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Some aspects of conjugation in the genus tremella dill. ex Fr. Flegel, Timothy William 1968

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SOME  ASPECTS OF  GENUS  CONJUGATION  TREMELLA  DILL.  EX  IN  THE  FR.  by  TIMOTHY B.Sc.j  A  THESIS THE  WILLIAM  University  of B r i t i s h  SUBMITTED  Columbia^  IN P A R T I A L  REQUIREMENTS MASTER in  FLEGEL  FOR OF  THE  1965  FULFILMENT DEGREE  OF  SCIENCE  the Department of BOTANY  We  accept  this  thesis  as c o n f o r m i n g  to the  required: -g£andar.jd#/  THE  UNIVERSITY  OF  BRITISH  September  t  1968  COLUMBIA  OF  In  presenting  an  advanced  the I  Library  further  for  this degree shall  agree  scholarly  by  his  of  this  written  thesis  in p a r t i a l  f u l f i l m e n t of  at  University  of  the  make that  i t freely  permission  purposes  may  representatives. thesis  for  be It  financial  available for  by  the  is understood gain  Department Columbia  for  extensive  granted  permission.  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  British  shall  reference  Head  be  requirements  Columbia,  copying  that  not  the  of  and  of my  I agree  for that  Study.  this  thesis  Department  copying  or  allowed  without  or  publication my  ABSTRACT  C u l t u r a l studies were c a r r i e d out with haploid strains of T r e m e l l a mesenterica F r .  }  T\ encephala P e r s . and T. subanomala C o k e r to deter-  mine the conditions and c o u r s e of conjugation.  Under the conditions of  the experiments, the optimum pH for growth was and so also was  4. 7 for a l l three species  the optimum for conjugation in m i x e d isolates of T.  and _T. m e s e n t e r i c a .  A time lapse sequence was  photographed  c o u r s e of conjugation in m i x e d isolates of T\ m e s e n t e r i c a .  encephala  to follow the  Conjugation  hormones such as those r e p o r t e d for T. m e s e n t e r i c a by Bandoni (1965) were demonstrated  for the other two species.  d i a l y s i s m e m b r a n e into agar.  These hormones passed through  E x t r e m e l y dilute suspensions of T. me sen-  t e r i c a haplonts were pulse exposed to s e m i p u r i f i e d hormone extracts.  These  suspensions were f i l t e r e d and the c e l l s were observed on the d r i e d f i l t e r c l e a r e d with g l a c i a l acetic acid. r e m o v a l of the hormone.  Conjugation tube production terminated with  Growth and conjugation in. T. m e s e n t e r i c a were  unaffected by the antibiotic cycloheximide.  -iii-  TABLE  OF  CONTENTS  Introduction  1  M a t e r i a l s and Methods  6  Results  12  Discussion  .  28  Summary  37  Bibliography  39  Appendix  43  -iv-  LIST O F  TABLES  Table 1.  N a m e s and contents of m e d i a used.  44  Table 2.  B u f f e r s used i n p H studies.  46  Table 3.  C r o s s i n g scheme used for p H v e r s u s conjugation on s o l i d media.  47  Table 4.  E s t i m a t i o n of growth of T. m e s e n t e r i c a and T. encephala at various pH values.  13  Tables 6-10.  O p t i c a l density of T r e m e l l a cultures at v a r i o u s p H values^ m e a s u r e d at 12, 24 and 36 hours.  Table 11.  Induction of conjugation tubes on d i a l y s i s prints of T. subanomala isolates.  20  Table 12.  Induction of conjugation tubes on d i a l y s i s prints of T. encephala isolates.  20  Table 13.  Conjugation between T. m e s e n t e r i c a isolates 2259-6 and 2259-7 on solid m e d i u m at various p H values.  18  Table 14.  Conjugation between T. encephala isolates 2392-1 and 2392-7 on s o l i d medium at various pH values.  18  48-52  - V-  LIST O F  FIGURES AND  PLATES  FIGURES F i g u r e 1.  Flow scheme of p r e p a r a t i o n of m e d i u m for pH-growth t r i a l s .  53  F i g u r e 2.  Apparatus used to mount the agar plug for m i c r o s c o p i c time-lapse sequence.  10  F i g u r e 3.  P r e p a r a t i o n of c e l l s for hormone pulsing experiments.  54  F i g u r e s 4-8.  F i n a l optical density of haplont cultures of T r e m e l l a isolates at various p H values.  F i g u r e 9.  Conjugation between T. m e s e n t e r i c a isolates i n liquid m e d i u m at various p H values.  19  F i g u r e 10.  O p t i c a l density of cultures of T\_ m e s e n t e r i c a isolate 2259-7 incubated with and without cycloheximide for 24 hours.  24  14-16  PLATES Plate 1.  T i m e lapse photographs of conjugation in T. m e s e n t e r i c a f r o m 45 to 165 minutes.  21  Plate 2.  T i m e lapse photographs of conjugation i n T. m e s e n t e r i c a f r o m 180 to 480 minutes.  22  P l a t e s 3-5.  Photographs of c e l l s f r o m hormone pulsing t r i a l s .  25-27  - vi-  ACKNOWLEDGMENT  The author wishes to thank the following people for their a s s i s t a n c e during the r e s e a r c h reported in this thesis and during the preparation of the thesis itself. Dr. R. J. Bandoni, P r o f e s s o r , to whom, as s u p e r v i s o r of this t h e s i s , I a m m o s t indebted. Dr.  B. E. Tregunna, A s s i s t a n t P r o f e s s o r , who, upon reading the  original manuscripts, Dr.  made many useful suggestions.  T. B i s a l p u t r a , A s s o c i a t e P r o f e s s o r , who f r e e l y offered the  f a c i l i t i e s of his l a b o r a t o r y and d a r k r o o m for preparation of the plates. J. A.  B e r r y whose ideas and constant m o r a l support were invaluable.  Roxanne Walls and Douglas Muth who helped with the typing and preparation of the plates. And,  to other faculty, staff and students in the Department of  Botany who have a s s i s t e d the author during this work.  -1-  INTRODUCTION Fungi of the genus T r e m e l l a D i l l , ex F r . have a dimorphic ilative growth f o r m .  assim-  Growth in the haploid phase of the life c y c l e i s rep-  resented by yeastlike budding c e l l s and i n the dikaryotic phase, by hyphae. K o b a y a s i and Tobaki (1965) l i s t s e v e r a l other genera of the o r d e r T r e m e l l a l e s which show the same growth pattern. of the o r d e r U s t i l a g i n a l e s .  It i s exhibited a l s o by some genera  The budding phase i n the life c y c l e of these  o r g a n i s m s a r i s e s f r o m germinating b a s i d i o s p o r e s ( T r e m e l l a l e s ) or s p o r i d i a (Ustilaginales) on a r t i f i c i a l media.  T h i s phase i n the life c y c l e is terminated  by conjugation or fusion of the yeastlike c e l l s and the initiation of the d i k a r y otic phase.  Sexual factors and the morphogenetic transition between the  haploid and dikaryotic growth f o r m s of the genus T r e m e l l a a r e the subjects of this investigation. In order to gain a full h i s t o r i c a l perspective in this study, one must f i r s t examine the e a r l y literature concerned with the smuts (Ustilaginales). These o r g a n i s m s , because of their importance as p a r a s i t e s of c o m m e r c i a l l y cultivated plants, have r e c e i v e d m u c h c l o s e r attention over the years than m e m b e r s of the T r e m e l l a l e s .  However, that the two groups are c l o s e l y  related was f i r s t proposed by P a t o u i l l a r d g i c a l studies of Neuhoff (1924).  (1900)  and v e r i f i e d by the cytolo-  Since that time the two groups have, by the  m a j o r i t y of m y c o l o g i s t s , been c l a s s i f i e d together in the Subclass  Hetero-  basidiomycetidae. F u s i o n s between the yeastlike s p o r i d i a of smut fungi were f i r s t  -2-  a c c u r a t e l y d i a g r a m e d by Tulasne (1847), de B a r y (1853), and von Waldheim (1869) as observations of the stages i n smut spore germination.  Brefeld  (1883, 1895) extended the work to show this phenomenon i n a l a r g e number of smutspecies  3  and his excellent drawings a r e r e p r o d u c e d i n the r a t h e r r e c e n t  publication of F i s c h e r and Holton (1957). fusion p r o c e s s was  De B a r y (1884) proposed that this  sexual i n nature and thus named it conjugation, but he  opposed by his student B r e f e l d on this point.  was  De B a r y defended his point of  view i n detail and, as a r e s u l t of the d i s c o v e r i e s to be l i s t e d in the following paragraphs, his view was proven c o r r e c t .  F i s c h e r and Holton (1957) deal with  the de B a r y - B r e f e l d c o n t r o v e r s y i n detail. Illustrations of the above authors show s p o r i d i a separated by some d i s tance fusing v i a conjugation tubes or p r o c e s s e s ,  Kneip (1919) further showed  that these fusions were of a specific rather than a r a n d o m nature.  