a , the 2 0 C treatment was performed i n darkness.  Treated and u n t r e a t e d s c l e r o t i a i n the  4 C i n c u b a t o r 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 I I a t 4 C and matured a t 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 a t 15 C. (3)  F r e e z i n g of S c l e r o t i a V i a b l e s c l e r o t i a were f r o z e n a t - 5 C f o r two weeks on  water agar p l a t e s .  They were then p l a c e d on f r e s h p l a t e s and  incubated a t 1 5 C.  A germination r a t e of 40$ was observed, 3 0 $  l e s s than the 4 G p r e t r e a t e d C. (1)  control.  Other P h y s i c a l F a c t o r s Drying of S c l e r o t i a V i a b l e s c l e r o t i a were a i r d r i e d a t 2 0 C f o r 2 5 days and  then p l a c e d on water agar a t 4 G f o r g e r m i n a t i o n .  A f t e r 30  days, 5 5 $ of the s c l e r o t i a had produced b a s i d i o c a r p s .  A 95$  germination r a t e o c c u r r e d i n the c o n t r o l . (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 a t 1 5 C were removed from the  colony and washed i n f l a s k s of s t e r i l e water a t 2 0 C.  To  simulate the l e a c h i n g e f f e c t of r a i n , the f l a s k s were s e t on a r e c i p r o c a t i n g shaker f o r 48 hours.  The washed s c l e r o t i a were  p r e t r e a t e d a t 4 C f o r 5 days and incubated on water agar a t 15 C. A f t e r 3 6 days, 7 1 $ germination had o c c u r r e d , the same as the unwashed c o n t r o l . (3)  S c l e r o t i u m Diameter I t i s known that the l a r g e r s c l e r o t i a of C i a v i c e p s  purpurea  -49(Fr.) T u l . a r e more v i a b l e than s m a l l e r ones (Cooke and M i t c h ell,  I966).  S c l e r o t i a from s e v e r a l c u l t u r e s of T. erythropus  were measured and a r e c o r d kept of t h e i r germination to d e t e r mine i f a s i m i l a r c o r r e l a t i o n e x i s t e d . diameters g r e a t e r than 0.7 mm, v i a b i l i t y was e v i d e n t .  Of the s c l e r o t i a with  no c o o r e l a t i o n between s i z e and  S c l e r o t i a measuring l e s s than 0.7  were seldom observed to germinate.  In nature, and i n c u l t u r e ,  the average s c l e r o t i u m diameter was g r e a t e r than 1.0 (4)  mm  mm.  I l l u m i n a t i o n of S c l e r o t i a S c l e r o t i a grown to m a t u r i t y i n the l i g h t were a b l e to  germinate when p l a c e d on water agar and incubated i n darkness. Although the sporophores that arose i n darkness u s u a l l y 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 c o n d i t i o n s . D.  Medium Composition  Effects  S c l e r o t i a were formed  i n c u l t u r e on a v a r i e t y of media a t  s e v e r a l 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 e q u a l l y v i a b l e . a t 10 C germinated p o o r l y rate  S c l e r o t i a grown on Medium B  ( l e s s than 5$) i n c o n t r a s t to a high  (95$) 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 e x t r a c t of wheat germ were equal i n v i a b i l i t y  to those produced on Medium C.  V a r y i n g the C/N r a t i o from 19:1 to.190:1 i n Medium A ( r e f e r to s e c t i o n VI) d i d not a l t e r the germination r a t e of s c l e r o t i a produced a t 15 C. The s c l e r o t i a that formed on water agar were small (0.50.8 mm)  and had a low germination r a t e .  Whether t h e i r low  v i a b i l i t y was an e f f e c t of s i z e o r of n u t r i e n t s was not determined.  -50E.  In S i t u Germination Stage I I I s c l e r o t i a that had developed on a colony on  Medium A or B d i d not u s u a l l y germinate i n p l a c e .  If l i f t e d  from the colony and r e l o c a t e d on the same p l a t e , no germination resulted.  Placement of a square of d i a l y s i s tubing between  the s c l e r o t i u m and the parent colony d i d not encourage ination.  germ-  Exceptions to t h i s f a i l u r e to germinate i n s i t u  were noted i n s e v e r a l month o l d c o l o n i e s grown on Media A and C.  F e r t i l e b a s i d i o c a r p s arose from s c l e r o t i a i n c u l t u r e s on  Medium C when i n c u b a t e d a 4 C a f t e r maturation a t 15 C.  Seven  month o l d c u l t u r e s on Medium A grown a t k C produced b a s i d i o c a r p s from s c l e r o t i a ;  the b a s i d i o c a r p s l a c k e d heads and were s t e r i l e .  S c l e r o t i a whose development  was a r r e s t e d between stage  II and I I I o f t e n gave r i s e to f e r t i l e sporophores i n c u l t u r e . T h i s o c c u r r e d most commonly on media w i t h a low C/N  ratio i n  c u l t u r e s i n c u b a t e d a t 10 and 15 C. VIII. A.  B a s i d i o c a r p Formation and Growth  General Observations In nature, b a s i d i o c a r p s of T. erythropus are known to  a r 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 Columbia.  British  However,, i n c u l t u r e , b a s i d i o c a r p s were produced from  s c l e r o t i a , d i r e c t l y from the colony s u r f a c e , and from the edges of the inoculum p l u g .  Often a medium that d i d not support  f r u i t i n g from the agar s u r f a c e induced some sporophore development from the inoculum b l o c k .  When plugs were i n v e r t e d  prior  to i n o c u l a t i o n , the appearance of b a s i d i o c a r p s was delayed, and the f i r s t the agar.  f r u c t i f i c a t i o n arose from the lower s i d e , n e a r e s t  T h i s o r i e n t a t i o n e f f e c t appeared to be a r e s u l t of  -51the much denser m y c e l i a l mat  formed on the upper s u r f a c e of  a culture. Small sporophores arose from inoculum plugs on water agar p l a t e s and sometimes developed i n the agar away from the i n o c ulation point.  On more f a v o r a b l e media b a s i d i o c a r p s arose  d i r e c t l y from the agar as w e l l as from the inoculum p l u g . O c c a s i o n a l l y sporophores were produced from lumps i n the colony.  sclerotium-like  These i l l - d e f i n e d lumps were r e p o r t e d  to be a b o r t i v e s c l e r o t i a i n c u l t u r e s of T. s c l e r o t i o i d e s Donald,  (Mac-  193^).  Since i t was sclerotia,  p o s s i b l e to o b t a i n b a s i d i o c a r p s without  the e f f e c t s of n u t r i t i o n a l and environmental f a c t o r s  on b a s i d i o c a r p f o r m a t i o n c o u l d be i n v e s t i g a t e d f r e e from the i n t e r f e r e n c e of t h i s p h y s i o l o g i c a l l y a c t i v e B.  Morphology  structure.  of the Mature B a s i d i o c a r p  Corner ( 1 9 5 0 ) has d e s c r i b e d the s t r u c t u r a l d e t a i l s of mature T. erythropus b a s i d i o c a r p s . of T. i s h i k a r i e n s i s ,  Except f o r the p o s s i b i l i t y  the many recombinations and synonyms a s s o c -  i a t e d w i t h other Typhulas are not found i n T. e r y t h r o p u s . The c h a r a c t e r i s t i c c o l o r i n g of the head and s t a l k , the general spore dimensions, and the s t r u c t u r e of the s c l e r o t i u m make t h i s an e a s i l y i d e n t i f i a b l e s p e c i e s .  Lehfeldt  (I923) found  this  s p e c i e s convenient to use because of the c e r t a i n t y of i t s identification.  I chose i t f o r the same reason.  B a s i d i o c a r p s were t y p i c a l l y unbranched, branching was  occasionally present.  but i n c u l t u r e  These branched  fructifica-  t i o n s were always found on p l a t e s i n c o n d i t i o n s u n f a v o r a b l e f o r development  ( i . e . on water agar or i n d a r k n e s s ) .  b a s i d i o c a r p s were seen i n f i e l d  collections.  No  branched  -52C.  B a s i d i o c a r p Development Sporophores f o l l o w e d  the same p a t t e r n of development  r e g a r d l e s s of t h e i r source of i n i t i a t i o n the agar s u r f a c e , or inoculum p l u g s ) .  ( i . e . from s c l e r o t i a ,  F o r convenience,  development has been d i v i d e d i n t o f o u r stages, IV.  At stage I  I I , I I I , and  the b a s i d i o c a r p 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. little  I,  their  This body elongates  with  i n c r e a s e i n diameter (0.2 mm), assuming a g r a d u a l l y  tapered  c a n d l e - l i k e shape up to 1.2 mm l o n g .  Stage I I commences  when the base of the young b a s i d i o c a r p begins to darken ( f i g . 31). T h i s darkening i s caused by the sheathing  c e l l s of the s t a l k  becoming t h i c k - w a l l e d and pigmented, much l i k e the c r u s t that forms on the s u r f a c e of o l d e r c o l o n i e s . sporophore elongates  The tapered  stage  II  f o r 3 6 - 4 8 hours when the upper 0.4 mm  suddenly s w e l l s to d e l i m i t a w e l l - d e f i n e d c l a v a t e head ( f i g . 35). The appearance o f the head i n d i c a t e s the onset of stage I I I . No hymenium i s present and  a t t h i s time.  head development continue.  Sporophore  elongation  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).  f o r 10-15 days under f a v o r a b l e  S p o r u l a t i o n continues  c o n d i t i o n s of temperature and humidity. the stage I V b a s i d i o c a r p continues  During t h i s  to elongate  interval  (see l a t e r f o r  details)'. Sporophores past m a t u r i t y and  collapsed.  took on a watery appearance  The head of a f a l l e n sporocarp gave r i s e to a  d i k a r y o t i c mycelium or was converted  to a s c l e r o t i u m on the  agar s u r f a c e . The II  spread  zone of e n c r u s t i n g hyphae that f i r s t appeared i n stage up the s t a l k a few mm behind the apex of the growing  -53-  basidiocarp.  Mature sporophores a r e 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 t h a t  a r o s e from a s c l e r o t i u m p o s s e s s e d 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 t h a t were produced from the mycelium the r e d d i s h - b r o w n s t a l k .  