In other  words, fusion would occur only between c e r t a i n compatible s p o r i d i a .  Ben-  saude (1918) and Kneip (1913a, 1913b, 1922) by determining the nuclear behav i o r i n a number of hyphal Homobasidiornycetes, dies i n the smuts. fusions was  l e d the way to s i m i l a r stu-  In this manner the f u l l sexual significance of s p o r i d i a l  i l l u c i d a t e d (summary, F i s c h e r and Holton, 1957).  Subsequent  to this work, the fusion phenomenon was used to advantage i n determining the distribution of sex compatibility factors i n smuts (Bauch 1922, Holton 19 32, Sleumer 1932).  L a t e r works (Holton 1953, 1953a; Rowell 1955) even opposed  individual p a i r s of s p o r i d i a of T i l l e t i a species using a m i c r o m a n i p u l a t o r o b s e r v e d suxual fusion v i a conjugation tubes.  and  -3-  Bauch (1925) hypothesized that conjugation i n Ustilago was  mediated  by specific c h e m i c a l agents which induced the production of conjugation tubes,, He devised a s e r i e s of experiments to prove the existence of these agents,,  However, he could not, i n any of his various attempts,  chemical  obtain, f r o m  sporidial c u l t u r e s , extracts capable of inducing conjugation tubes i n compatible sporidia,,  Although he could not obtain experimental proof, he i n s i s t e d  upon the p r e s e n c e of stimulating substances and argued that i t was i n e x p e r i m e n t a l technique that prevented their isolation,,  limitations  In spite of this  i n i t i a l work on the physiology of conjugation and on diffuseable agents m e d i a ting conjugation i n smuts, further r e s e a r c h i s l a c k i n g  0  In 1853 Tulasne a c c u r a t e l y d i a g r a m e d the b a s i d i a , b a s i d i o s p o r e s and germinating b a s i d i o s p o r e s of T r e m e l l a m e s e n t e r i c a and T, v i o l a c e a  8  Brefeld  (1908) r e p o r t e d , f o r T r e m e l l a , the extended c u l t u r e of b a s i d i o s p o r e s as yeastlike budding colonies on a r t i f i c i a l m e d i a .  He too d i a g r a m e d the germination  of b a s i d i o s p o r e s and their yeastlike offspring. nificance of this germination phenomenon was  The fact that the sexual s i g not understood i s pointed out  in Kneip's work of 19 28 where he stated that for the T r e m e l l a l e s the o r i g i n of the d i k a r y o t i c phase was  not yet known.  C y t o l o g i c a l studies of Dangeard  (1895), Neuhoff (19 24) and Whelden (19 34) dealt with m e m b e r s of the T r e m e l laceae.  Although they c l a r i f i e d the nuclear condition of the hyphae, b a s i d i o -  spores and conidia of the T r e m e l l a s studied, they failed to determine the o r i g i n of the dikaryotic hyphae of the fruiting body.  Developmentally  and  c u l t u r a l l y , the group r e c e i v e d little attention i n the following years and not  -4-  until 1961 did Bandoni note the initiation of the dikaryotic phase i n T r e m e l l a encephala  by the fusion of g e r m tubes of yeastlike haploid c e l l s  a  Later  (19t>3) he gave a detailed account of sexual fusion i n _T. m e s e n t e r i c a „  At  this time he t e r m e d the fusion p r o c e s s e s " c o n j u g a t i o n t u b e s " and noted that their production was artificial media,  dependent upon which mating strains were m i x e d on  K o b a y a s i and T o b a k i (1965) also r e p o r t e d conjugation i n a  c u l t u r a l study of T r e m e l l a f u c i f o r m i s and H o l t e r m a n i a c o r n i f o r m i s ( T r e m e l laceae).  In 1965 Bandoni pointed out that conjugation tubes i n T r e m e l l a  m e s e n t e r i c a were produced by budding c e l l s of one mating type i n response to specific c h e m i c a l stimulation by another,,  He showed the c h e m i c a l s to  be continuously produced by each mating type without stimulation by another, T h e s e substances were diffuseable a c r o s s a d i a l y s i s membrane, nonvolatile, and stable to autoclaving,, gation hormones,  Bandoni t e r m e d these c h e m i c a l substances conju-  L e v i (1956) r e p o r t e d a s i m i l a r conjugation phenomenon i n  liquid cultures of Sac char omyces c e r e v i s i a e and proposed the presence of conjugation hormones.  He was able to demonstrate  induction of conjugation  tubes i n minus c e l l s when placed on agar where plus c e l l s had been growing, but not the r e v e r s e .  D i f f u s i o n a c r o s s a c o l l o d i o n m e m b r a n e was  once but was not repeatable.  He was  demonstrated  not able to p r e p a r e an active f i l t r a t e .  In fungi, there a r e no other known p a r a l l e l s to the conjugation hormone s y s t e m d e s c r i b e d here, in fungi,  M a c h l i s (1966) gave a short review on sex hormones  Raper (1967) gave a m o r e r e c e n t review of the sex hormones known  for fungi and it includes the work with T r e m e l l a m e s e n t e r i c a ,  -5-  The p r e s e n t study was  initiated to determine m o r e precisely the con-  ditions and c o u r s e of the conjugation p r o c e s s i n the genus T r e m e l l a .  It i s  hoped that a c l e a r e r understanding of the m e c h a n i s m of conjugation w i l l lead to a c l o s e r a n a l y s i s of the genetics of sexuality i n this group of organisms,,  -6-  M A T E R I A L S AND  METHODS  The three species of T r e m e l l a used i n this work were T. encephala P e r s . , T. m e s e n t e r i c a F r . , and T. subanomala Coker.  The specific  compatible haploid isolates were p r o v i d e d by Dr. R. J. Bandoni and bore the following numbers: T. encephala  -  RJB  2392-1 and -7  T. m e s e n t e r i c a -  RJB  2259-6 and -7  T. subanomala  RJB  2327-1 and -8  -  The i n i t i a l m e d i a employed for maintaining the strains was m o d i f i e d malt-peptone agar (MMP)  but this was  used f o r conjugation t r i a l s .  later changed to the s a m e  solid media  Because conjugation proceeded p o o r l y on  specific m e d i a were devised f o r each of the species studied. l i s t e d , with names and contents, i n T a b l e 1 (Appendix).  MMP,  These a r e  M e d i a m o r e than one  week old were n o t used because of reduced growth and conjugation. The liquid m e d i a used i n growth t r i a l s at v a r i o u s pH values were s p e c i a l l y p r e p a r e d to avoid c a r a m e l i z a t i o n of the sugars and breakdown of vitamins during autoclaving.  A d i a g r a m showing the composition and prep-  aration of this m e d i u m i s given i n F i g u r e 1 (Appendix).  The inoculum used  came f r o m r a p i d l y growing shake cultures no m o r e than two days old.  Best  r e s u l t s were obtained when an amount of i n o c u l u m was used to have the i n i t i a l optical density of the t r i a l s a p p r o x i m a t e l y 0.10.  The test tubes were put on  a shaker at 15 to 20° C and m e a s u r e d at 12, 24 and 36 hours. m o n i t o r e d by change i n optical density m e a s t i r e d at 560 nm Spectronic 20 spectrophotometer.  Growth was  on the Bausch& Lomb  M i c r o s c o p i c examination for b a c t e r i a l  -7-  .contamination of the cultures and t r i a l s was  c a r r i e d out at a l l stages of  experimentation. P r e p a r a t i o n of solid m e d i a for pH studies followed the same procedure as with liquid media. of 1. 5% w/v  and was  The agar was  added to achieve a final concentration  autoclaved with the glucose-yeast extract solution.  B u f f e r s were p r e p a r e d as 1 M  stocks and were o r i g i n a l l y diluted to  give final concentration of 0. 001 or 0. 004 M.  However, this buffer concen-  tration did not maintain constant pH in solid m e d i a and was 0. 05 M.  thus changed to  A t this concentration, the pH values held within - 0. 1 of a pH unit.  The composition and pH of the buffers used are given in Table 2 (Appendix). A l l m e a s u r e m e n t s were made on the R a d i o m e t e r pH m e t e r M o d e l 28 which gave a reproducibility  of - 0. 02 of a pH unit.  The inorganic salt solution employed was and the source of the vitamins B i o t i n , Thiamine, N u t r i t i o n a l B i o c h e m i c a l s Inc. and r e f r i g e r a t e d .  Vogel's solution (Hood 1966) P y r i d o x i n e , and Inositol  was  The stocks were p r e p a r e d in 4 0 % ethyl a l c o h o l  The complete v i t a m i n stock for T. encephala was  kindly  provided by M. Shand. Conjugation proceeded v e r y slowly and p o o r l y with T.  subanomala  even with the v i t a m i n f o r t i f i e d m e d i a used for T. m e s e n t e r i c a and T.  encephala.  However, our isolates of T\_ subanomala grew in close a s s o c i a t i o n with Diatrype bullata on Salix sp.  Hence, extracts of the Diatrype and attached Salix bark  were employed and found to a c c e l e r a t e conjugation. p r e p a r e d in the following way:  The extracts were  4. 5 g r a m s d r y weight of Diatrype  -8-  and attached bark were ground up in a m o r t a r with sand and two 50 ml. lots of 4 0 % ethyl alcohol.  The supernatant solution was  decanted, pooled,  f i l t e r e d , and r e f r i g e r a t e d in a tightly stoppered bottle.  T h i s extract and  a l l vitamins used were added cold to the autoclaved medium. l i s t e d in Table 1 with m e d i a components.  Quantities are  The s m a l l amount of ethanol  added did not appear to affect the c e l l s . M e d i a of v a r i o u s pH values (as above ) were also used to determine the optimum pH range for conjugation.  Since T. encephala  and T. subanomala  did not conjugate in liquid m e d i a , it was not possible to determine the extent of conjugation accurately. quantitative ly.  Results for these species are always given s e m i -  On solid m e d i a ,  c e l l s were grown for two days at a l l the pH  values and then c r o s s e d at v a r i o u s pH values. Table 3 (Appendix). enterica  The scheme i s shown in  Conjugation proceeded w e l l in liquid culture for T.  mes-  and quantitative counts of conjugation were made using the haemo-  cytometer. Conjugation t r i a l s were o r i g i n a l l y c a r r i e d out by making an X on the agar with one of the a r m s of the X for each mating strain.  T h i s c r o s s was  covered  with a cover slip and observed four to sixteen hours later for the presence of conjugation tubes.  T h i s worked w e l l for T. m e s e n t e r i c a . However, T. enceph-  ala and T. subanomala did not conjugate under a c o v e r s l i p .  Consequently, future  conjugation t r i a l s were executed i n the following manner. S m a l l numbers of each mating type were thoroughly m i x e d on the agar with a s t e r i l e inoculating -loop. -After-four to sixteen hours for T. m e s e n t e r i c a and two days for T\ ala and T. subanomala , the c r o s s e s were viewed m i c r o s c o p i c a l l y .  enceph-  The m a j o r i t y  -9-  of c e l l s were s c r a p e d away using a c o v e r s l i p and the r e m a i n i n g c e l l s were c o v e r e d with another. dish.  Observations were thus made d i r e c t l y i n the p e t r i  T h i s method was r e p o r t e d by Bandoni (pers. comm. 1964) for  T. encephala. The p r e s e n c e of conjugation hormones and, at the same time, their diffusibility ,  was  determined using d i a l y s i s membrane.  These m e m b r a n e s  were p l a c e d on agar and inoculated with the v a r i o u s mating strains.  Removal  of the m e m b r a n e after one of two weeks also r e m o v e d the c e l l s which had never been i n d i r e c t contact with the medium.  Other c e l l s could be inocula-  ted on the d i a l y s i s p r i n t to determine the effect of any diffusible p r e s e n t i n the agar.  substances  B e f o r e autoclaving, the wet d i a l y s i s m e m b r a n e  was  stretched over one a l u m i n u m r i n g and held i n place by a slightly l a r g e r conc e n t r i c a l u m i n u m r i n g (as an e m b r o i d e r y hoop). and autoclaved.  The m e m b r a n e was  trimmed  B y doing this, the m e m b r a n e could be moulded into a cup  shape which retained c e l l s inoculated i n the centre.  When the m e m b r a n e  was  p l a c e d on agar, the a l u m i n u m rings were removed. A time lapse study was  c a r r i e d out to determine the c o u r s e of conjuga-  tion after compatible isolates of T. m e s e n t e r i c a had been m i x e d together. Suspensions o f young r a p i d l y - g r o w i n g c e l l s of each type were m i x e d and poured on an agar plate.  A f t e r fifteen minutes, the suspension was  poured  out of the p e t r i dish and the container was left inverted for twenty minutes. A n agar plug was then r e m o v e d and set up for viewing i n the apparatus shown in F i g u r e 2,  The m e d i u m was kept damp during the observations by the  -10-  addition of s t e r i l e d i s t i l l e d water at the base of the agar plug. photograph was taken 45 minutes after m i x i n g the c e l l s .  The f i r s t  Subsequent  photo-  graphs were taken at fifteen minute i n t e r v a l s except for the final photograph which was taken after an i n t e r v a l of 195 minutes, that i s , 8 hours after the beginning of the experiment.  COVERSLIP AGAR BLOCK PLEXIGLASS 1  MICROSCOPE SLIDE  F i g . 2. Apparatus used to mount the agar plug for m i c r o s c o p i c t i m e - l a p s e sequence.  F o l l o w i n g the time lapse study, a pulsing experiment was c a r r i e d out. C e l l s of a single mating type were p r e p a r e d a c c o r d i n g to the flow scheme i n F i g u r e 3 (Appendix).  T h e y were exposed  for v a r y i n g lengths of time to  active hormone extract of the opposite mating type and then f i l t e r e d i n two 2 m l . lots through one inch, five m i c r o n pore-sized, triacetate G e l m a n f i l t e r s . These f i l t e r s were then washed three times with f r e s h , s t e r i l e m e d i u m to  -11-  remove  remaining hormone  other was  One  filter was  immediately  i n c u b a t e d t o a t o t a l o f 24 h o u r s o n M C M ,  experiment,  A t the c o n c l u s i o n of the  slide.  It a p p e a r e d as though the  f i l t e r s m i g h t be k e p t f o r an u n l i m i t e d l e n g t h of t i m e a n d after clearing with glacial acetic acid.  The  b y J . A. The  T.  camera.  The  was  cycloheximide  Concentrations  For  observed  Orthomat pro-  conjugation in  i t s value as  an  the g r o w t h s t u d i e s the q u a n t i -  c a s e i n h y d r o l y s a t e i n the M C M  w e r e doubled.  spectrophotometer.  as w i t h the p u l s i n g t r i a l s above.  i n c u b a t e d 24 h o u r s b e f o r e f i l t r a t i o n a n d purchased  were  o f 0. 0 5 t o 2 0 0 0 m i c r o g r a m s o f c y c l o h e x -  were used.  m e a s u r e d u s i n g t h e S p e c t r o n i c 20  was  on g r o w t h and  i n v e s t i g a t e d i n o r d e r to d e t e r m i n e  of l i q u i d M C M  ties of g l u c o s e and  were conducted  an  immediately  Berry.  investigatory tool. imide per ml.  with  dried  h o r m o n e e x t r a c t foi' these pulse t r i a l s was  effect of the antibiotic  mesenterica  observed  c e l l s thus p r e p a r e d  and p h o t o g r a p h e d i n d a r k p h a s e on a L e i t z m i c r o s c o p e  vided  the  p i e c e s of the a i r d r i e d f i l t e r s w e r e c l e a r e d by p l a c i n g t h e m i n  g l a c i a l a c e t i c a c i d on a m i c r o s c o p e  microscope  a i r d r i e d and  Conjugation  Controls, and  observation.  Growth  The  trials  trials  were  cycloheximide  u n d e r the t r a d e n a m e A c t i - d i o n e f r o m the U p j o h n  Company.  was  -12-  RESULTS The  c u l t u r a l c o n d i t i o n s f o ra l lthe h a p l o i d T r e m e l l a  isolates,  -and d u r i n g c o n j u g a t i o n , w e r e c r i t i c a l t o t h e c o n j u g a t i o n p r o c e s s .  before  Old  vegetative c u l t u r e s gave little o r no conjugation when c r o s s e d on f r e s h m e d i u m . T.  encephala  a n d T.  subanomala  w e r e quite sensitive i n this r e s p e c t  haplonts m o r e than three days o l dgave quite r e d u c e d conjugation when Isolates one w e e k o l d o r m o r e w o u l d n o t conjugate.  T, m e s e n t e r i c a  since crossed. was  l e s s s e n s i t i v e i n that c u l t u r e s up to one w e e k c o u l d be c r o s s e d w i t h o n l y a s m a l l d e c r e a s e i nthe extent of conjugation. A l l isolates grew well on M M P this m e d i u m .  and reserve  Working stocks were maintained  stocks w e r e cultured on  either on M M P  or  conjuga-  tion m e d i u m w i t h no d i f f e r e n c e i n conjugation r e s u l t s upon m a t i n g . crosses  c a r r i e d out on M M P  were only a limited success  a n d w e r e c o m p l e t e l y u n s u c c e s s f u l w i t h T. e n c e p h a l a A  A  simpler nitrogen source  g r o w t h a n d c o n j u g a t i o n w i t h T. m e s e n t e r i c a subanomala .  growth.  MCM  b u t n o t w i t h T.  However, various modifications of M C M  growth and conjugation for the latter two species.  a more  subanomala.  