The temperature of i n c u b a t i o n and  the medium c o m p o s i t i o n a f f e c t e d p i g m e n t a t i o n . are d e t a i l e d i n a l a t e r  had  These e f f e c t s  sub-section.  The l e n g t h of b a s i d i o c a r p s d u r i n g d i f f e r e n t s t a g e s of development was r e l a t i v e l y c o n s t a n t . measured from 0 . 1 III  to 1.2  from 3 . 2 to 6 . 3 mm,  mm,  Stage I carpophores  s t a g e II from 1 . 2  to 3 . 2 mm,  and s t a g e IV from 6 . 3 t o 2 5 mm.  head of a 2 5 mm b a s i d i o c a r p averaged 4 mm  stage The  i n length.  The d u r a t i o n of each s t a g e and, t h u s , the time from  initia-  t i o n t o m a t u r i t y were v a r i a b l e , i n f l u e n c e d by temperature and medium c o m p o s i t i o n . another  These e f f e c t s a r e p r e s e n t e d i n d e t a i l i n  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 a t 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 c l o s e to the i n o c ulum p l u g .  As the c o l o n i e s grew, b a 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 p o i n t ( f i g . 3 3 ) •  A  60 mm  c o l o n y i n c l u d e d 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 inner  diameter  circle. All  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 t h a t d e s c r i b e d f o r i s o l a t e D.  diameter  T-4.  E x p a n s i o n of the B a s i d i o c a r p The growth of the s t a g e I primordium i n t o a mature b a s i d i o -  c a r p was b r o u g h t about a l m o s t e n t i r e l y by the i n f l a t i o n of  -54pre-existing cells.  A f t e r e a r l y stage I, few new  a t the apex of the sporophore.  This was  c e l l s formed  demonstrated by  a p p l i c a t i o n of marks to sporophores and by m i c r o s c o p i c ination.  B a s i d i o c a r p s i n v a r i o u s stages  the  exam-  of development from  s c l e r o t i a and  inoculum plugs were marked with spots of b l a c k  ink or spores  of Equisetum along t h e i r l e n g t h .  between the spots was and  determined.  developed a t the same r a t e as those growth of a sporophore from 1 mm  Unmarked b a s i d i o c a r p s  t h a t were marked.  to 8.3  mm  a non-expanding trunk.  I or I I sporophore elongated head.  i s shown i n f i g u r e  The  t i p (0.2-0.4 mm)  little,  The  I I I and  independent of the e l o n g a t i o n of the  r e g i o n approximately IV sporocarps,  the head.  0.4  mm  behind  the growth zone was  l o n g body, stalk. c o n f i n e d to  the apex.  In  stage  l o c a t e d j u s t below  The  percentage of a b a s i d i o c a r p e l o n g a t i n g was  v e r s e l y p r o p o r t i o n a l to the t o t a l l e n g t h of the F i g u r e 25  stage  R e l a t i v e to the base, the growth zone t r a v e l e d up  the s t a l k .  illustrates this relationship.  of the l e n g t h of a young 2 mm  Less than 40$  i n the process  of  3°$)  70$  of the l e n g t h of a 7 mm  elongating. n a  d  in-  fructification.  Approximately  sporophore was  hyphal c e l l s a t the base (the remaining  The  mm  growth zone of a stage I I b a s i d i o c a r p was  mm  of a  c o n t r i b u t i n g only to the  When the head had been d e l i m i t e d as a 0.4  i t s growth was  tion.  The  Only the upper p a r t of a b a s i d i o c a r p expanded, l e a v i n g  behind  a 1-2  distance  measured d a i l y with an", o c u l a r micrometer,  the zone of expansion was  24.  The  ceased  The infla-  basidiocarp  was  elongation.  r a t e of e x t e n s i o n of the sporophore i n c r e a s e d as  s t r u c t u r e developed.  At 10 C, a stage IV b a s i d i o c a r p  a t an average r a t e of J.2 mm/24 hours; a stage  the  elongated  I grew a t  just  -55-  FACING PLATE 16 F i g u r e 2k:  The growth of a b a s i d i o c a r p from 1 mm t o 8.3 Note t h a t the l o w e r p a r t ceases e l o n g a t i o n b e f o r e 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.  mm.  8--  LENGTH mm  I  II  III  STAGE Figure  2k  IV  -561.1 mm/24 hours.  The e l o n g a t i o n r a t e s of the f o u r stages are  i l l u s t r a t e d i n f i g u r e 25. E.  Temperature E f f e c t s B a s i d i o c a r p s arose from inoculum plugs p l a c e d on Medium  A a f t e r 2-3 days i n c u b a t i o n a t 4, 10, and 15 C.  Since  very  vigorous sporophore p r o d u c t i o n occurred when the plugs were p l a c e d on Medium A with 1.0 g / l of c a s e i n h y d r o l y s a t e i n p l a c e of asparagine, temperature.  t h i s medium was used to study  the e f f e c t s of  B a s i d i o c a r p s were formed r a p i d l y a t 4 and 10 C;  stage IV sporophores were observed 6-8 days a f t e r i n o c u l a t i o n .  on the i n o c u l a t i o n p l u g  At 15 C, development was r e t a r d e d ,  r e q u i r i n g up to 10 days f o r b a s i d i o c a r p s to reach stage IV.  The  e f f e c t of temperature on the d u r a t i o n of each stage i s shown i n Table I I and f i g u r e 27.  These data r e p r e s e n t average v a l u e s .  Not a l l stage I p r i m o r d i a completed t h e i r development.  The  delay i n r e a c h i n g stage IV a t 15 C was seen i n the t r a n s i t i o n from stage I I to I I I .  Head formation f r e q u e n t l y was  delayed  or t o t a l l y i n h i b i t e d a t 15 C. The p r o d u c t i o n of b a s i d i o c a r p s from the s u r f a c e of the colony was l i m i t e d to a much narrower temperature range that f o r t h e i r development from i n o c u l a t i o n p l u g s .  than  On Medium A  and Medium A with c a s e i n h y d r o l y s a t e , sporophores were r a r e l y produced a t 0, 4, or 10 C.  B a s i d i o c a r p s that d i d develop a t  these temperatures u s u a l l y were very s h o r t and were composed of of a sub-globose head on a t i n y s t a l k . sporophores l a c k e d pigmentation. b a s i d i o c a r p s developed grown on both media.  These 1-2 mm high  fertile  At 15 C, however, t y p i c a l  f r e e l y from the m y c e l i a l mat of c u l t u r e s S i x t y or more sporophores commonly were  produced i n a s i n g l e p e t r i p l a t e .  These mature f r u c t i f i c a t i o n s  -57-  FACING PLATE  17  Growth of the B a s i d i o c a r p - 2 F i g u r e 25:  The r e l a t i o n s h i p of the stage of development to the r a t e of extension of b a s i d i o c a r p s produced a t 10 C.  Figure  26:  The c o r r e l a t i o n of b a s i d i o c a r p l e n g t h to the l e n g t h of the growth zone i n e l o n g a t i n g basidiocarps.  3.0  EXTENSION RATE MM  /24  N  2.0H  1.04  0-  STAGE Figure  25  80-  %OF LENGTH EXPANDING  6 0 D U  ,  40-I  20H 0  T"  0  7  BASIDIOCARP LENGTH mm Figure  26  8*  -58-  FACING PLATE Figure  27:  18  The e f f e c t o f t e m p e r a t u r e on t h e r a t e o f basidiocarp  development.  p h o r e s p r o d u c e d a t k C was at  10 C.  The r a t e  f o r sporo-  v e r y s i m i l a r to t h a t  Table  I I :  Duration carp  of  the  stages  of  Basidio-  development  DURATION OF STAGES, DAYS 0-I  Ml  ll-lll  lll-IV  15°  2-3  1.5  3  2  8.5-9.5  10°  2-3  1.5-2  1.5-2  6-8  4°  2-3  2-3  1.5  1-1.5 1-2  TEMP  0-IV  65-8.5  IVH  STAGE II-  0  3  4 5 6 AGE, DAYS F i g u r e 27  8  T-  9  -59were i n d i s t i n g u i s h a b l e from those a r i s i n g from  sclerotia.  B a s i d i o c a r p s d e v e l o p i n g from the colony s u r f a c e a t 18 C l a c k e d a hymenium.  At 20 C, no sporophores were produced.  The e f f e c t of temperature on b a s i d i o c a r p s produced from s c l e r o t i a was s i m i l a r to that observed on sporophores a r i s i n g from i n o c u l a t i o n p l u g s .  At 4 and 10 C, l a r g e , t y p i c a l carpo-  phores were produced from v i a b l e s c l e r o t i a .  However, b a s i d i o -  carps a r i s i n g from s c l e r o t i a a t 15 C f r e q u e n t l y grew to j u s t 5-12 mm i n l e n g t h and remained a t stage I I . When these stage II  sporophores were incubated a t 4 or 10 C they resumed d e v e l -  opment and reached stage IV. No d i f f e r e n c e s were noted between b a s i d i o c a r p s produced from s c l e r o t i a from c u l t u r e or from the field. F.  pH E f f e c t s The e f f e c t of pH on b a s i d i o c a r p f o r m a t i o n was determined  by counting the number of mature sporophores produced i n c u l tures on Medium A a t 15 C. t h i s time.  The f i n a l pH was a l s o determined a t  C u l t u r e s were grown f o r 31 days a t 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, r e s p e c t i v e l y . ing  o c c u r r e d where the i n i t i a l  b a s i d i o c a r p s formed per p l a t e of  pH was 5.5-6.0, w i t h 15-18 (fig.  28).  At an i n i t i a l pH  7.0. f r u i t i n g was l e s s vigorous and was n e g l i g i b l e on media  w i t h and i n i t i a l G.  Maximum f r u i t -  Photo  pH of 4.0, 5.0, 7.7, or 8.5.  Effects  B a s i d i o c a r p s of T. erythropus a r e 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  in  the zone o f e l o n g a t i o n , not a t the apex.  When the p o s i t i o n  -60-  FACING PLATE 1 9 Figure 2 8 :  The e f f e c t of i n i t i a l pH on b a s i d i o c a r p prod u c t i o n on Medium A a t 1 5 C. grown f o r 31 days.  C u l t u r e s were  CO  CO  CD  LO  O CM  O  LO  CO  CL LU CC H  <<  O cu Q CC CO LU < CL  CQ  LO  CL  3  -H  -61-  of the l i g h t source was toward the new 35).  shifted,  source was  the growth of the sporophore  evident i n l e s s than nine hours ( f i g .  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.  L i g h t p a s s i n g through r e d c e l l o p h a n e d i d not  evoke a growth adjustment. Exposure  to l i g h t appeared  opment of mature b a s i d i o c a r p s .  to be necessary f o r the d e v e l Dark-grown c u l t u r e s  started  from l i g h t - g r o w n m y c e l i a f r e q u e n t l y produced branched stage I I b a s i d i o c a r p s or d i d not produce any sporophores.  Sclerotia  grown to m a t u r i t y i n the l i g h t sometimes gave r i s e to f e r t i l e b a s i d i o c a r p s when germinated i n darkness, but most b a s i d i o c a r p s d i d 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 s u r f a c e i n dark-grown c u l t u r e s from dark-grown m y c e l i a . i n these c u l t u r e s .  started  No f e r t i l e sporophores were produced  A c o n t r o l of dark-grown mycelium  inoculated  onto a p l a t e and grown i n the l i g h t produced f e r t i l e H.  Gravity  sporophores.  Effects  Observations of b a s i d i o c a r p s produced i n darkness i n i n v e r t e d p e t r i p l a t e s showed no signs of a p o s i t i v e or negative g e o t r o p i c response.  Sporocarps arose a t v a r i o u s angles away  from the agar s u r f a c e .  Since l i g h t has been i m p l i c a t e d i n the  g e o t r o p i c response of some basidiomycetes (Taber, I 9 6 6 ) , grown c u l t u r e s were incubated a t s e v e r a l a n g l e s . t i o n of the b a s i d i o c a r p s was  noted.  No c u l t u r e d  The  light-  orienta-  sporophores  of t h i s s p e c i e s were g e o t r o p i c . I.  Carbon:Nitrogen  (C/N) R a t i o E f f e c t s  The asparagine c o n c e n t r a t i o n i n Medium A was give C/N media was  ratios  (g C/g N) of 3.8:1  5-5, n d a  to 190:1.  a d j u s t e d to  The pH of a l l  c u l t u r e s were incubated a t 15 C.  A f t e r 26  -62-  day s the number of stage IV b a s i d i o c a r p s was f i n a l pH of the media was trated i n figure 2 9 . C/N  determined.  The  counted:, and  r e s u l t s are  B a s i d i o c a r p p r o d u c t i o n was  r a t i o of 1 9 : 1 , w i t h an average of 4 0 stage  per p l a t e . of 9 . 5 : 1 and  F r u i t i n g v i g o r was 38:1.  one  of and  9.5:1,  highest at a  h a l f t h i s value a t C/N  ratios  At 6 2 : 1 only 1 5 sporophores were present,  of 1 9 : 1 to 1 9 0 : 1 was and  4.7:1,  illus-  IV b a s i d i o c a r p s  and at g r e a t e r than 9 5 : 1 no f r u i t i n g o c c u r r e d . of media with C/N  the  5.5.  The  Media with  had f i n a l pH values of  3.8:1  final  5.8,  pH  ratios 7.0,  8.0, respectively. There was  a good c o r r e l a t i o n between the degree of f r u i t i n g  and b a s i d i o c a r p l e n g t h .  The medium that y i e l d e d the maximum  number of b a s i d i o c a r p s a l s o produced the l o n g e s t ones as w e l l . B a s i d i o c a r p s produced a t a C/N  r a t i o of 1 9 : 1 were l o n g  s l e n d e r , whereas those a t 3 . 8 : 1 were short and Other i s o l a t e s grown on C/N  J.  9-5:1  T-4.  Nutrient Concentration E f f e c t s The  was  stout.  r a t i o s of 9 5 : 1 and  responded i n the same manner as i s o l a t e  and  c o n c e n t r a t i o n of glucose and asparagine  adjusted  A C/N  i n Medium A  to make media of 1 / 1 0 to 2X r e g u l a r s t r e n g t h .  r a t i o of 9 * 5 : 1 was  maintained  were grown a t 1 5 C f o r 2 6 days.  i n a l l media.  Cultures  Maximum b a s i d i o c a r p  production  from the colony s u r f a c e occurred on the 1 / 1 0 s t r e n g t h medium ( 1 . 0 g / l glucose,  0.2 g / l asparagine).  sporophores were formed per p l a t e . nutrient concentration increased. b a s i d i o c a r p s on 1 / 2 , f u l l , 40,  2 5 , 2 0 , and  On t h i s medium 6 0 mature  F r u i t i n g decreased  as  the  The average number of mature  1.5X, and  2X s t r e n g t h media  2 per p l a t e , r e s p e c t i v e l y .  was  Sporophores were  formed e a r l i e s t on media with low n u t r i e n t c o n c e n t r a t i o n s .  -63-  FACING PLATE 20 F i g u r e 29=  The e f f e c t of v a r i o u s C/N r a t i o s on b a s i d i o carp p r o d u c t i o n a t 15 C. asparagine  The c o n c e n t r a t i o n of  was a d j u s t e d while the glucose  c e n t r a t i o n was maintained  a t 10.0  g/1.  con-  C/N F i g u r e 29  -64In c o n t r a s t to the decrease i n b a s i d i o c a r p p r o d u c t i o n as the s t r e n g t h of the medium i n c r e a s e d , m y c e l i a l growth was most vigorous K.  on the high s t r e n g t h media.  Nitrogen  Utilization  The d i f f i c u l t i e s a l r e a d y mentioned with regard n u t r i t i o n of s c l e r o t i u m development apply well.  to n i t r o g e n  to t h i s s e c t i o n as  Of the compounds t e s t e d , DL-asparagine and c a s e i n h y d r o l -  ysate  (1.0 g/1) supported maximum b a s i d i o c a r p p r o d u c t i o n .  sporophores were formed on media made with DL-alanine, Ca(N0^)2« support fers.  Although n i t r a t e s were p o o r l y u t i l i z e d ,  some b a s i d i o c a r p p r o d u c t i o n  Few  KNO^, or  they d i d  a f t e r three s e r i a l  trans-  No b a s i d i o c a r p s were produced from c u l t u r e s on media made  with DL-methionine, L - ( - ) - p h e n y l a l a n i n e , sine.  L - p r o l i n e , or L - t y r o -  Ammonium c h l o r i d e and ammonium s u l f a t e a l s o supported  no f r u i t i n g .  The r a p i d drop i n pH, p r o h i b i t i v e to growth, prob-  a b l y was r e s p o n s i b l e f o r the l a c k of b a s i d i o c a r p s .  Cultures  grown on 2% m a l t - e x t r a c t  agar ( D i f c o ) , used i n p r e l i m i n a r y  work with T. erythropus,  d i d not produce carpophores.  Addition  of 2.0 g/1 of ammonium s u l f a t e o r ammonium t a r t r a t e induced the formation  of robust  sporophores on the medium.  I t i s expected  that ammonium s a l t s could be u t i l i z e d f o r b a s i d i o c a r p if  formation  the pH of the medium could be maintained a t a s a t i s f a c t o r y  level. L.  Vitamin  Requirements  No b a s i d i o c a r p s were produced from c u l t u r e s grown on Medium A prepared without the v i t a m i n stock s o l u t i o n .  Addition  of 100 ug/1 of thiamine to the medium i n p l a c e of the v i t a m i n stock s o l u t i o n r e s t o r e d normal sporophore p r o d u c t i o n . serial  Two  t r a n s f e r s v e r i f i e d that thiamine was the r e q u i r e d  vitamin.  -65FACING PLATE 21 B a s i d i o c a r p Development F i g u r e 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.  F i g u r e 31:  Xl.5  Stage I I b a s i d i o c a r p s d e v e l o p i n g from the i n o c u l u m p l u g on the c a s e i n h y d r o l y s a t e medium. C u l t u r e was  grown a t 15 C.  t r o p i c response. F i g u r e 32:  Note the s t r o n g photo-  X7.  Stage IV b a s i d i o c a r p s d e v e l o p i n g 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 a t 10 C on Medium A w i t h c a s e i n h y d r o l y s a t e . 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 c a s e i n h y d r o l y s a t e . Note development from a g a r s u r f a c e .  A few  stage  I I I b a s i d i o c a r p s a r e p r e s e n t , but most are  stage  II.  Apparent b r a n c h i n g  i s an o p t i c a l  caused by o v e r l a y i n g s p o r o p h o r e s . F i g u r e J>k:  Germinating  a t 4 C. F i g u r e 35'  Most  Germination  sporowas  Approx. normal s i z e .  Stage I I I b a s i d i o c a r p s . t w i c e (90° The  X0.8.  s c l e r o t i a on water a g a r .  phores a r e a t e a r l y stage IV.  illusion  P l a t e had been r o t a t e d  each time) from the o r i g i n a l  position.  second r o t a t i o n o c c u r r e d 9 hours b e f o r e  photograph was ium A a t 15  C.  made.  C u l t u r e was  the  grown on Med-  -66DISCUSSION  I.  General C o n s i d e r a t i o n s The c u l t u r a l s t u d i e s performed w i t h T. erythropus p r o v i d e d  data that may e l u c i d a t e the l i f e nature.  c y c l e of t h i s fungus i n  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 a b l e to complete i t s e n t i r e l i f e 10 C.  These temperatures a r e comparable  to those p r e v a l e n t  d u r i n g the growing season of the organism. are  c y c l e a t if -  I n nature, s c l e r o t i a  formed i n the w i n t e r but remain dormant throughout the  s p r i n g and summer.  They germinate i n the f a l l when the h a b i t a t  is  I t appears l i k e l y  c o o l and moist.  that s c l e r o t i u m  activ-  a t i o n o c c u r r s i n the l a t e s p r i n g when s c l e r o t i a a r e t u r g i d . D e s i c c a t e d s c l e r o t i a from c u l t u r e c o u l d not be a c t i v a t e d . Mature  s c l e r o t i a c o l l e c t e d on p e t i o l e s i n May 19?0, d i d not  germinate when incubated a t 4, 10, or 15 C or a f t e r a p r e t r e a t ment of 4 C f o r 5-1° days f o l l o w e d by 15 C i n c u b a t i o n .  Sclerotia  c o l l e c t e d i n August, 1970, produced b a s i d i o c a r p s r e a d i l y when incubated a t 4 C but not a t 15 C. Not a l l Typhulas a r e autumnal. s p r i n g and autumn (Corner, 1950).  Some s p e c i e s s p o r u l a t e i n Corner a s c r i b e d t h i s  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 s p r i n g , when they should otherwise germinate.  This explana-  t i o n does not agree w i t h my f i e l d o b s e r v a t i o n s . Mature  sclerotia  of  T.  T. erythropus were present on p e t i o l e s by February.  s c l e r o t i o i d e s , another autumnal Acer p e t i o l e s .  s p e c i e s , a l s o was observed on  S c l e r o t i a of t h i s s p e c i e s were a l s o mature  b e f o r e the end of w i n t e r . II.  Mating System and Monokaryons T. erythropus appears to be f a c u l t a t i v e l y  homothallic.  -67In the absence of a compatible mating s t r a i n , s i n g l e - s p o r e h a p l o i d c u l t u r e s are a b l e to become d i k a r y o t i z e d .  However,  b e f o r e d i k a r y o t i z a t i o n , s i n g l e - s p o r e mycelia a r e p o t e n t i a l l y heterothallic.  In i t s h e t e r o t h a l l i c r e a c t i o n s , T. erythropus  is tetrapolar.  