than the v i t a m i n - f r e e c a s e i n hydro-  l y s a t e u s e d i n this m e d i u m was unable to support  good  a n d T.  mesenterica  s e a r c h for a m o r e defined m e d i u m for both growth and conjugation  yielded MCM.  T.  w i t h T.  However,  inclusive v i t a m i n stock was the n e c e s s a r y  n o m a l a , the additive was extracts of Diatrype  gave good  encephala  a n d  eventually gave F o r T. e n c e p h a l a  a d d i t i v e , a n d f o r T.  suba-  bullata.  With the conjugation m e d i a employed, age was an important  ,  factor.  M e d i a m o r e than two w e e k s o l dgave quite r e d u c e d g r o w t h and conjugation.  -13-  H e n c e , the c l o s e r together the t i m e of p r e p a r a t i o n and use of the m e d i u m , the better w e r e the r e s u l t s . The m e t h o d of c a r r y i n g out c r o s s e s was a l s o c r i t i c a l f o r the conjugat i o n r e s u l t s and was d i s c u s s e d i n d e t a i l i n the M a t e r i a l s and Methods. The o r i g i n a l pH v e r s u s growth study was executed on s o l i d m e d i a w i t h T. m e s e n t e r i c a and T. encephala.  The r e s u l t s a r e g i v e n i n Table 4.  The  pH v l a u e s o r i g i n a l l y intended w e r e not m a i n t a i n e d so that the f i n a l m e a s u r e d pH i s shown.  The r e s u l t s f o r the two s p e c i e s w e r e e s s e n t i a l l y the same  and gave good growth f r o m pH 4 to pH 7.  Only at pH 7. 8 and above was  growth  visibly less.  T. m e s e n t e r i c a  T.  encephala  F i n a l pH  Growth  F i n a l pH  Growth  4.0 5. 2 5.8 7.0 7.8 8. 8  -H-+  4. 3 5. 2 5.4 7.0 8. 0 9.0  +++ +++ +++ +++ ++  +++ +++ +++ ++  +++ ++  good growth no growth but c e l l s s t i l l l i v i n g n o t i c e a b l e i n h i b i t i o n of growth  Table 4. E s t i m a t i o n of growth of T. m e s e n t e r i c a and T. encephala at v a r i o u s pH v a l u e s .  R e s u l t s f r o m the l i q u i d m e d i a t r i a l s at v a r i o u s pH values a r e given i n t a b u l a r f o r m i n T a b l e s 6 to 10 (Appendix). graphed  as growth c u r v e s  O r i g i n a l l y these r e s u l t s w e r e  with optical density versus time.  However, in  -14-  e v e r y case the f i n a l o p t i c a l density was g r o w t h rate.  a d i r e c t r e f l e c t i o n of the r e l a t i v e  F o r e x a m p l e , the highest f i n a l o p t i c a l d e n s i t y (average of four  readings) i n a set of t r i a l s was  given by the c e l l s with the highest g r o w t h rate.  Consequently, f o r e a s i e r i n t e r p r e t a t i o n , the f i n a l o p t i c a l d e n s i t y m e a s u r e m e n t s f o r each pH have been a v e r a g e d and a r e shown g r a p h i c a l l y i n F i g u r e s 4 to 8.  The b a s e l i n e f o r the b a r s i n each of the i l l u s t r a t i o n s m a r k s the  o p t i c a l d e n s i t y r e s u l t i n g f r o m the i n o c u l u m at the beginning of the e x p e r i m e n t .  T. SUBANOMALA t3£7-l  ho  OA 0.8  O.D.  0 7  04 o.s  0.4 0.3 0.2  u  0.1 S.O  4.7  8.4  —  7.2  I  —  8.0  10. t  pH  F i g . 4. F i n a l o p t i c a l d e n s i t y of haplont c u l t u r e s of T. subanomala i s o l a t e at v a r i o u s pH values.  -15-  T. MESENTERICA  2259-6  i.o 0.9  O.D.  o.e • .  0.7  0.6  o.s 0.4 . 0.5  -  0.2  XL  0.1  2.1  4.7  7.2  II 8.0  U 10.2  F i g . 5. F i n a l optical density of -haplont cultures of T. m e s e n t e r i c a isolate at various p H values.  t;0  T. MESENTERICA 2259-7  0.9 O.S  O.D.  0.7 " 0.« ' 0.8 0.4 as -  0.2 0.1 -  XL  XL —  i  —  8.4  7.2  8.0  10.2  »H F i g . 6. F i n a l optical density of haplont cultures of T. m e s e n t e r i c a isolate at various pH values.  -16-  T. ENCEPHALA  1.0  2392-1  0.9 0.8 " 0.0.  0  7  0.6 0.5 0.4  '  0.3 0.2  JL  0.1 • 2.1  7.2  5.4  tr  8.0  10.2  pH  Fig.  7.  F i n a l optical density of haplont  c u l t u r e s o f IV. e n c e p h a l a  isolate at  -various p H values.  1.0  T.ENCEPHALA  0.9 • 0.8  2392-7  0.7 •  O.D.  0.8 0.5 0.4 0.3 0.2 • 0.1 -  n 2.1  "LT 3.0  4.7  5.4  7.2  — i — 8.0  >H  Fig.  8.  F i n a l optical density of haplont  c u l t u r e s o f T. e n c e p h a l a various p H values.  isolate at  — i — 10.2  -17-  In these l i q u i d m e d i u m t r i a l s , as w i t h the s o l i d m e d i u m , m a x i m u m g r o w t h was i n the a c i d i c range.  F o r a l l the s p e c i e s and t h e i r h a p l o i d  i s o l a t e s the o p t i m u m p H f o r growth was i n the v i c i n i t y of p H 4. 7.  However,  d i s t i n c t d i f f e r e n c e s a p p e a r e d at p H values away f r o m the o p t i m u m pH.  In  T. m e s e n t e r i c a there was a n o t i c e a b l e i n c r e a s e i n growth at p H 8. 0 after a c o n s i d e r a b l e d e c r e a s e at p H 7. 2.  T. encephala  and T. subanomala , on the  other hand, showed an i n c r e a s e at p H 7, 2 after a c o n s i d e r a b l e d e c r e a s e at p H 5. 4.  F o r each s p e c i e s then, there was a s t i m u l a t i o n of growth i n the  s l i g h t l y a l k a l i n e r e g i o n after a n o t i c e a b l e i n h i b i t i o n at a l o w e r pH.  There  appear to be two d i s t i n c t m o d i f i c a t i o n s of a b a s i c b i n o d a l g r o w t h pattern.  At  _pH 10. 2, f i n a l o p t i c a l d e n s i t y readings below the s t a r t i n g o p t i c a l density m u s t have r e s u l t e d f r o m h y d r o l y s i s of some of the i n o c u l a t e d c e l l s . The f i r s t e x p e r i m e n t s to d e t e r m i n e the effect of p H on conjugation w e r e c a r r i e d out on s o l i d m e d i a .  The r e s u l t s f o r T. m e s e n t e r i c a  and T. enceph-  a l a a r e given i n Tables 13 and 14. C o m p a r i s o n to the g r o w t h studies shows that p H values favourable f o r vegetative growth w e r e a l s o f a v o u r a b l e f o r conjugation.  H o w e v e r , f o r T. encephala , c e l l s w h i c h had been i n c u b a t e d a t p H 9. 0  (since they d i d not grow) conjugated  w e l l at p H 7. 0.  Conjugation t r i a l s i n l i q u i d m e d i u m w i t h T. m e s e n t e r i c a a r e given i n F i g u r e 9.  A t each p H where conjugation o c c u r r e d , a t y p i c a l c e l l i s a l s o drawn  to show that t h e r e was a qualitative as w e l l as a quantitative d i f f e r e n c e w i t h r e s p e c t to the conjugation tubes. than 2 c e l l lengths.  Those tubes at p H 3.0 w e r e s e l d o m longer  Those at p H 4.7 w e r e 5 c e l l lengths or longer and those  at p H 5. 4 w e r e only s l i g h t l y s h o r t e r than those at p H 4. 7.  A t p H 8. 0 the tubes  -18-  Pinal growing pH 4.0 4.0  7.0  7.8  8.8  8.8  +++ +++  5.8  S  5.8  +++  5.2 a o.  5.2  ++++ ++++  ++++  1  7.0  •H CO U O  ++++  ++++  ++++  7.8  ++  8.8  -  rH  Pint  ++  _  +++ +  -  8.8  ++++  excellent conjugation  +++  good conjugation  ++  f a i r conjugation  +  very few conjugation  -  no conjugation tubes  tubes  Table 13. Conjugation between T. m e s e n t e r i c a isolates 2259-6 and 2259-7 on solid m e d i u m at various p H values.  Pinal growing pH 4.3 4.3  ++++  8.0  9.0  9.0  +++  ++++  +++  ++++  ++  7.0 ++++  5.4 7.0  5.4  -  5.2  Pinal crossing pH  5.2  ++++  +++  +++  ++  ++  8.0  +  9.0  -  9.0  -  excellent conjugation  +++  good conjugation  ++  f a i r conjugation  +++ +  -  -  +  very few conjugation  -  no conjugation tubes  tubes  Table 14. Conjugation between T. encephala isolates 2392-1 and 2392-7 on solid m e d i u m at various p H values.  were v e r y short and m o s t l y branched.  It was interesting to note the lack of  conjugation at p H 7. 2 but its o c c u r r a n c e at p H 8. 0.  100  T. MESENTERICA 22596 + 22597  • 0  •o %  TO  CONJ. TUBES  a o  60  60 40 30 20 10  1  2.1  1"  3.0  —I— 4.T  8.4  T.2  Jl 8.0  10.2  pH  F i g . 9. Conjugation between T. m e s e n t e r i c a isolates i n liquid m e d i u m at various p H values.  The results of experiments to determine the p r e s e n c e of diffuseable agents governing conjugation are given in T a b l e s 11 and 12 for T. encephala and T. subanomala wherever  respectively.  It can be seen that tubes were present  one isolate was put down on the d i a l y s i s print of a conpatible isolate  T h i s was not the case when an isolate was put down on its own d i a l y s i s print.  -20-  On dialysis  No.of  On dialysis  membrane  plates  2327-1 a n d 2327-8  2 3  2327-1 2327-8  + +  2327-1  2 3  2327-1 2327-8  +  2 3  2327-8 2327-1  +  2327-8  +  Table  11.  print  tubes  conjugation tubes  present  conjugation tubes  absent  Induction of conjugation tubes on dialysis  p r i n t s o f T. s u b a n o m a l a  isolates.  