Four mating s t r a i n s were i d e n t i f i e d from the  nine monosporic i s o l a t e s d e r i v e d from i s o l a t e T-4. patibility  system i n  o n l y one o t h e r Typhula i s known;  carnata i s also t e t r a p o l a r Lehfeldt  The incom-  (Rj^ed,  T. i n -  I969).  ( I 9 2 3 ) , a student of Kniep,  crossed s i n g l e - s p o r e  m y c e l i a of T. erythropus and observed f u s i o n and clamp formation i n s u c c e s s f u l matings.  He c o u l d not determine  were b i p o l a r o r t e t r a p o l a r .  Kniep  i f this species  (I928) r e p o r t e d the formation  of h a p l o i d and " d i p l o i d " f r u c t i f i c a t i o n s by c u l t u r e s of the same s p e c i e s . were a c t u a l l y  I t i s not known i f these h a p l o i d f r u c t i f i c a t i o n s homodikaryotic.  In Sistotrema brinkmanni  three "subspecies" are r e c o g n i z e d ,  one h o m o t h a l l i c , one w i t h b i p o l a r h e t e r o t h a l l i s m , and one w i t h t e t r a p o l a r h e t e r o t h a l l i s m (Lemke, I 9 6 9 ) .  The same s i t u a t i o n  may e x i s t i n T. erythropus, but f u r t h e r study i s needed. Noble (1937) was a b l e to o b t a i n only f i v e monosporic i s o l a t e s of T. t r i f o l l i • c o u l d not determine  She demonstrated h e t e r o t h a l l i s m but  i f the s p e c i e s were b i p o l a r o r t e t r a p o l a r .  Lemke ( 1 9 6 9 ) noted that dikaryons c o u l d be homokaryotic  or  h e t e r o k a r y o t i c and used the terms "homodikaryotic" and "heterod i k a r y o t i c " to denote these s i t u a t i o n s . used i n the present Homodikaryotic  His terminology  was  study. c u l t u r e s of T. erythropus grew more s l o w l y  than h e t e r o d i k a r y o n s .  T h i s slow growth r a t e 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 i n o c u l a t i o n would seem  -68to  be f a v o r i n g outbreeding. D i k a r y o t i z a t i o n of the monokaryotic  observed  c o l o n i e s was  i n the young b a s i d i o c a r p s that arose i n the center of  the colony.  L a t e r , clamp connections were formed on the  hyphae a t the margin of the colony.  The h o m o d i k a r y o t i z a t i o n  process might be s i m i l a r to t h a t d e s c r i b e d i n Taphrina i960).  first  (Kramer,  In t h i s genus the d i k a r y o t i c c o n d i t i o n a r i s e s as a  r e s u l t of the m i t o t i c d i v i s i o n of the s i n g l e h a p l o i d nucleus d u r i n g the germination of a b l a s t o s p o r e o r an ascospore. Typhula mycelium, l i k e the Taphrina b l a s t o s p o r e , can produce h a p l o i d c e l l s f o r an indeterminate p e r i o d .  Under s u i t a b l e  d i t i o n s a homodikaryon i s i n i t i a t e d , and the l i f e be  slower growth r a t e of monokaryons i s common i n the  h i g h e r basidiomycetes  (Fincham  and Day, I 9 6 3 ;  Raper,  I966).  should be emphasized that a l l nine s i n g l e - s p o r e i s o l a t e s  were slow growing, and a l l developed w i t h i n one week of each o t h e r . f a c u l t a t i v e homothallism ter  c y c l e can  completed. The  It  con-  b a s i d i o c a r p s and clamps  This would suggest  that t h i s  i s a w e l l e s t a b l i s h e d g e n e t i c charac-  i n T. erythropus and that h o m o d i k a r y o t i z a t i o n was not a  r e s u l t of a contamination w i t h a compatible mating s t r a i n . T. t r i f o l i i (Noble, 1937).  produced  haploid basidiocarps i n culture  These sporophores  a t u r e d i k a r y o t i c sporophores. per basidium.  had the appearance of m i n i -  Four b a s i d i o s p o r e s were  The b a s i d i o s p o r e s were approximately  the s i z e of b a s i d i o s p o r e s from the d i k a r y o n .  produced  one h a l f  Only three of  Noble's f i v e h a p l o i d i s o l a t e s formed b a s i d i o c a r p s i n c u l t u r e . These same three a l s o formed h a p l o i d s c l e r o t i a i n c u l t u r e .  -69J-« erythropus b a s i d i o s p o r e s from b a s i d i o c a r p s formed on s i n g l e - s p o r e c u l t u r e s were equal i n s i z e to normally basidiospores.  Hanna  (I928)  produced  r e p o r t e d that spores from h a p l o i d  and d i k a r y o t i c f r u c t i f i c a t i o n s of Coprinus lagopus were of equal  size.  Ill.  M y c e l i a l Growth, D i k a r y o t i c The g e n e r a l m o r p h o l o g i c a l f e a t u r e s of m y c e l i a l growth of  T. erythropus a r e s i m i l a r to those r e p o r t e d f o r T. s c l e r o t i o i d e s by MacDonald ( 1 9 3 * 0 •  The growth r a t e of 2-3 mm/day i n c r e a s e i n  colony diameter i n c u l t u r e s of T. erythropus was l e s s than the r a t e s recorded f o r other s p e c i e s .  D e j a r d i n and Ward  (1971)  noted r a t e s of 3.5 mm/day f o r T. i n c a r n a t a , 3.8 mm/day f o r T. i d a h o e n s i s , and 4.4 mm/day f o r T. t r i f o l i i .  An undetermined  Typhula s p e c i e s showed a r a t e o f 8 mm/day (Lockhart, T. erythropus was a b l e to grow a t temperatures 20 C.  I967). from 0 to  B a s i d i o s p o r e germination o c c u r r e d over the same range.  The p s y c h r o p h i l i c nature of many Typhula s p e c i e s i s w e l l  docu-  mented (MacDonald, 1934; Tasugi, 1935; Remsberg, 1940; T e r u i , 1941;  Ekstrand, 1 9 5 5 ; Tomiyama, 1 9 5 5 ; Potatosova, 1960a; Jack-  son, I 9 6 3 ; Lockhart, 196?; Corner, 1970; D e j a r d i n and Ward, I 9 7 I ) .  Doubtless, t h i s a b i l i t y  to grow a t very low temperatures  when other f u n g i a r e i n h i b i t e d i s important to t h e i r mode of existence. P s y c h r o p h i l y i s not uncommon i n the f u n g i .  Other psychro-  p h i l i c s p e c i e s , e x c l u d i n g Typhulas, amd t h e i r optimum temperatures f o r growth i n c l u d e S c l e r o t i n i a b o r e a l i s , 0 C (Ward, I966), Phacidium i n f e s t a n s , 1 5 C (Perhson, 1948), 1 5 C (Cochrane,  I958),  Herpotrichia nigra,  and an u n i d e n t i f i e d basidiomycete, 12 -  17 C (Ward, e t a l , I 9 6 I ) .  -70Th e optimum pH f o r m y c e l i a l growth of T. erythropus 4-6. i  n  was  D e j a r d i n and Ward ( I 9 7 I ) r e p o r t e d maximum l i n e a r e x t e n s i o n  5!*  trifolii,  T. i n c a r n a t a , and T. i d a h o e n s i s a t pH  5-7.  T h e i r work v e r i f i e d Tasugi's (1935) r e p o r t on the optimum pH for  T. i n c a r n a t a (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  d e f i c i e n c y of t h i s type i s common i n the h i g h e r basidiomycetes 1 9 5 8 ) , but has not been i n v e s t i g a t e d i n other s p e c i e s  (Cochrane, of  Typhula.  The scant growth that d i d occur i n the apparent  absence of thiamine probably can be a t t r i b u t e d to i m p u r i t i e s in  the other i n g r e d i e n t s  of the medium.  glucose f r e q u e n t l y are contaminated  Agar, asparagine,  with b i o l o g i c a l l y  i c a n t amounts of the v i t a m i n (Cochrane,  and  signif-  I958).  Asparagine and c a s e i n h y d r o l y s a t e as n i t r o g e n sources supported maximum m y c e l i a l growth.  They were a l s o s u p e r i o r to  other sources f o r s c l e r o t i u m and b a s i d i o c a r p p r o d u c t i o n . N i t r a t e s d i d not support the f o r m a t i o n of a dense m y c e l i a l  mat.  The r a p i d drop i n pH to i n h i b i t o r y l e v e l s d u r i n g ammonium u t i l i z a t i o n by T. erythropus has been noted i n many other f u n g i (Apparao,  1956;  Cochrane, 1958;  i n i t i a l growth that was  Ward, 1964;  Curren, I 9 6 8 ) .  made on media with ammonium s a l t s  dense and comparable to that w i t h a s p a r a g i n e .  I t appeared  The was that  ammonium would have supported the p r o d u c t i o n of a dense m y c e l i a l mat  had the pH not changed.  IV.  S c l e r o t i u m Development The development of s c l e r o t i a of T. erythropus was  to  that d e s c r i b e d f o r other Typhulas  1934;  Remsberg, 1940).  (deBary,  1887;  similar  MacDonald,  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 in The  days to reach m a t u r i t y a f t e r i n i t i a t i o n comparison to the 14-16  (MacDonald, 1 9 3 4 )  days r e q u i r e d "by T. erythropus.  same f o u r stages of development were noted d u r i n g 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 t h e i r f o r m a t i o n by T. erythropus. maturing  exudation of l i q u i d  from  f i r s t r e p o r t e d by deBary i n I 8 8 7 .  s c l e r o t i a was  and l a t e r i n v e s t i g a t o r s  He  (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 the compaction  The  mainly water that was  of the s c l e r o t i u m .  erythropus s c l e r o t i a was  evaporated  c r y s t a l l i n e d e p o s i t remained.  expelled during  When the l i q u i d from  T.  on a g l a s s s l i d e , a white  Remsberg observed  the same phe-  nomenon i n o t h e r Typhula s p e c i e s . The e f f e c t s of temperature difficult of  to a s s e s s .  A d e f i n i t e i n t e r a c t i o n between  i n c u b a t i o n and the C/N  S c l e r o t i u m p r o d u c t i o n was C/N  of 9 5 : 1 ; a t 4 and  D e j a r d i n and Ward  on s c l e r o t i u m p r o d u c t i o n were temperature  r a t i o of the medium was e s t a b l i s h e d . maximal a t 1 $ C on a medium with a  1 0 C, the optimum C/N was  (I97I) mentioned  9.5:1.  that the g r e a t e s t number of  s c l e r o t i a were formed 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 (note h i g h C/N)  a t temperatures  above 1 0 C.  I f the  same temperature-C/N r e l a t i o n s h i p e x i s t s f o r the s p e c i e s i n t h e i r study as f o r T. erythropus, t h e i r o b s e r v a t i o n i s of value without more experimental d a t a . for  MacDonald's  (193*0  same may  be  said  o b s e r v a t i o n that maximal s c l e r o t i u m pro-  d u c t i o n by T. s c l e r o t i o i d e s was The i n c u b a t i o n temperature  a t 1 3 - 1 5 C on  PDA.  a l s o i n f l u e n c e d the time to  s c l e r o t i u m i n i t i a t i o n and to m a t u r i t y . most r a p i d a t 1 0 and  The  little  These processes were  1 5 C, s c l e r o t i a appearing 8 - 1 2 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. otium f o r m a t i o n o c c u r r i n g 5-14 s p e c i e s she examined.  Remsberg (1940) r e p o r t e d s c l e r days a f t e r i n o c u l a t i o n i n the  14  MacDonald (193^) r e c o r d e d 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 D e j a r d i n and Ward (1971) 4 days f o r T. t r i f o l i i and T. i d a h o e n s i s and 6 days f o r T. i n c a r n a t a .  The l a t t e r s p e c i e s was  to r e q u i r e 7-10 days O n  r e p o r t e d by J a c k s o n  (I963)  PDA.  The c o a l e s c e n c e and f u s i o n of s c l e r o t i a a t l o w e r temperat u r e s i n T. e r y t h r o p u s was other species.  d e s c r i b e d p r e v i o u s l y by Remsberg f o r  T h i s e f f e c t was  e v i d e n t o n l y when c u l t u r e s were  grown on a r i c h medium o r one w i t h a low C/N  ratio.  Corner  (1950) proposed t h a t 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  p r o b a b l y 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 . i s 40:1  to 50:1  The C/N  r a t i o of f o r e s t  litter  (Brock, I 9 6 6 ) .  Townsend (1957) examined s c l e r o t i u m f o r m a t i o n i n s e v e r a l s p e c i e s of f u n g i 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 r e c o g n i z e d t h r e e d i f f e r e n t s t a g e s of  sclerotium formation: i n i t i a t i o n , maturation. ments.  growth to f u l l s i z e ,  and  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 -  These s t a g e s correspond  present i n v e s t i g a t i o n .  to stages I a , l b , and I I i n the  Stage I I I of the T. e r y t h r o p u s  sclero-  tium i s e q u i v a l e n t to the "mature s c l e r o t i u m " i n Townsend s f  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 f o r m a t i o n i n T. e r y t h r o p u s •  On Medium A  prepared  w i t h g a l a c t o s e , s c l e r o t i a d i d not d e v e l o p 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 p r o 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 s o u r c e .  -73Except f o r the a b o r t i v e s c l e r o t i a that developed on Medium A at  1 5 C, stage I I s c l e r o t i a , once formed, continued  on to  stage  III. Townsend ( 1 9 5 7 ) and  others  (Hawker, 1 9 5 0 ; Cochrane, 1 9 5 8 )  have r e p o r t e d that f a c t o r s f a v o r i n g v e g e t a t i v e growth a l s o favor sclerotium production. c u l t u r e s of T. erythropus The  This c o r r e l a t i o n was  grown on media of v a r i o u s  r i c h e s t medium supported  the optimum temperature f o r m y c e l i a l growth and 1 5 C.  strengths.  maximal s c l e r o t i u m p r o d u c t i o n  the h i g h e s t r a t e of l i n e a r e x t e n s i o n of hyphae.  formation was  observed i n  and  Furthermore, sclerotium  This would i n d i c a t e that s c l e r o t i u m f o r -  mation can be c l a s s i f i e d as v e g e t a t i v e growth i n c o n t r a s t to r e p r o d u c t i v e growth.  Cochrane ( 1 9 5 8 ) suggested t h a t ,  s c l e r o t i u m development, as a prelude may  to sexual  reproduction,  be expected to be i n f l u e n c e d by the same f a c t o r s . "  ever, he presented  no  evidence  i n support  of t h i s .  How-  Such f a c -  t o r s as h i g h e r temperatures, high s t r e n g t h media, and C/N  "...  high  r a t i o s at 1 5 C, which f a v o r e d s c l e r o t i u m p r o d u c t i o n i n  T. erythropus  f r e q u e n t l y were i n h i b i t o r y to b a s i d i o c a r p f o r -  mation. Wheeler and  Waller  Sclerotium r o l f s i i  (I965) found that the i n i t i a t i o n of  Sacc. s c l e r o t i a i n c u l t u r e 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 d i d not appear u n t i l the p l a t e was lium. pus .  This e f f e c t was  not observed i n c u l t u r e s of T.  Sclerotium i n i t i a t i o n ,  completed before The  f i l l e d with myce-  growth, and maturation  the p l a t e was  erythro-  were o f t e n  covered.  s t i m u l a t o r y e f f e c t of wheat germ on s c l e r o t i u m f o r -  mation was  evident i n two ways.  First,  s c l e r o t i a were formed  -74e a r l i e r on Medium C than on any other media.  I t has been  shown p r e v i o u s l y that f a c t o r s f a v o r i n g m y c e l i a l growth a l s o favor sclerotium production.  The short l a g p e r i o d f o r the  m y c e l i a l growth r a t e was r e f l e c t e d i n the more r a p i d appearance on mature s c l e r o t i a on Medium C. increase  The second e f f e c t was to  the number of s c l e r o t i a produced per p l a t e .  These  b e n e f i c i a l e f f e c t s of wheat germ could r e s u l t from the i n c l u s i o n of new i n g r e d i e n t s i n t o the medium ( i . e . , vitamins,  sugars,  amino a c i d s ) , the a l t e r a t i o n of the p r o p o r t i o n of the other i n g r e d i e n t s , o r both.  The f a c t o r o r f a c t o r s i n wheat germ  were water s o l u b l e , heat s t a b l e , and d i a l y z a b l e . V.  Sclerotium  Germination  A l l previously reported  attempts to germinate  sclerotia  of Typhulas were done by p l a c i n g them on moist s t e r i l e or s o i l .  The use of water agar f o r t h i s purpose i n the present  i n v e s t i g a t i o n was s u p e r i o r to previous reasons:  sand  methods f o r three  1) i t f a c i l i t a t e d o b s e r v a t i o n s ,  e a s i l y reproducible,  2) r e s u l t s were  3) n u t r i t i o n a l f a c t o r s were minimized.  No dormancy p e r i o d o r s p e c i a l treatment was necessary f o r s c l e r o t i u m germination i n any species p r e v i o u s l y Mature s c l e r o t i a of T. t r i f o l i i colony  tested.  germinated r e a d i l y on the parent  but r e q u i r e d an undetermined r e s t i n g p e r i o d when sown  on moist s o i l . ( N o b l e , 1937).  I t was not s t a t e d i n e a r l i e r  p u b l i c a t i o n s i f s c l e r o t i a produced a t temperatures s i m i l a r to those i n the f i e l d were as v i a b l e as those produced a t the optimum temperature f o r m y c e l i a l growth.  I found that  of T. erythropus produced a t 4 C or from f i e l d  sclerotia  collections i n  May d i d not germinate under c o n d i t i o n s f a v o r a b l e f o r the germi n a t i o n of s c l e r o t i a grown a t 10 and 15 G.  The 4 C grown  -75s c l e r o t i a r e q u i r e d treatment a t 15-20  C f o r germination to  occur. A good c o r r e l a t i o n between s c l e r o t i u m s i z e and has been shown i n C l a v i c e p s purpurea  viability-  (Cooke and M i t c h e l l ,  I966).  Large s c l e r o t i a of t h i s s p e c i e s were s i g n i f i c a n t l y more v i a b l e than s m a l l e r s c l e r o t i a .  This r e 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 sclerotia  (0.7 mm)  that never  germinated.  Corner (1950) i n t e r p r e t e d the Typhula s c l e r o t i u m as an a d a p t a t i o n to low temperature, but my r e s u l t s i n d i c a t e  that  i t s f u n c t i o n i s to c a r r y the fungus through the warm, dry summer.  During t h i s time no f l e s h y , newly f a l l e n p e t i o l e s are  a v a i l a b l e to support m y c e l i a l growth. 20 C, m y c e l i a l growth was  At temperatures above  essentially n i l .  Species of t h i s  genus, i n c l u d i n g T. erythropus, w i l l grow a t very low tures.  In r e g i o n s w i t h very severe w i n t e r s the s c l e r o t i u m  have the d u a l f u n c t i o n of o v e r w i n t e r i n g and At l e a s t  First,  oversummering.  s c l e r o t i a produced a t 4 C norm-  a l l y r e q u i r e d a treatment of 15-20  C f o r germination to occur.  Ten to f o u r t e e n days a t the e l e v a t e d temperature were ent to ensure g e r m i n a t i o n .  by t h e i r f r e q u e n t exposure Only 6-10  suffici-  The o c c a s i o n a l 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  to room temperature d u r i n g examina-  month o l d 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 t h i s was time of heat treatment was I t was  may  two phases of s c l e r o t i u m germination were i n f l u -  enced by temperature.  tion.  tempera-  sporadic.  A minimum  not determined.  noted that s c l e r o t i a produced a t k, 10, and 15 C  -76-  had the areas of b a s i d i o c a r p o r i g i n e s t a b l i s h e d w i t h i n them during sclerotium formation. a prelocation occurred.  Corner ( 1 9 5 0 )  suggested that such  The e f f e c t i v e n e s s of the 1 5 - 2 0 C  treatment on s c l e r o t i a grown a t k C seems to be i n b r i n g i n g the  b a s i d i o c a r p p r i m o r d i a to a l e v e l of d i f f e r e n t i a t i o n com-  p a r a b l e to that i n 1 5 C grown s c l e r o t i a .  Cross s e c t i o n s of  s c l e r o t i a produced a t 1 0 and 1 5 C f r e q u e n t l y 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. produced a t 4 C.  No such area were noted i n s c l e r o t i a  Thus, while the l o c a t i o n of the b a s i d i o -  carp o r i g i n may be p h y s i o l o g i c a l l y determined d u r i n g s c l e r o t i u m f o r m a t i o n a t k C, m o r p h o l o g i c a l d i f f e r e n t i a t i o n a p p a r e n t l y does not occur u n t i l the s c l e r o t i u m i s exposed of  to temperatures  1 0 C or higher. S c l e r o t i a had to be t u r g i d f o r the heat a c t i v a t i o n to be  effective.  S c l e r o t i a of C l a v i c e p s purpurea r e q u i r e a c o l d  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  ( M i t c h e l l and Cooke, I 9 6 8 ) .  The second temperature e f f e c t concerned the germination of  s c l e r o t i a produced a t 1 5 C and of a c t i v a t e d s c l e r o t i a grown  at  4 C.  