On dialysis  No. of  On dialysis  membrane  plates  print  Conjugation tubes  2329-1 a n d 2329-7  2 3  2392-1 2392-7  + +  2392-1  2 3  2392-1 2392-7  +  2 3  2392-7 2392-1  2392-7  +  Table  12.  _  +  conjugation tubes  present  conjugation tubes  absent  Induction of conjugation  p r i n t s o f T. e n c e p h a l a  Photographs f r o m the time ica  Conjugation  a r e s h o w n i n P l a t e s 1 a n d 2.  tubes o n dialysis  isolates.  l a p s e s t u d y o f c o n j u g a t i o n i n T. m e s e n t e r T h e f i r s t p h o t o g r a p h w a s t a k e n 45 m i n u t e s  after the cells had been m i x e d together.  A t 90 m i n u t e s c e l l #2 b e g a n t o  -21-  Plate 1. T i m e lapse photographs of conjugation i n T. m e s e n t e r i c a f r o m 45 to 165 minutes.  2  45 min.  C e l l Number 3  Mm.  60 mm.  7 5 min.  * M 90 min.  105 min. 0  120 min.  135 min.  150 min.  165 mm.  p*  1  -22-  Plate 2. T i m e lapse photographs of conjugation in T. m e s e n t e r i c a f r o m 180 to 480 minutes.  C e l l Number 3  180  4  B K  min.  195 min.  210 min. •  225 min.  If'** £3 240 min.  255 min.  270 min.  285 min. o *  480 min.  fig >JS  must  l  5  -23-  p r o d u c e a c o n j u g a t i o n tube.  A t t h i s t i m e c e l l #1 w a s  just detaching a  a n d a t 120 m i n u t e s , i t t o o b e g a n t o p r o d u c e a c o n j u g a t i o n t u b e . a r o s e at the s a m e p o i n t w h e r e the b u d h a d j u s t d e t a c h e d .  bud,  T h i s tube  C e l l #3,  from  45 m i n u t e s t o 255 m i n u t e s w e n t t h r o u g h one c o m p l e t e b u d d i n g s e q u e n c e b e g i n n i n g a " c o n j u g a t i o n tube a t 285 m i n u t e s . b u d a t 6 0 m i n u t e s , c e l l #3 b e g a n a n e w 210 m i n u t e s .  before  A f t e r d e t a c h m e n t of its  original  o n e a t 105 m i n u t e s a n d c o m p l e t e d i t b y  T h i s g a v e , f o r t h a t b u d , a g e n e r a t i o n t i m e o f 2 1/2 h o u r s  d e t a c h m e n t of the p r e v i o u s d a u g h t e r c e l l to its own g r o w t h of the c e l l f r o m a p p e a r a n c e  detachment.  to d e t a c h m e n t took 13/4  The  hours.  actual The  c o n j u g a t i o n t u b e i n c e l l #3 a r o s e a t t h e s a m e p o i n t w h e r e t h e d a u g h t e r had arisen.  C e l l s #4 a n d #5,  t h r o u g h the whole sequence  with and without a bud r e s p e c t i v e l y ,  p r o d u c i n g n e i t h e r new  from  cells  went  buds nor tubes.  P h o t o g r a p h s f r o m t h e h o r m o n e p u l s i n g t r i a l s a r e g i v e n i n P l a t e s 3 t o 5. C o n j u g a t i o n t u b e s w e r e f i r s t s e e n i n h o r m o n e e x p o s u r e s o f 120 m i n u t e s o c c u r r e d with a l l subsequent exposures. the  the  W i t h i n c r e a s i n g t i m e of e x p o s u r e  length of the c o n j u g a t i o n tubes i n c r e a s e d .  b r a n c h i n g was  common.  and  A t 8 hours and  beyond,  In the t r i a l s i m m e d i a t e l y d r i e d a f t e r e x p o s u r e to  h o r m o n e , buds d i d not a p p e a r at the ends of the c o n j u g a t i o n t u b e s .  However,  w i t h r e m o v a l o f t h e h o r m o n e a n d i n c u b a t i o n t o 24 h o u r s , r e v e r s i o n t o b u d d i n g was  general. Results f r o m growth studies using cycloheximide are given i n Figure  10,  T h e y s h o w t h a t c y c l o h e x i m i d e h a d l i t t l e e f f e c t o n t h e g r o w t h o f T. m e s e n t e r i c a . A l t h o u g h t h e r e was  s o m e d e c r e a s e i n g r o w t h r a t e , the a n t i b i o t i c was  ineffective  -24-  in stopping growth. cycloheximide  Conjugation  t r i a l s i n the p r e s e n c e o f 2000 ug. of  p e rm l . ofconjugation m e d i u m  showed that p r o d u c t i o n of  conjugation tubes was unaffected b y the antibiotic.  T h e length of the conjuga-  tion tubes i n both t r i a l s a n d c o n t r o l s w a s the same.  O.D."  O NO CYCLOHEXIMIDE & CYCLOHEXIMIDE 2 MO./ML.  10  It  14  16  18  20  22  24  TIME (HRS.)  F i g . 10.  Optical density ofcultures of  T. m e s e n t e r i c a  isolate  2259-7  with and without cycloheximide  incubated f o r 24 h o u r s .  -25-  P L A T E  A  3  - C e l l s i n c u b a t e d 30 m i n u t e s w i t h  hormone,  filtered, washed and incubated a further 2 3 h o u r s a n d 30 m i n u t e s w i t h o u t  B  hormone.  - C e l l s i n c u b a t e d 60 m i n u t e s w i t h . h o r m o n e , filtered, washed and incubated a further 23 h o u r s w i t h o u t  C  hormone.  - Cells incubated 2 hours with hormone, filtered, washed and incubated a further 22 h o u r s w i t h o u t  D  hormone.  - Cells incubated 4 hours with  hormone,  filtered and washed.  E  - Cells incubated 4 hours with  hormone,  filtered, washed and incubated a further 20 h o u r s w i t h o u t  hormone.  -26-  P L A T E  4  Cells incubated 6 hours with hormone, filtered and washed.  G  - Cells incubated 6 hours with  hormone,  filtered, washed and incubated a-further 18 h o u r s w i t h o u t  H  hormone  - Cells incubated 8 hours with hormone, filtered and washed.  C e l l s i n c u b a t e d 10 h o u r s w i t h h o r m o n e , filtered and washed.  J  - C e l l s i n c u b a t e d 10 h o u r s w i t h  hormone,  filtered, washed and incubated a further 14 h o u r s w i t h o u t  hormone.  -27-  PL.ATE  K  5  - C e l l s i n c u b a t e d 24 h o u r s w i t h filtered and washed.  hormone,  -28-  DISCUSSION The choice of m e d i a f o r culturing fungi, as pointed out by L i l l y and Barnett (1951), must be made on the basis of the purpose for which the m e d i u m is to be used.  These authors and Cochrane (1959) demonstrate that  nutritional requirements  may  v a r y widely for even a single o r g a n i s m at  different stages i n its life c y c l e . nutritional requirements  In other words, conclusions about the  of a single o r g a n i s m must be drawn with caution,  and generalizations f r o m one fungus to another are quite tenuous.  Such studies  must be c a r r i e d out with scrupulous precaution against contamination and close r e g a r d to the interaction of experimental v a r i a b l e s . r e s e a r c h reported here, it was nutritional requirements  Consequently, i n the  not the author's intention to determine the  of the fungi studied.  Rather, the purpose was  to  find a m e d i u m which gave good conjugation and which, at the same time, a known composition.  had  However, the m e d i a f i n a l l y used were semisynthetic  since, in addition to known components, they a l l contained components f r o m natural sources. The three species of T r e m e l l a studied grew p o o r l y with ammonium nitrate as the nitrogen source but grew well when v i t a m i n - f r e e c a s e i n hydrolysate was  used instead.  Hence, under the c u l t u r a l conditions used,  these fungi were unable to synthesize one or m o r e amino acids f r o m an inorganic nitrogen source.  L i l l y (1965) c o n s i d e r s it doubtful that there are  many fungi which cannot use inorganic nitrogen.  He feels that a specific amino  -29-  a c i d requirement i s m o s t often the case.  Such an amino a c i d could be  c o n s i d e r e d as a growth factor i n the widest sense ( F r i e s 1965).  From  the present study it i s i m p o s s i b l e to say whether these T r e m e l l a s have an obligate organic nitrogen requirement  or a specific amino a c i d  requirement.  The v i t a m i n s , biotin, thiamine, pyridoxine and i n o s i t o l used as growth f a c t o r s for T. subanomala and T. m e s e n t e r i c a have been c o m m o n l y r e p o r t e d as r e q u i r e d by other fungi ( L i l l y and Barnett 1951, Cochrane 1958 and 1965).  The authors suggest that these vitamins function as coenzyme  components. was  Fries  Although a r e a d i l y available and m o r e i n c l u s i v e v i t a m i n stock  used to promote conjugation i n T. encephala, no c u l t u r a l study  was  conducted to determine which of the vitamins i n the stock were essential. It i s c e r t a i n only that the four vitamins and m e d i u m used for the other two T r e m e l l a s were not sufficient to promote good conjugation. no attempt was  F o r T\_ subanomala,  made to identify the growth factor which came f r o m the extracts  of Diatrype bulatta.  Unknown growth factors in n a t u r a l extracts are common  in studies with fungi and sections are devoted to them i n the works of each of the above authors. Boyle (1924) and P r a t t (1924a and 1924b) reported that m e d i a f r o m aged cultures of F u s a r i u m T h i s inhibition was the medium.  sp. were capable of inhibiting its own  growth.  