The c e l l  carp primordium  or c e l l s  that d i f f e r e n t i a t e d i n the b a s i d i o -  p r i o r to germination seldom developed f u r t h e r  when l e f t a t 1 5 C.  Rather, most r a p i d germination r e s u l t e d  when s c l e r o t i a were incubated a t k C.  At the reduced  t u r e , b a s i d i o c a r p f o r m a t i o n was s t i m u l a t e d . the  tempera-  This would e x p l a i n  e f f e c t i v e n e s s of the 5 - 7 day treatment a t k C on the i n -  creased germination r a t e of 1 5 C grown s c l e r o t i a .  When these  s c l e r o t i a were r e t u r n e d to 1 5 C to germinate, the germination process had a l r e a d y been t r i g g e r e d .  Continued sporophore  devel-  -77opment a t 15 C was Potatosova  another matter, and i t i s d i s c u s s e d l a t e r .  (1960a) r e p o r t e d a temperature  e f f e c t on  otium germination s i m i l a r to the one d i s c u s s e d above. of  T. v a r i a b i l i s ,  T. t r i f o l i i ,  scler-  Sclerotia  T. i n c a r n a t a , and T. i d a h o e n s i s  were p l a c e d outdoors i n pots of s t e r i l e sand and covered w i t h glass.  F r u c t i f i c a t i o n s d i d not appear u n t i l the o u t s i d e temp-  e r a t u r e s had dropped s e v e r a l degrees. ilis  and T. t r i f o l i i  Sporophores  of T. v a r i a b -  were produced a t 8-15.5 C, of T. i n c a r n a t a  at- 1.4-13.5 C, and of T. i d a h o e n s i s a t 1.4-4.6 G. T. erythropus s c l e r o t i a produced a t 10 C germinated  on  water agar a t 10 C, although more r a p i d germination o c c u r r e d at VI.  4 C. B a s i d i o c a r p Growth and Development Remsberg (1940) and Tasugi ( I 9 2 9 ,  considerable d i f f i c u l t y  1935)  encountered  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 s p e c i e s .  They found that  UV r a d i a t i o n  (265O-325O &), which was  Pyrex glassware, was heads.  necessary f o r the development  In the absence  s t a l k s were produced.  T. t r i f o l i i  variabilis  of  of f e r t i l e sterile  However, not a l l Typhulas r e q u i r e  (Noble, 1937;  T. s c l e r o t i o i d e s  T.  sporophores  T. erythropus belongs to t h i s  s p e c i e s a b l e to s p o r u l a t e under  this  (MacDonald,  Potatosova, 1960a), and  (Potatosova, 1960a) produced f e r t i l e  when grown under g l a s s .  through  of t h i s q u a l i t y UV l i g h t , long,  wavelength of UV to s p o r u l a t e . 1934),  not t r a n s m i t t e d  group  glass.  The b a s i d i o c a r p s of T. erythropus are s t r o n g l y t r o p i c , as are those of T. s c l e r o t i o i d e s  (MacDonald,  C o n s i d e r i n g the m i c r o h a b i t a t , the advantage  photoI934).  to the fungus i n  p o s s e s s i n g t h i s p o s i t i v e p h o t o t r o p i c response i s obvious. In  -78the shaded l e a f l i t t e r a p o s i t i v e l y p h o t o t r o p i c would grow toward a l i g h t source,  sporophore  a v o i d i n g contact with  moist d e b r i s p i l e d around the s c l e r o t i u m .  A negative  the geo-  t r o p i c response would reduce the e f f e c t i v e n e s s of spore d i s charge by d r i v i n g the b a s i d i o c a r p i n t o the l i t t e r sclerotium.  The  sporophore of T. erythropus  above the  i s ensured of  maximum e f f i c i e n y i n spore r e l e a s e by growing toward open areas i n the l i t t e r where b a s i d i o s p o r e s d i s p e r s e d by  the wind.  MacDonald  are most l i k e l y  (193^)  to be  commented that  the  b a s i d i o c a r p s of T. s c l e r o t i o i d e s are n e g a t i v e l y g e o t r o p i c , he presented  no  but  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 p h o t o t r o p i c response. carp toward a new  I t was  growth adjustment of a b a s i d i o -  l i g h t source occurred  t i o n , below the head. stimulus was  The  I t was  i n the zone of  elonga-  not determined where the  light  received. not c o n c l u s i v e l y demonstrated that l i g h t  required f o r basidiocarp i n i t i a t i o n . the f o r m a t i o n  of the head and  from the mycelium.  L i g h t was  was  necessary f o r  hymenium on b a s i d i o c a r p s  arising  However, s c l e r o t i a grown i n c o n d i t i o n s , o f  l i g h t were sometimes able to produce f e r t i l e b a s i d i o c a r p s when germinated i n darkness. for  Thus i t seems that the l i g h t  stimulus  head development might be s t o r e d i n the s c l e r o t i u m  an emergent b a s i d i o c a r p had  reached stage I I .  noted that s c l e r o t i u m germination were independent The  and  I t was  until previously  b a s i d i o c a r p development  events.  development of the T. erythropus  a r b i t r a r i l y d i v i d e d i n t o f o u r stages sporophore, head formation,  and  basidiocarp  of primordium,  sporulation.  A  was  headless  similar  -79approach u s i n g f o u r stages  has been f o l l o w e d i n s t u d i e s of  carpophore development i n Agaricus (Bonner e t a l , 1956; Gruen, I963). recognized  seven stages  bisporus  (Lange) Imbach  Komagata and Okunishi  (I969)  i n development of carpophores of  Coprinus kimurae. In I887, deBary r e c o g n i z e d  that the growth zone i n sporo-  phores of T. v a r i a b i l i s was l i m i t e d to the apex. b e l i e v e d that c e l l d i v i s i o n continued  Moreover, he  i n the growing t i p u n t i l  the b a s i d i o c a r p had a t t a i n e d i t s f u l l l e n g t h .  He a l s o noted  that no f u r t h e r augmentation occurred a t the base of the MacDonald (±93^) commented that growth was a p i c a l  sporophore. in  the sporophore of T. s c l e r o t i o i d e s . Corner (1950) s t a t e d , i n r e f e r e n c e to Typhula sporophores,  that,  "... a p i c a l growth may be a r r e s t e d very e a r l y and i n f l a -  t i o n may be so prolonged  that the f r u i t - b o d i e s appear to emerge  as though from a button-stage,  as i n t y p i c a l a g a r i c s , but d e v e l -  opment i s never r e a l l y i n d i r e c t w i t h a p e r i o d of a p i c a l growth f o l l o w e d by a separate  p e r i o d of expansion."  cate that the p e r i o d of c e l l p e r i o d of c e l l  division.  c e l l d i v i s i o n has ceased. erythropus,  This would  indi-  i n f l a t i o n overlaps p a r t of the  Further,  i n f l a t i o n continues  after  What Corner s a i d i s true f o r T.  but he misjudged the nature  and the d u r a t i o n of the  o v e r l a p 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  slight inflation. d u r i n g stages  When the b a s i d i o c a r p elongates r a p i d l y  II-IV, i t i s as a r e s u l t of i n f l a t i o n .  new  c e l l s a r e formed a t a low r a t e i n the head r e g i o n .  new  c e l l s produce b a s i d i a on the slowly growing head.  However, These Basidio-  -80carp e l o n g a t i o n and basidium formation  a r e concurrent  events  that cease a t approximately the same time. There appears to be no s i g n i f i c a n t d i f f e r e n c e between the development of the T. erythropus a g a r i c sporophore. Agaricus  sporophore and that of an  Bonner et a l ( 1 9 5 6 ) marked carpophores of  b i s p o r u s with dots o f carmine and noted the r e g i o n  of b a s i d i o c a r p expansion and the movement of t h i s zone up the s t i p e of a d e v e l o p i n g  basidiocarp.  ( 1 9 2 4 ) and B o r r i s s (193*+)»  They found, as had B u l l e r  that maximum e l o n g a t i o n occurs i n  the upper p a r t of the s t i p e , below the cap.  This r e g i o n i s  r e s p o n s i b l e f o r the great i n c r e a s e i n the h e i g h t of a carpophore.  A f t e r the 2 cm button  the s t i p e , and c e l l sion. stipe  stage,  no new c e l l s a r e formed i n  i n f l a t i o n accounts f o r b a s i d i o c a r p expan-  The expansion of the a g a r i c p i l e u s i s independent of elongation. In stage I T. erythropus  basidiocarps,  0 . 5 mm below the t i p i s the growth zone.  the r e g i o n 0 . 2 -  As t h i s zone moves  upward, i t i n c r e a s e s to a 1 - 2 mm l o n g r e g i o n t r a v e l i n g away from the p r e v i o u s l y expanded lower p a r t of the s t a l k . formation  Head  and expansion a r e independent of s t a l k growth.  In both a g a r i c and Typhula f r u c t i f i c a t i o n s ,  the l e n g t h  of the growth zone (zone of expansion) i s i n v e r s e l y p r o p o r t i o n a l to the height of the b a s i d i o c a r p . l e n g t h of a t a l l ,  A s m a l l e r percentage of the  o l d e r sporophore i s expanding than t h a t of  a s h o r t , younger sporophore.  No new c e l l formation  occurs a t  the base of e i t h e r type of b a s i d i o c a r p . The  time from i n i t i a t i o n to maturity  not known f o r most s p e c i e s of Typhula.  of b a s i d i o c a r p s i s  Corner ( 1 9 5 0 )  a time of 1 5 - 2 0 days f o r T. v a r i a b i l i s , and MacDonald  recorded (1934)  -81-  reported  1-4  days f o r growth to f u l l  This time i n t e r v a l temperature. present full  5-6  i n T. erythropus  At 4 and  sclerotloides.  i s dependent upon i n c u b a t i o n  10 G, a s p o r u l a t i n g b a s i d i o c a r p i s  days a f t e r  the appearance of stage I .  s i z e r e q u i r e s another 3-5  frequently inhibited;  s i z e of T.  days.  Growth to  At 15 C, s p o r u l a t i o n i s  sporophores do not exceed stage I I i n  t h e i r development. Basidiocarp production  was  not  so s u b j e c t to the  a c t i o n between i n c u b a t i o n temperature and influenced sclerotium formation.  C/N  ratios  interthat  At temperatures below 15  sporophores seldom were produced from the colony  C,  surface.  S i m i l a r responses to i n c u b a t i o n temperature were observed i n all  sporophores, r e g a r d l e s s of t h e i r o r i g i n  colony  (i.e.,  s u r f a c e , or inoculum p l u g ) .  