a t t r i b u t e d to toxic metabolic products accumulating i n  T h e y t e r m e d these toxic metabolites staling substances  reviewed s i m i l a r work by other authors.  and  It is questionable whether the  reduction in conjugation between c e l l s of a.ged cultures of T r e m e l l a i s a  -30-  parallel situation.  A l t h o u g h , f o r two  of c o n j u g a t i o n a f t e r t w o  species, cultures showed  d a y s , there was  not a c o n c u r r e n t  inhibition  i n h i b i t i o n of g r o w t h .  A l s o , c e l l s w e r e a l w a y s t r a n s f e r r e d to f r e s h m e d i u m f o r m a t i n g .  These  two  occur in  facts suggest that changes w h i c h s e l e c t i v e l y affect conjugation,  c e l l s of o l d c u l t u r e s .  These changes may  be  due  to s t a l i n g  substances.  C o c h r a n e (1958) c a l l s f o r c a u t i o n i n t h e i n t e r p r e t a t i o n o f d a t a f r o m experiments. of t h e c e l l a n d cell.  He  optimum pH  growth curve".  on the s u r f a c e m e t a b o l i c  f a c t o r i n the e n v i r o n m e n t m a y  e x p l a i n s ; " The  pH  can  of m e d i a a n d  considered  v i e w the r e s u l t s of the p H  studies  w i t h the m e d i a and  C a u t i o n m u s t a l s o be solid media trials.  The  Greater  inadequacies  tolerances are  of the u n e v e n e x p o s u r e of c o l o n y The  the  of T a b l e rough  preferences",  as a p p r o x i m a t i o n s and  as  limited  With this precaution in m i n d ,  h e r e o n l y as g i v i n g the  buffers  3  optimum  used.  o b s e r v e d i n i n t e r p r e t a t i o n of the r e s u l t s f r o m  i s i l l u s t r a t e d by c o m p a r i s o n of the p H liquid media.  values  a p o s s i b l e i n d i c a t i o n of e c o l o g i c a l  t h e s e f i g u r e s m u s t be  for conjugation  change  v i e w e d with s o m e caution; at best they offer a  to the c o n d i t i o n s of the p a r t i c u l a r e x p e r i m e n t " . one  reactions  In a d i s c u s s i o n a c c o m p a n y i n g a t a b l e of  f o r v a r i o u s f u n g i , he  t h e r e f o r e , be  "...  the a f f e c t of p H  " t h a t a l m o s t any  values  guide to c h o i c e and  discusses  the s u b s e q u e n t e f f e c t u p o n the e n t r y of v a r i o u s n u t r i e n t s i n t o the  states  s h a p e of the p H  should,  He  pH  of t h i s t e c h n i q u e versus  for precise  g r o w t h r e s u l t s on  solid  s h o w n by the f u n g i on a g a r  c e l l s to the s u p p o r t i n g  b i n o d a l g r o w t h p a t t e r n f o r the  species  studies and  because  medium.  s t u d i e d h e r e has  also been  -31-  r e p o r t e d by F r i e s (1956) for Coprinus.  With that fungus, the pattern  r e s u l t e d f r o m p r e c i p i t a t i o n of m i n e r a l ions at various pH values and their subsequent unavailability to the organism.  F u r t h e r experimentation  needed to determine i f such is the situation with -Conjugation i n T. m e s e n t e r i c a was growth.  is  Tremella.  m o r e sensitive to pH than  was  Where m i n i m a l growth o c c u r r e d , no conjugation o c c u r r e d ,  and  this might be expected for the other two T r e m e l l a s would they a l s o conjugate in l i q u i d culture.  The conjugation t r i a l s on solid m e d i a , on the other hand,  showed wider tolerance.  A g a i n , this is probably due to the uneven exposure  of c e l l s to the substrate.  High conjugation for T. encephala and not for  T. m e s e n t e r i c a when c r o s s e d on agar at pH 7.0 after incubation at pH  9.0,  can be understood by r e f e r e n c e to their r e s p e c t i v e behavior i n liquid medium. A t pH 7. 2 T. encephala grows v e r y well and T. m e s e n t e r i c a neither grows w e l l nor  conjugates.  A knowledge of conjugation hormones leads to speculation as to their mode of action.  It has been shown here that with i n c r e a s i n g time of  exposure to the hormone, tube length i n c r e a s e d .  A l s o , it was  shown that,  with r e m o v a l of the hormone, tube growth r e v e r t e d to budding.  Consequently,  the conjugation hormone was  Bandoni (1965)  indicated that this was  consumed by the stimulated c e l l s .  the probable situation.  He felt that there was  a  c o r r e l a t i o n between time of incubation on d i a l y s i s m e m b r a n e s and tube length of isolates placed on the d i a l y s i s print.  With i n c r e a s i n g incubation time, the  final'tube "length i n c r e a s e d and the c e l l s eventually r e v e r t e d to budding.  He  wrote, ' 'Since tube length and length of the f i r s t incubation p e r i o d seemed  -32-  to be c o r r e l a t e d where isolates were used consecutively, the hormones must be u t i l i z e d or a l t e r e d during tube growth.  T h i s would tend to substan-  tiate Gwynne- Vaughan s view (1928) that differences between strains of a 1  heterothallic fungus might lie in the ability of each to synthesize  some  substance, or to p e r f o r m one step in the synthesis of a substance, r e q u i r e d for development' ' . Since the time of Gwynne - Vaughan, Jacob and Monod (1961) have introduced the m o d e l of enzyme induction and r e p r e s s i o n .  In this model,  c o n t r o l of enzyme production at the genetic l e v e l o c c u r s through the. i n t e r action of three genetic components, namely, regulator, operator, s t r u c t u r a l genes.  and  C h e m i c a l compounds ( r e p r e s s o r s ) , originating f r o m the  regulator, either p e r m i t of inhibit the e x p r e s s i o n of an operator and s t r u c t u r a l genes it controls (an operon).  Interaction of r e p r e s s o r s  the with  substrates (effectors) r e v e r s e s the foregoing situation to ' 'open up' ' the p r e v i o u s l y inhibited operon (induction) or to ' 'close down' ' a p r e v i o u s l y functioning operon (repression). D i c k (1965) invoked a modified  Jacob and Monod m o d e l of induction  to explain the action of the incompatibility factors of Schizophyllum commune. T h i s he  did  with r e s e r v a t i o n , indicating that models involving zymogens  might also be invoked.  However, he favoured the m o d e l involving operons  because of the m u l t i p l i c i t y of functions c o n t r o l l e d by the incompatibility l o c i . He proposed that active r e p r e s s o r s f r o m A and B l o c i of compatible n u c l e i would be inactivated by their mutual interaction.  P r e l i m i n a r y work by  this author indicated fundamental protein differences between different  -33-  homokaryons as well as between homokaryons and dikaryons.  This  indicated synthesis of completely new proteins upon f o r m a t i o n of dikaryons. R a p e r (1966) also used this m o d e l to explain the function of incompatibility factors i n determining the morphology of SchizophyHum commune. E s s e r and Kuenen (1967) have reviewed work strongly genetically inducible  enzymes in fungi.  indicative of  However, in a section on  morphogenesis where they reviewed attempts to demonstrate  induction  phenomena, they stated, ' 'a definite m o d e l of the chain of b i o c h e m i c a l and b i o p h y s i c a l p r o c e s s e s which connects the genetic i n f o r m a t i o n to c e l l , tissue, and organ differentiation is not yet available' ' . It is difficult to say how close the compatibility system in S c hi z ophy Hum is to that in T r e m e l l a .  Schizophyllum is tetrapolar with the A incompatibility  factor controlling nuclear a s s o c i a t i o n , conjugate d i v i s i o n , clamp initiation and c l a m p septation. (Raper 1966).  The B factor controls nuclear m i g r a t i o n and clamp fusion,  T h i s fungus is hyphal and hyphal fusion o c c u r s r e g a r d l e s s of  the incompatibility factors.  Bandoni (1963) showed that T r e m e l l a m e s e n t e r i c a  had a. m o d i f i e d tetrapolar incompatibility system.  Conjugation was controlled  by two equally distributed a l l e l i c factors A and a.  E s t a b l i s h m e n t of a  dikaryon (as evidenced by c l a m p connections) depended upon a multiple a l l e l i c B factor which segregated independently of the A's. , mating types.  T h i s gave many different  Bandoni (pers. comm.) was unable to establish common-A,  common-B or c o m m o n - A B heterokarycns i n T. m e s e n t e r i c a , pseudoclamps in matings occurrance  of this type involving T. encephala.  but did observe The  of common-B heterokaryons in this latter species indicates  -34-  conjugation tubes i n T r e m e l l a m i g h t fuse randomly.  