The  optimum pH f o r b a s i d i o c a r p formation  s u r f a c e was  6.0.  This c o n t r a s t s with  m y c e l i a l growth and  The  optimum C/N  The  colony  relevance  fats  of a low  i n T. erythropus  g i v e s the C/N  r a t i o of  of  C/N  19:1.  15=1  (Madelin,  r a t i o f o r f r u i t i n g from  the  to f r u i t i n g from s c l e r o t i a on maple  i s not  entirely  clear.  Brock  (I966)  r a t i o of "microorganisms" as approximately  the Typhula s c l e r o t i u m i s r i c h i n s t o r e d glycogen ( S c u r t i and  pH  from c u l t u r e s of  and a l a n i n e were the main c o n s t i t u e n t s  p e t i o l e s i n the f i e l d  Since  internal  r a t i o f o r b a s i d i o c a r p p r o d u c t i o n was  Coprinus lagopus grown on a medium with a C/N  The  colony  7.0.  Madelin r e p o r t e d maximum sporocarp formation  when glucose  from the  the lower pH optima f o r  sclerotium formation.  mature s c l e r o t i a i s near  1956).  sclerotia,  Converso, I965K  the C/N  10:1. and  of a s c l e r o t i u m i s  -82probably  higher than 1 0 : 1 .  culture. support  This would agree with data from  S c l e r o t i a contained  s u f f i c i e n t r e s e r v e m a t e r i a l s to  the p r o d u c t i o n of up to e i g h t b a s i d i o c a r p s without  exogenous n u t r i e n t  an  supply.  Maximum sporophore formation from the medium occurred on a 1/10 s t r e n g t h medium.  This response i s i n accord w i t h the  u s u a l d i s t i n c t i o n s noted between v e g e t a t i v e and r e p r o d u c t i v e growth (Cochrane, I 9 5 8 ) .  Reproduction  i n T. erythropus i s  f a v o r e d by a weak medium, and v e g e t a t i v e growth, i n c l u d i n g s c l e r o t i u m formation, VII.  i s f a v o r e d by a r i c h medium.  I n t e r a c t i o n of N u t r i t i o n a l / E n v i r o n m e n t a l  Factors  As r e s u l t s from experiments with T. erythropus  and other  f u n g i have shown, a s i n g l e i n g r e d i e n t of the medium does not operate  independently  of the other i n g r e d i e n t s , t h e i r  propor-  t i o n s , environmental c o n d i t i o n s , o r the phase of growth of the organism.  The e f f e c t s of temperature, pH, the molecular  con-  f i g u r a t i o n and c o n c e n t r a t i o n of carbon and n i t r o g e n sources, the C/N r a t i o , v i t a m i n s , interrelated.  s a l t s , and f u n g a l response are complexly  In f i g u r e 36 I have attempted to d e p i c t  relationships graphically.  Manipulation  these  of one f a c t o r changes  the optimum l e v e l of the other 6 f a c t o r s .  The p a r t i c u l a r  f u n g a l s t r u c t u r e c i t e d i n the c h a r t may be the mycelium, the s c l e r o t i u m , o r the b a s i d i o c a r p i n any stage of i t s development. Temperature appears to be the most important factor. and  single  Although a d e f i n i t e c o r r e l a t i o n between temperature  the optimum C/N r a t i o f o r s c l e r o t i u m p r o d u c t i o n was noted,  t h i s 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 .  C u l t u r e s on high or low C/N r a t i o media produced only  sclerotia  -83FACING PLATE 22 The I n t e r r e l a t i o n s h i p of N u t r i t i o n a l and  Environmental  F a c t o r s and Fungal Development F i g u r e J>6: A change i n any of the 7 f a c t o r s w i l l  influ-  ence the uptake of c e r t a i n substances, the optimum c o n c e n t r a t i o n of c e r t a i n substances, or the morphological response of the fungus. Each f a c t o r i n the hexagon a f f e c t s and i s a f f e c t e d by the o t h e r s , and a l l may  operate  as l i m i t i n g f a c t o r s i n the development of a p a r t i c u l a r fungal structure.  particular fungal structure  Figure 3 6  -84when incubated at temperatures p r e v a l e n t d u r i n g formation  sclerotium  i n nature.  The uptake of carbon and n i t r o g e n compounds by c e r t a i n f u n g i has been found to be a f f e c t e d by temperature I968).  to  I t i s p o s s i b l e that the e f f e c t i v e C/N  the hyphae of T. erythropus  (Burnett,  ratio available  when grown on Medium A at 15 C  i s d i f f e r e n t than that i n c u l t u r e s incubated a t 4 or 10 The  C.  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 a t 15  could have been the r e s u l t of s e v e r a l f a c t o r s . seen to be very important  The  pH  C  was  i n s c l e r o t i u m formation a t 15  C.  C u l t u r e s grown on media w i t h a pH of 4 or 5 d i d form some sclerotia.  The uptake of organic and  i n o r g a n i c n i t r o g e n com-  pounds by c e r t a i n f u n g i i s dependent upon pH and  the  t i o n of the compounds ( F r i e s , 1956;  Nicholas,  I966).  Jones,  I963;  concentra-  Amino a c i d s y n t h e s i s i n some f u n g i has been r e p o r t e d  be a l i m i t i n g f a c t o r at higher temperatures  ( D e v e r a l l , 1966).  C e r t a i n compounds are not as r e a d i l y u t i l i z e d as Thus, a C/N  to  r a t i o of 40:1-50:1 c i t e d f o r l i t t e r  others.  (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 s u b s t r a t e 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 u n a v a i l -  able to the organism. Inorganic  s a l t s and vitamins have been r e p o r t e d to be  l i m i t i n g f a c t o r s under c e r t a i n c o n d i t i o n s of growth 1958;  Casselton,  I966).  The  (Cochrane,  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 f a c t o r s must be examined i n a two-way r e l a t 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 Hawker (1957) and requirements and  c y c l e of an organism.  Cochrane (1958) r e p o r t e d that the  nutritional  optimum p h y s i c a l c o n d i t i o n s o f t e n d i f f e r i n  -85i n d i f f e r e n t stages VIII. The  of development of the same s p e c i e s .  Taxonomic Aspects and  small s i z e and  makes morphological premium.  Implications  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 c h a r a c t e r i s t i c s of taxonomic value a t a  Corner ( 1 9 5 0 , 1 9 7 0 ) uses head s i z e and  badidiospore  shape,  dimensions, s t a l k l e n g t h and width, c o l o r a t i o n ,  s u b s t r a t e , number of sporophores per s c l e r o t i u m , and  sclerotium  s t r u c t u r e as major taxonomic c r i t e r i a i n d i s t i n g u i s h i n g s p e c i e s . The  v a l i d i t y of s c l e r o t i a l c h a r a c t e r s f o r taxonomic perposes  was  negated by Rj^ed (I969) when he s u c c e s s f u l l y crossed  carnata with two  Corner ( 1 9 5 0 ,  T. graminum.  T. i n -  1 9 7 0 ) had p l a c e d these  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 d i f f e r e n c e s . The  f a i l u r e of s u c c e s s f u l mating between the monokaryotic  i s o l a t e s of T. erythropus  and  the l i m i t s of both s p e c i e s .  T. s c l e r o t i o i d e s helped  Corner ( 1 9 5 0 ) s t r e s s e d t h a t ,  f o r the red-brown c o l o r of the stem and ropusj  Although the f r u c t i f i c a t i o n s are s i m i l a r ,  sclerotioides."  the s c l e r o t i a l s t r u c -  species i s quite d i f f e r e n t .  The  hyphae of T. s c l e r o t i o i d e s are t h i c k - w a l l e d and erythropus  are t h i n - w a l l e d .  t h i c k n e s s of the cortex  with age. 2 5 mm,  those of  T.  and  differ.  s t a l k was  study showed that  not constant  but  the  increased  While a s p o r u l a t i n g b a s i d i o c a r p grew from 6 . 3 to  the head l e n g t h i n c r e a s e d from 1.2 to 4 . 0 mm.  a t i o n and carps  sclerotial  A l s o , the s u r f a c e p a t t e r n  C u l t u r a l data from the present l e n g t h of the head and  "But  s c l e r o t i u m i t jjT. e r y t h -  would h a r d l y be d i s t i n g u i s h a b l e from T.  ture of the two  to d e f i n e  s t a l k p r o p o r t i o n s were r e l a t i v e l y  produced from s c l e r o t i a .  Color-  constant  in basidio-  However, when s c l e r o t i u m - f r e e  sporophores were formed on a medium with a low  C/N  ratio,  the  -86s t a l k s were t h i c k and  s h o r t and were a p l a i n brown c o l o r .  The number of b a s i d i o c a r p s per s c l e r o t i u m 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 r a r e l y two  to three, up to 8  sporophores were produced from c u l t u r e d s c l e r o t i a .  Sclerotia  that gave r i s e to t h i s number of b a s i d i o c a r p s were no l a r g e r than those from f i e l d Basidiospore  collections.  s i z e from c u l t u r e d sporophores of T. e r y t h -  ropus v a r i e d from 6-8 JJL X 2-3.6 )i.  Ekstrand  c o n s i d e r a b l e importance on the length/width spores  of two  s p e c i e s he d e s c r i b e d , and,  ( 1 9 5 5 ) has  placed  r a t i o of b a s i d i o -  i n f a c t , the  were d i s t i n g u i s h e d by t h i s s i n g l e c h a r a c t e r .  W.  species  C. MacDonald  ( I 9 6 I ) d i d not agree and r e l e g a t e d both to synonomy with i d a h o e n s i s , a s p e c i e s d e s c r i b e d by Remsberg. genus can be d i v i d e d i n t o a l a r g e - s p o r e d spored  group; T. erythropus  Species  T.  i n this  group and a s m a l l -  i s i n the l a t t e r .  MacDonald (193*+) thought t h a t , "spore s i z e may  Although J . A. t u r n out to be  the only s t a b l e c h a r a c t e r i n the group," the v a r i a t i o n i n c e r t a i n s p e c i e s i s so great that t h i s p r e d i c t i o n i s u n l i k e l y . A s i n g l e b a s i d i o c a r p of T. p h a c o r r h i z a produced spores i n g from 1 1 - 2 0 ^ x k.7-?.5p r a t i o v a r i e d from 1.6 The nature  to  (Corner,  1950).  The  measur-  length/width  2.9.  of the s u b s t r a t e i n i d e n t i f y i n g  saprophytic  s p e c i e s 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 than an i n d i c a t i o n of s u b s t r a t e s p e c i a l i z a t i o n  (Corner,  data 1950).  