In such a c a s e , the  i n c o m p a t i b i l i t y s y s t e m i n T r e m e l l a w o u l d be c l o s e t o that i n S c h i z o p h y l l u m . It w o u l d d i f f e r b y h a v i n g a m o r e e x t e n d e d f u n c t i o n f o r t h e A l o c u s t h a n Schizophyllum.  In the latter organism,  one m i g h t speculate that the gentic  sites governing hyphal f o r m a t i o n i nhaplonts a r e no longer (evolutionarily speaking) Regardless  by the A locus and consequently  of the p r e c e e d i n g  controlled  function continuously.  c o n j e c t u r e about the affinities of  incompatibility i n Tremella and Schizophyllum, work  does  there i sevidence  from  this  that c o n j u g a t i o n tube p r o d u c t i o n i n T r e m e l l a i sa n e x a m p l e of genetic  induction.  It w a s h o p e d t h a t c y c l o h e x i m i d e w h i c h i n h i b i t s p r o t e i n s y n t h e s i s  i n s o m e o t h e r o r g a n i s m s ( K e r r i d g e 1958) w o u l d d o t h e s a m e i n T r e m e l l a mesenterica.  T h i s w o u l d have p r o v i d e d a m e a n s of d e t e r m i n i n g  o r n o t c o n j u g a t i o n tube p r o d u c t i o n was dependent upon protein. RNA  Other antibiotics interrupting the chain  whether  newly synthesized  of events  from  messenger-  t r a n s c r i p t i o n to p r o t e i n f o r m a t i o n at the r i b o s o m e m i g h t give definitive  results i nthis The  direction.  only extensively studied morphogenetic change which c o m e s close  to the one s t u d i e d h e r e i s t h e i n t e r c o n v e r s i o n of b u d d i n g a n d h y p h a l  forms  of C a n d i d a a l b i c a n s a n d M u c o r spp. , d e s c r i b e d b y N i c k e r s o n a n d h i s c o - w o r k e r s ( r e v i e w s , N i c k e r s o n 1963,  R o m a n o 1965).  Falcone  a n d N i c k e r s o n (1959)  d e v i s e d a n hypothesis to e x p l a i n the i n t e r c o n v e rsion of yeast and hyphal  forms.  T h i s m o d e l p r o p o s e d that the m o r p h o l o g i c a l d i f f e r e n c e s s e e n w e r e b a s e d upon the c h e m i c a l nature of the c e l l w a l l .  A predominance of disulfide  bonds  -35-  between p r o t e i n m o i e t i e s of protein-manan complexes, supposedly •hyphal morphology.  determined  In budding morphology, the disulfides were supposed  to be maintained in the reduced  sulfhydryl state.  The interconver sion of  sulfhydryl''. and disulfide bonds was p r o p o s e d to be mediated by a protein disulfide reductase.  Budding or hyphal morphology, then, would u l t i m a t e l y  depend upon the presence or absence of protein disulfide reductase.  Nickerson  and Falcone (1956a) have shown protein disulfide reduction by cell-free extracts of S a c c h a r o m y c e s c e r e v i s i a e .  Following this they (1956b) demon-  strated high reduction activity with extracts f r o m yeast-forrn Candida but low activity in hyphal Candida .  They have not isolated and c h a r a c t e r i z e d a  specific enzyme. It i s attractive to apply simultaneously the F a l c o n e - N i c k e r s o n (1959) and the Jacob-Monod (1961) models to conjugation tube production in T r e m e l l a mesenterica.  The budding c e l l s would have high protein reductase activity.  Introduction of conjugation hormone f r o m a compatible mating type would produce the following changes.  The operon producing m - R N A for protein  disulfide reductase would be r e p r e s s e d .  Without the enzyme, disulfide  bonds would.form between the c e l l - w a l l protein moieties and hyphal m o r p h o l ogy would ensue.  T h i s m o d e l could be further s i m p l i f i e d by suggesting that  the hormones themselves are inactive r e p r e s s o r s produced by the A locus of each mating type.  Combination of hormones f r o m compatible mating types  would produce an active r e p r e s s o r molecule.  T h i s r e p r e s s o r would " shut  down" the operon with the s t r u c t u r a l gene for protein disulfide reductase in both mating types.  When the dikaryon was  e s t a b l i s h e d , the r e p r e s s o r would  -36-  be continuously produced i n t r a c e l l u l a r l y and hyphal morphology would be maintained. A f t e r the preceeding  speculation, there is one final consideration to  be made which is m u c h less speculative. time-lapse photography.  This pertains to the results f r o m  A s reported for apiculate yeasts by Streiblova et  a l (1964), T r e m e l l a c e l l s in these photographs f o r m e d s u c c e s s i v e buds at the same point.  The observation that conjugation tubes a l s o a r i s e f r o m the  same place, shows the v e r y unique nature of this budding point.  Bartnicki-  G a r c i a (1963), studying d i m o r p h i s m in M u c o r r o u x i i , proposed that filamentous growth was  e s s e n t i a l l y the same as yeastlike growth except that the f o r m e r  is p o l a r i z e d and the latter is not.  He  s a i d , ' ' P e r i o d i c a l l y , during vegetative  growth of M u c o r by either elongation or u n i f o r m expansion, a point is reached whereupon new  centres of morphogenesis are c r e a t e d m o r e or less perpen-  dicular to the c e l l surface: incipient protruberances  are f o r m e d which,  con-  tinuing the o r i g i n a l pattern of development, give r i s e to either l a t e r a l branches or s p h e r i c a l buds.  Hence, hyphal branches and yeastlike buds, although  m o r p h o l o g i c a l l y different, may  be c o n s i d e r e d p h y s i o l o g i c a l l y equivalent.  The c e l l u l a r m e c h a n i s m s determining appearance of new genesis may  centers of morpho-  be s i m i l a r , if not identical for both m o r p h o l o g i c a l types. . . ' '.  The fact that buds and conjugation tubes in T r e m e l l a a r i s e f r o m the same place shows that the c e l l u l a r m e c h a n i s m s for morphogenesis are at least centered at a specific point.  Consequently, this region of the c e l l should  r e f l e c t its unique p h y s i o l o g i c a l nature through u l t r a s t r u c t u r a l specialization. M o r e o v e r , the u l t r a s t r u c t u r e should change with impending changes i n morphology.  In c o n c l u s i o n t h e n , t h e c o n j u g a t i o n s y s t e m i n T r e m e l l a simple, yet specifically induced change.  and genetically determined  T h i s s y s t e m has the advantage over  other s i m i l a r  involves a  morphological morphogenetic  s c h e m e s d i s c u s s e d above i n that i t i s n a t u r a l l y r a t h e r than a r t i f i c i a l l y Therefore,  the phenomenon of conjugation i n T r e m e l l a  induced.  provides an excellent  o p p o r t u n i t y to d e t e r m i n e w h e t h e r t h e o r i e s of m o r p h o g e n e s i s d e r i v e d v i a a r t i f i c i a l s y s t e m s a r e a p p l i c a b l e t o a n a t u r a l one.  SUMMARY (1)  C u l t u r a l conditions to obtain good conjugation i n T r e m e l l a  mesenterica, (2) T.  T.  suspensions logical  are described. encephala  and  i s demonstrated.  A time lapse sequence i s presented  p r o d u c t i o n i n T. m e s e n t e r i c a (4)  a n d T\_ s u b a n o m a l a  T h e p r e s e n c e o f c o n j u g a t i o n h o r m o n e s i n T.  subanomala (3)  encephala  s h o w i n g c o n j u g a t i o n tube  .  A method i s described f o r experimentally handling very  dilute  of c e l l s a n d then r e c o v e r i n g t h e m e a s i l y to o b s e r v e m o r p h o -  changes. (5)  A h o r m o n e p u l s i n g study i s d e s c r i b e d w h i c h shows that  tion h o r m o n e i s used up by s t i m u l a t e d c e l l s .  It i s s h o w n t h a t  conjuga-  continuous  p r e s e n c e of h o r m o n e i s r e q u i r e d to m a i n t a i n hyphal m o r p h o l o g y i n h a p l o i d cells.  -38-  (6)  G r o w t h i n T\_ m e s e n t e r i c a  i s shown to be e s s e n t i a l l y u n a f f e c t e d  by the a n t i b i o t i c c y c l o h e x i m i d e i n c o n c e n t r a t i o n s up to 2000 m i c r o g r a m s millilitre. (7)  A testable h y p o t h e s i s of e n z y m e  r e p r e s s i o n i s f o r m u l a t e d to  e x p l a i n t h e a c t i o n o f t h e c o n j u g a t i o n h o r m o n e s i n T.  mesenterica.  pe  -39-  BIBLIOGRAPHY B a n d o n i , R. J . 1961. T h e g e n u s N a e m a t e l i a . Naturalist.  The A m e r i c a n  Midland  66: 319-328.  B a n d o n i , R. J . 1 9 6 3 . C o n j u g a t i o n i n T r e m e l l a m e s e n t e r i c a . J o u r n a l of Botany. B a n d o n i , R. J . 1 9 6 5 .  Secondary  mesenterica.  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Peptone Agar  a g a r ; 1000 m l . d i s t i l l e d  Subanomala  yeast  SCM  0. 5 g. g l u c o s e ;  water.  1 g. c a s e i n  Conjugation  h y d r o l y s a t e ; 15 g. a g a r ; 10 m l .  Medium  o f t h e f o u r - v i t a m i n s t o c k * ; 20 m l . V o g e l ' s s t o c k * * ; 10 m l .  Diatrype  e x t r a c t ; m a k e u p t o 1000 m l . w i t h distilled Mesenterica  M C M  water.  0. 5 g. g l u c o s e ; 1 g. c a s e i n  Conjugation  h y d r o l y s a t e ; 10 m l . o f t h e f o u r -  Medium  v i t a m i n s t o c k * ; 20 m l . V o g e l ' s s t o c k * * ; 15 g„ a g a r ; m a k e u p t o 1000 m l , w i t h d i s t i l l e d  Encephala  E C M  water.  0. 5 g. g l u c o s e ; 1 g. c a s e i n  Conjugation  h y d r o l y s a t e ; 10 m l . c o m p l e t e  Medium  v i t a m i n s t o c k * * ; 20 m l . V o g e l ' s s t o c k * * ; 15 g. a g a r ; m a k e u p t o 1000 m l . w i t h d i s t i l l e d  water.  L i q u i d m e d i a w e r e t h e s a m e a s t h e a b o v e m i n u s t h e 15 g. o f a g a r ,  * 50 u g . B i o t i n ; 1 m g .  P y r i d o x i n e ; 1 mg.  T h i a m i n e ; 50 m g .  Inositol; made up  t o 100 m l . w i t h 4 0 % e t h y l a l c o h o l . ** A s u s e d f o r U s t i l a g o h o r d e i b y C..H,  Hood; see f o l l o w i n g page.  -45-  Vitamin stock  solution  Thiamin  100 m g .  Riboflavin  50 m g .  Pyridoxin C a l c i u m pantothenate  50 m g . 200 mg.  p-Amino benzoic acid  50 m g .  Nicotinic acid Choline chloride  200 m g . 200 mg.  Inositol Folic acid D i s t i l l e d w a t e r toone l i t r e . to o n e l i t r e of m e d i u m .  Vogel's Add  solution  3  stirring: 1 2 3 g.  z  (anhyd. )  2 5 0 g.  NH4NO3 (anhyd. )  100 g.  2  P 0  4  solution  t i m e s 50 ( m o d i f i e d b y l a c k o f b i o t i n ) .  successively with Na citrate •2H O K H  400 mg. 50 m g . 10 m l . o f t h i s  MgS0 '7H 0 4  10 g.  2  CaCl * 2H O Trace element solution Distilled water 2  z  Chloroform  5 g. 5 ml. 7 50 m l . 2ml.  - T o b e d i l u t e d 50 f o l d w i t h d i s t i l l e d w a t e r b e f o r e u s e . - Store atr o o m temperature.  Trace  elements Citric acid*H 0  5 g.  ZnS0 *7H 0 Fe(NH ) . (S0 ) »6H 0  5 g. 1 g.  2  4  2  4  2  4  2  2  CuS0 «5H 0  0. 2 5 g.  MnS0 -H 0  0. 0 5 g.  H3BO3 (anhyd. )  0. 0 5 g.  Na Mo0 '2H 0 Chloroform Distilled water  0. 0 5 g. 1 ml. 95 m l .  4  2  4  2  2  4  2  - Store atr o o m temperature.  -46-  T A B L E  2  Material Used Citric acid  pH  *  3. 0 4.7 5. 4  Monobasic sodium phosphate  2, 1 7. 2 12. 0  Sodium carbonate  Tris-carboxymethyl aminomethane  * A l ladjustments H C 1 o r 10 N  o f the 1 M  NaOH.  10. 2  8. 0  stocks were made with either  concentrated  -47-  T A B L E  3  C r o s s i n g S c h e m e on Solid  Growing 3. 0 3.0  4.7  7.2  X  4.7  X  10.2  X  X  X  X  X X  8. 0  X  10. 2 12. 0  8.0  X X  X  pH  X  5.4 7. 2  5.4  Medium  X X  X  X  -48-  T A B L E  T. m e s e n t e r i c a  6  2259-6  Optical Density 0 Hours 0. 0 9 7  0.108  0. 114  0. 108  0. 108  0. 0 9 2  0. 0 7 6  12 H o u r s  24 H o u r s  36 H o u r s  0. 137  0. 1 8 7 .  0. 2 0 1  0.143  0.174  0.194  0. 137  0. 1 7 4  0. 194  0. 137  0. 181  0. 2 0 1  0.194  0..328  0.495  0. 201  0. 3 3 7  0. 4 6 9  0. 187  0. 3 6 7  0. 4 6 9  0.194  0. 3 3 7  0.469  0. 2 0 1  0. 3 9 8  0. 5 2 3  0. 2 0 1  0. 3 6 7  0. 6 9 9  0. 2 0 1  0.482  0. 6 0 2  0.194  0.469  0.699  0.168  0.284  0.377  0. 168  0. 2 4 4  0. 4 0 9  0. 1 7 4  0. 2 6 0  0.398  0.168  0. 2 7 6  0.387  0.119  0.125  0.119  0. 119  0. 119  0.119  0. 119  0. 119  0. 119  0. 119  0.119  0.125  0. 119  0. 1 4 3  0. 174  0.125  0.143  0.194  0.125  0.155  0.168  0.119  0.155  0.161  0.071  0. 0 6 6  0. 0 6 6  0. 0 7 1  0. 0 7 1  0. 0 6 1  0.071  0.066  0.061  0.071  0.066  0.056  -49-  T A B L E  7  T. m e s e n t e r i c a  2259-7  Optical Density pH  0 Hours  2.1  3. 0  0. 0 5 1  0. 0 5 1  13 H o u r s  25 H o u r s  0.061  0. 0 8 1  0.092  0. 0 6 1  0.081  0. 0 8 1  0.061  0.081  0.081  0.061  0. 0 7 6  0.092  0.076  0.131  0.187  0. 0 7 6  0.119  0.215  0.076  0. 131  0. 2 0 8  0. 119  0. 2 2 9  0.081  0.149.  0. 2 8 4  0. 0 8 1  0.115  0. 2 8 4  0. 0 8 1  0. 131  0. 2 4 4  0. 0 8 1  0. 131  0.252  0.076  0. 125  0. 2 2 9  0.081  0.143  0.181  0. 0 7 6  0. 137  0.237  0.081  0. 125  0. 2 4 4  0. 0 7 6 4.7  5.4  7 .  2  8. 0  -10. 2  0.051  0. 0 4 6  0.051  0. 0 4 6  0.056  37 H o u r s  .  0. 0 6 1  0. 0 6 1  0.061  0. 0 6 1  0.061  0. 0 6 6  0.056  0.071  0. 0 7 1  0.056  0. 0 6 1  0.066  0.056  0.076  0.125  0.051  0.086  0.125  0.056  0. 0 8 6  0.114  0. 0 6 1  0. 0 8 1  0.119  0. 0 3 2  0.041  - 0. 0 3 6  0. 0 3 2  0.032  0. 0 3 6  C.032  0.036  0.032  0. 0 3 6  0. 0 3 6  0. 0 3 2  -  50-  T A B L E  T. e n c e p h a l a  Optical 0 Hours 0. 114  0. 131  0. 125  0.125  0. 131  0. 119  0.097  13 H o u r s  8  2392-1  Density 25 H o u r s  36 H o u r s  0. 0 6 1 0. 0 6 1 0. 161  0. 2 2 9 0. 2 4 4 0. 2 0 8  0.301 0. 301 0. 301  0. 155  0. 215  0. 310  0. 319  0. 6 0 2  0. 8 2 4  0. 301  0. 5 3 8  0. 8 5 4  0. 301  0. 5 8 5  0. 8 2 4  0. 301  0. 5 6 9  0.921  0. 310  0. 6 7 8  1. 0 9 7  0. 2 8 4  0.602  0.959  0.284  0.585  1.046  0. 310  0.495  1. 0 0 0  0. 181  0. 2 2 2  0. 2 6 8  0. 174  0. 215  0. 2 7 6  0. 181  0. 2 2 2  0. 2 6 8  0. 181  0. 2 2 2  0. 2 6 8  0. 2 6 0  0. 4 3 2  0. 5 0 9  0. 2 4 4  0. 3 9 8  0. 4 9 5  0. 2 6 0  0. 4 3 2  0.569  0. 2 4 4  0.398  0.538  0. 215  0.222  0. 215  0. 2 2 2  0. 215  0. 2 0 8  0. 215  0. 2 0 8  0. 2 0 8  0. 2 2 2  0. 2 0 8  0. 215  0.071  0. 0 5 1  0.056  0. 0 6 1  0.056  0.056  0. 0 7 1  0. 0 5 6  0.056  0.066  0. 0 6 1  0. 0 6 1  T A B L E  T. e n c e p h a l a  9  2392-7  Optical  Density  0 Hours  12. 5 H o u r s  24 H o u r s  0. 0 8 1  0. 0 9 7  0.114  0.149  0.097  0.119  0. 155  0. 0 9 7  0.125  0.161  0. 0 9 7  0.125  0.161  0.143  0.244  0.409  0.137  0.220  0.409  0. 137  0. 2 6 0  0.409  0.137  0.252  0. 4 2 0  0.081  0. 0 8 1  0. 0 8 1  0.086  0. 0 7 1  0.081  36 H o u r s  0.149  0.252  0. 5 5 2  0.149  0. 2 8 4  0.509  0.161  0. 3 3 7  0. 6 7 8  0. 155  0.337  0.745  0.125  0.168  0. 2 8 4  0. 119  0. 168  0.268  0. 131  0.174  0.276  0. 119  0.174  0.268  0. 125  0.215  0.357  0. 125  0.208  0.347  0. 125  0.222  0.347  0. 131  0.194  0. 3 5 7  0.102  0. 108  0.119  0.108  0. 114  0. 125  0. 1 0 2  0.114  0.119  0.108  0. 114  0. 125  0.056  0.051  0.041  0. 0 5 6  0. 0 5 1  0. 0 4 1  0. 0 5 6  0.051  0.041  0. 0 7 1  0.061  0. 0 4 1  T A B L E  T.  10  subanomala  Optical 0 Hours 0. 125  0.131  0.149  0. 155  0. 155  0.114  Density  13 H o u r s  24 H o u r s  36. 5 H o u r s  0.194  0. 2 2 9  0. 2 8 4  0.194  0. 2 2 2  0. 2 7 6  0.194  0. 2 4 4  lost  0.194  0. 2 2 9  lost  0. 3 3 7  0/550  0.745  __ 0. 3 4 7  0. 5 2 3  0. 7 4 5  0. 3 2 8  0.149  2327-1  0. 4 9 5  0. 7 9 6  0. 3 3 7  "~  0. 4 8 2  0. 7 7 0  0. 3 5 7  0. 5 2 3  1. 0 0 0  0. 3 5 7  0. 5 3 8  0. 6 5 8  0. 3 5 7  0. 5 5 2  0.921  0. 3 5 7  0. 5 3 8  0. 7 9 6  0. 2 6 8  0. 2 8 4  0. 301  0. 2 6 8  0. 2 8 4  0. 310  0. 2 6 8  0. 2 8 4  0. 301  0. 2 6 8  0. 2 8 4  0. 3 4 9  0. 3 1 9  0. 4 8 2  0. 7 7 0  0. 310  0. 4 8 2  0. 6 5 8  0. 3 1 9  0. 4 6 9  0. 6 5 8  0. 3 2 8  0. 4 6 9  0. 6 5 8  0. 319  0. 2 9 2  0. 2 8 4  0. 319 0. 310 0. 2 8 4  0. 2 9 2  0. 301  0. 2 9 2  0. 301  0.187  0. 301  "0.081  "0.086  0. 0 7 6  0.086  0. 0 8 1  0. 0 7 1  0. 0 9 7  0. 0 8 1  0. 0 7 6  0. 0 8 1  0. 0 9 2  0. 0 8 1  -53-  FIGURE  1.  F l o w s c h e m e of p r e p a r a t i o n of m e d i u m f o r p H - g r o w t h t r i a l s .  1500 m l .  100 m l .  distilled  water  water  1. 5 g.  t  soytone  9 f l a s k s w i t h 140  3 g. g l u c o s e a n d  ml.  1. 5 g. y e a s t e x t r a c t .  each  7. 5 m l .  buffer s t o c k to  each flask and pH  distilled  adjust  with IN N a O H IN  or  HC1.  Autoclave  Autoclave  Inoculate (except blank) with 10 m l .  10 m l .  of i n o c u l u m .  t o 12 t e s t t u b e s  for each  pH.  M e a s u r e four tubes at 12, 24 a n d  36 h o u r s .  -54-  FIGURE  3.  P r e p a r a t i o n of c e l l s f o r h o r m o n e p u l s i n g e x p e r i m e n t s .  Rapidly growing  inoculum  *  I  Determine, with sterile the dilution the o p t i c a l  d e n s i t y t o 0. 0 2 .  Adjust sufficient to o p t i c a l  medium,  factor to get  inoculum  d e n s i t y 0. 0 2  with sterile  medium  v 0. 0 4 m l . o f t h i s  3. 5 m l . o f s t e r i l e  solution.  medium.  0. 1 m l . o f h o r m o n e e x t r a c t . (Total v o l u m e now 4 m l . )  t E x p o s e , then filter and w a s h in two 2 m l .  lots.  

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