Thus, n e a r l y a l l c u r r e n t l y used taxonomic c r i t e r i a i n t h i s genus are q u i t e v a r i a b l e . many s p e c i e s , we  U n t i l crosses are made between  cannot know the l i m i t s of morphological  var-  - 8 7 -  i a t i o n within a single species. In a d d i t i o n to the problem of s p e c i e s determination, 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 Species  i s questionable.  of both genera produce b a s i d i o c a r p s from the colony  s u r f a c e without s c l e r o t i a  (Koske and P e r r i n , I 9 7 I ) .  have been found i n the l i f e  c y c l e of P. p e t a s i t i d i s  I97O) and P. s e t i p e s (Koske and P e r r i n ,  I97I).  Sclerotia (Corner,  -88BIBLIOGRAPHY Apparao, A. 1 9 5 6 . The r o l e o f pH i n n i t r o g e n u t i l i z a t i o n "by P i r i c u l a r i a o r y z a e . E x p e r i m e n t i a , 1 2 : 2 1 5 - 2 1 6 . deBary, A. I 8 8 7 . Comparative morphology and b i o l o g y o f f u n g i , mycetozoa, and b a c t e r i a . Oxford Univ. P r e s s ( C l a r e n don), London. ;  B e r t h i e r , J . I 9 6 7 . Une n o u v e l l e c l a v a r i a c e e a scle"rote: P t e r u l a s c l e r o t i c o l a nov. sp. B u l l . Soc. M y c o l . F r . 8^:731-737.  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Kramer, C. L. i 9 6 0 . M o r p h o l o g i c a l development and n u c l e a r b e h a v i o r i n the genus T a p h r i n a . M y c o l o g i a , j52:295-320. Leach, C. M. I 9 6 2 . S p o r u l a t i o n o f d i v e r s e s p e c i e s of f u n g i under n e a r - u l t r a v i o l e t r a d i a t i o n . Can. J . B o t . , 4 0 : 1 5 1 - 1 6 1 L e h f e l d t , W. 1923. Uber d i e Entstehung des Paarkernmycels b e i h e t e r o t h a l l i s c h e n B a s i d i o m y c e t e n . Hedwigia, 6 4 : 3 0 - 5 1 . Lehmann, H. I 9 6 5 . Untersuchungen uber d i e Typhula-Faule des G e t r e i d e s . I . Zur P h y s i o l o g i e von Typhula i n c a r n a t a L a s c h ex F r . P h y t o p a t h o l . ,.Z. , 5 3 : 2 5 5 - 2 8 8 . Lemke, P. A. 1969. A r e e v a l u a t i o n o f h o m o t h a l l i s m , h e t e r o t h a l l i s m and the s p e c i e s concept i n S i s t o t r e m a b r i n k m a n n i . Mycologia, 61:57-76. L i l l y , V. G. and H. L. B a r n e t t . 1 9 5 1 . P h y s i o l o g y o f f u n g i . M c G r a w - H i l l Book Co., New Y o r k . L o c k h a r t , C. L. I 9 6 7 . E f f e c t s o f temperature and v a r i o u s COg and Og c o n c e n t r a t i o n s on the growth o f Typhula s p . , a p a r a s i t i c fungus o f s t r a w b e r r y p l a n t s . Can. J . P l a n t Sci.,  4_7_:450-452.  MacDonald, J . A. I 9 3 4 . The l i f e h i s t o r y and c u l t u r a l c h a r a c t e r i s t i c s o f Typhula g y r a n s . Ann. A p p l . B i o l . , 21:590-613.  MacDonald, W. C. I 9 6 I . A r e v i e w o f t h e taxonomy o f some low temperature f o r a g e pathogens. Can. P I . D i s . Surv., 41:256-260.  M a d e l i n , M. F. 1956. S t u d i e s on the n u t r i t i o n o f Coprinus lagopus F r . , e s p e c i a l l y as a f f e c t i n g f r u i t i n g . Ann. B o t . (N.S.) (London), 2 0 : 3 0 7 - 3 3 0 . M a r t i n , G. W. 1952. R e v i s i o n o f the n o r t h c e n t r a l Treme l l a l e s . U n i v . Iowa S t u d . N a t u r . H i s t . , 19:1-122. M i t c h e l l , D. T. and R. C. Cooke. 1968. Some e f f e c t s o f temperature on g e r m i n a t i o n and l o n g e v i t y o f s c l e r o t i a i n C l a v i c e p s p u r p u r e a . Trans,. B r i t . M y c o l . S o c , 5 1 : 721-729. N i c h o l a s , D. J . D. I 9 6 6 . U t i l i z a t i o n o f i n o r g a n i c n i t r o g e n compounds, I n G. C. A i n s w o r t h and A. S. Sussman (ed.) The f u n g i , V o l . I , Academic P r e s s , New York.  -91Noble, M. 1937. The morphology and c y t o l o g y of Typhula t r l f o l i l Host. Ann. B o t . (N.S.) (London), .1:67-98. Perhson, S. 0. 1948. S t u d i e s on the growth p h y s i o l o g y of P h a c i d i u m i n f e s t a n s K a r s t . P h y s i o l . P l a n t a r u m , I-.38-56. P o t a t o s o v a , Mme E. G. 1960a. ( C o n d i t i o n s o f g e r m i n a t i o n of the s c l e r o t i a of the f u n g i of the genus T y p h u l a ) . Zasch. R a s t . , Moskva, 5_:40-. . 1960b. ( T y p h u l o s i s o f w i n t e r c r o p s ) . Trud. vses I n s t . Zasch. R a s t . , 14:135-142. . 1960c. (Fungi of the genus Typhula i n the USSR) B o t . Zhur., 43_: 567-572. P r o t o s e n k o , E. P. I967. Typhula b o r e a l i s E k s t r a n d na Tyul'panakh v S.S.S.R. (Typhula i d a h o e n s i s on t u l i p i n U.S.S.R.). M i k o l . i F i t o p a t o l . , 1 : 1 0 7 - 1 0 9 . Raper, J . R. I966. G e n e t i c s of s e x u a l i t y i n the h i g h e r f u n g i . Ronald P r e s s , New York. Remsberg, R. A. 1940. M y c o l o g i a , 32:52-96.  S t u d i e s i n the genus Typhula.  Roed, H. I969. On the r e l a t i o n s h i p between Typhula graminum. K a r s t . and T. i n c a r n a t a L a s c h ex F r . F r i e s i a , 2:219-225. S c u r t i , J . and L. Converso. I 9 6 5 . M i c r o s c o p i c and u l t r a m i c r o s c o p i c s t r u c t u r e of s c l e r o t i a of Typhula sp. C a r y l o g i a , 18:263-284. Taber, W. A. I 9 6 6 . Morphogenesis i n b a s i d i o m y c e t e s . I n G. C. A i n s w o r t h and A. S. Sussman (ed.) The f u n g i , V o l . I I , Academic P r e s s , New York. T a s u g i , H. 1929. On the snow-rot fungus, Typhula graminum K a r s t e n , of graminaceous p l a n t s . J . Imper. A g r i c . E x p t . S t a . , 1:41-56. . 1935. On "the p h y s i o l o g y o f Typhula graminum K a r s t . J . Imper. A g r i c . E x p t . S t a . , 2:443-456. T e r u i , M.  1941.  Trans. Sapporo Nat. H i s t . S o c ,  17:41-.  Tomiyama, K. 1952. S e v e r a l f a c t o r s a f f e c t i n g the Typhula snow b l i g h t of w i n t e r wheat. Ann. P h y t o p a t h . Soc. Japan, 16:113-116. . 1955. S t u d i e s on the snow b l i g h t d i s e a s e of w i n t e r c e r e a l s . Rep. Kokkaido A g r i c . E x p t . S t a . , 47:1-234. Townsend, B. B. and H. J . W i l l e t s . 1954. The development o f s c l e r o t i a o f c e r t a i n f u n g i . Trans. B r i t . M y c o l . S o c , ^2:213-221.  -92(64) Townsend, B. B. 1957. N u t r i t i o n a l f a c t o r s i n f l u e n c i n g the p r o d u c t i o n of s c l e r o t i a by c e r t a i n f u n g i . Ann. Bot. (N.S.) (London), 2:153-166. (65)  Ward, E. W. B., J . B. Lebeau, and M. W. Cormack. I96I. The grouping of i s o l a t e s of a low temperature b a s i d i o mycete on the b a s i s of c u l t u r a l behavior and pathogeni c i t y . Can. J . Bot., 39:297-306.  (66) Ward, E. W. B. 1964. The u t i l i z a t i o n of n i t r o g e n compounds, e s p e c i a l l y ammonia, by a low temperature basidiomycete. Can. J . Bot., 42:1071-1086. (67)  . I966. P r e l i m i n a r y s t u d i e s on the p h y s i o l o g y of S c l e r o t i n i a b o r e a l i s , a h i g h l y p s y c h r o p h i l i c fungus. Can. J . Bot., 44:237-246.  (68) Wheeler, B. E. J . and J . M. Waller. I 9 6 5 . The p r o d u c t i o n of s c l e r o t i a by S c l e r o t i u m r o l f s i i , I I . the r e l a t i o n s h i p between m y c e l i a l 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.  -93APPENDIX A Collection (1)  Data  Typhula erythropus F r i e s (a) i s o l a t e T-4; UBC c u l t u r e  c o l l e c t i o n # 5OI9  L o c a t i o n : U.B.C. campus, Vancouver, B.C. Date: 3 0 September, I969 H a b i t a t : on p e t i o l e s  of Acer macrophyllum  (b) i s o l a t e s T - l o ^ and T-18 ; UBC c u l t u r e 2  # 5020, 5021  L o c a t i o n : U.B.C. campus, Vancouver, B.C. Date: 1 5 October, I97O H a b i t a t : on p e t i o l e s  of Acer macrophyllum  (c) i s o l a t e T - 2 9 ; UBC c u l t u r e  # 5022  L o c a t i o n : U.B.C. campus, Vancouver, B.C. Date:  1 2 November, 1 9 7 0  H a b i t a t : on p e t i o l e s ( 2 ) Typhula s c l e r o t i o i d e s  of Acer macrophyllum  (Pers.) F r i e s  (a) i s o l a t e T - 1 9 ; UBC c u l t u r e  # 5023  L o c a t i o n : U.B.C. campus, Vancouver, B.C. Date: 1 3 October, I97O H a b i t a t : on p e t i o l e s  of Acer macrophyllum  D r i e d specimens o f a l l c o l l e c t i o n s have been d e p o s i t e d i n the U.B.C. m y c o l o g i c a l herbarium.  -94APPENDIX  B  C u l t u r e Media Medium A substance  amount  glucose  10.0 g  asparagine  2.0 g  KH P0i).  1.0 g  2  MgS0^-?H 0  0.5 g  vitamin stock s o l n .  1.0 ml  micro-elements stock s o l n .  2.0 ml  2  agar (K&S b r a n d , h i g h g e l s t r e n g t h ) 12.0 g, d i s t i l l e d water  1.0 1  vitamin stock s o l u t i o n thiamine  5 0 mg  pyridoxine  0 , 5 mg  inositol  2 5 mg  biotin  2 5 ug  D i s s o l v e i n 5°0 ml o f 20$ e t h a n o l , s t o r e i n refrigerator. m i c r o e s s e n t i a l elements s t o c k s o l u t i o n Fe(N0 )3'9H 0  181 mg  ZnSO^*7H20  110 mg  3  2  51 mg  MnS0^.'4H 0 2  D i s s o l v e i n 1 5 0 ml d i s t i l l e d w a t e r .  Add IN  H S0i|, u n t i l s o l u t i o n becomes c o l o r l e s s . 2  add  Then  110 mg CuS04'5H 0 and 100 ml d i s t . w a t e r . 2  Store i n r e f r i g e r a t o r .  -95Medium B Same as Medium A except asparagine c o n c e n t r a t i o n i s 0.2 g/1 i n s t e a d of 2.0 g/1. Medium C Make up Medium A l a c k i n g asparagine.  Pour 20 ml of  hot s t e r i l e medium i n t o 90 mm p e t r i p l a t e s 0.12-0.17 g of a u t o c l a v e d  containing  wheat germ f l a k e s ( R o c k h i l l  "brand, Wild Rose M i l l s , Vancouver, B. C ) . Malt  e x t r a c t - y e a s t extract-peptone  substance malt e x t r a c t yeast  extract  (MYP)  amount (Difco) (Difco)  peptone agar  agar  (K&S brand)  d i s t i l l e d water  7.5 g 0.5 g 1.0 g 12.0 g 1.0 1  Water Agar 1.2$ K&S brand high g e l s t r e n g t h agar i n d i s t i l l e d water.  

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