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UBC Theses and Dissertations

Somatic recombination in Ustilago hordei during the parasitic phase on barly Megginson, Fiona Gertrude Ariel 1973

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SOMATIC RECOMBINATION IN USTILAGO HORDEI DURING THE PARASITIC PHASE ON BARLEY  FIONA G.A. MEGGINSON B.Sc. Honours, U n i v e r s i t y o f E d i n b u r g h ,  1971  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  i n t h e Department of Genetics  We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA May, 1973  In p r e s e n t i n g an the  advanced degree at Library  I further for  this thesis  shall  the  of  this thesis  written  University  of  permission for  s c h o l a r l y p u r p o s e s may his  f u l f i l m e n t of  representatives.  be  for f i n a n c i a l gain  C\  £"A/  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  the  It i s understood  ETICS Columbia  shall  the  requirements  Columbia,  for reference  extensive  g r a n t e d by  permission.  Department o f  British  make i t f r e e l y a v a i l a b l e  agree that  by  in partial  that  not  and  copying of Head o f my  be  I agree  that  study.  this  thesis  Department  copying or  for  or  publication  allowed without  my  i ABSTRACT  The q u e s t i o n i n v e s t i g a t e d i n t h i s study  was  whether or not somatic recombination can occur i n U s t i l a g o h o r d e i w h i l s t i t i s i n the p a r a s i t i c phase on b a r l e y . investigation  was  carried  out i n two  The  parts.  In the f i r s t and major p a r t , b a r l e y seeds were inoculated a r t i f i c i a l l y  w i t h mixtures of n u t r i t i o n a l l y  d e f i c i e n t mutants of U. h o r d e i . way  T h i s was  done i n such a  t h a t i n f e c t i o n of the mature p l a n t s c o u l d o n l y occur  i f a t l e a s t one recombination event had taken p l a c e a s e x u a l l y , between a t l e a s t two formation.  One  i n f e c t i v e d i k a r y o n s , p r i o r to t e l i o s p o r e  smutted  p l a n t was  found.  of the t e l i o s p o r e s from t h i s i n f e c t i o n was The demonstrate  second p a r t of the study was  Detailed  analysis  carried  out.  designed to  t h a t more than one i n f e c t i v e d i k a r y o n can  s i m u l t a n e o u s l y occupy the h o s t .  A g a i n , mixtures of  n u t r i t i o n a l l y d e f i c i e n t mutants were used to i n o c u l a t e b a r l e y seeds but t h i s time so t h a t the o r i g i n of t e l i o s p o r e s from any i n f e c t i o n c o u l d be t r a c e d back to the p a r e n t a l types i n the i n f e c t i v e d i k a r y o n . I t was  concluded t h a t somatic recombination can  i n f a c t occur w h i l s t the fungus o c c u p i e s the host  tissue.  ii TABLE OF CONTENTS Page LITERATURE REVIEW  1  INTRODUCTION  8 11  MATERIALS AND METHODS  11 11 .11  A. Strains 1. Ustilago hordei... 2, Hordeum valgare  .12 12 .1^ 14 .14-  B. Culture of the fungus 1. Media 2. Growth of the fungus 3. Techniques a. Mating type tests b. Random spore analysis and r e p l i c a p l a t i n g c. Tetrad analysis  .1415  C. Host inoculation and planting.. .16 1. Techniques 16 a. Basic method of seed inoculationJ.6 b. Experimental plan 17 2. Rationale .22 25  RESULTS  .25  A. Somatic recombination experiment 1. Harvesting... 2. Random spore analysis  .25 ..........25 .29  3. Tetrad analysis B. Multiple i n f e c t i o n experiment. DISCUSSION BIBLIOGRAPHY  *  ^0 .32 51  iii LIST OF TABLES Table  Page No.  1.  Mutants of y_. h o r d e i used i n t h i s study............. 13  2.  P l a n f o r seed i n o c u l a t i o n i n somatic recombination  3.  experiment  •  ,  P l a n f o r seed i n o c u l a t i o n i n m u l t i p l e i n f e c t i o n 20  experiment. 4.  P r e l i m i n a r y random spore a n a l y s i s o f smut from head 501..  5.  26  Second random spore a n a l y s i s o f smut from head 501  6.  T e t r a d a n a l y s i s o f 501  ..2? teliospores  after 29  micromanipulation. 7.  Random spore a n a l y s i s made from the m u l t i p l e 31  i n f e c t i o n experiment 8.  9. 10.  18  P o s s i b l e ways i n which somatic recombination  could  have occurred to g i v e r i s e to t e l i o s p o r e s 501  37  P o s s i b i l i t i e s f o r 501  39  teliospore formation..  Table showing the m e i o t i c s e g r e g a t i o n s from germination o f t e l i o s p o r e types  expected kl  iv LIST OF FIGURES Page F i g u r e 1.  No.  Steps i n the p a r a s e x u a l c y c l e as they may  occur i n U. h o r d e i  Pontecorvo)  (after 4  V  ACKNOWLEDGEMENTS  I would l i k e t o express my thanks t o Dr. C l a y t o n Person both f o r s u g g e s t i n g the problem and f o r h e l p f u l discussion. laboratory  I am a l s o g r a t e f u l to him f o r the use o f h i s facilities.  Thanks a r e due, i n a d d i t i o n , t o Dr. Tony G r i f f i t h s for  h e l p and encouragement d u r i n g the e a r l y stages o f the  p r o j e c t when Dr. Person was away i n A u s t r a l i a . The author was enabled to c a r r y out t h i s work by a s c h o l a r s h i p awarded under the Commonwealth S c h o l a r s h i p and F e l l o w s h i p P l a n .  1.  LITERATURE REVIEW  The extreme v a r i a b i l i t y amongst p l a n t pathogenic f u n g i has been known f o r a l o n g time.  Even where normal  sexual r e p r o d u c t i o n i s i m p o s s i b l e because  the a l t e r n a t e  host i s absent or mating types are i n c o m p a t i b l e , the pathogenic organisms variability. may  o f t e n continue to show e x t e n s i v e  T h i s i s e s p e c i a l l y t r u e o f the smuts, which  be more v a r i a b l e than any other group of p l a n t  genic f u n g i  (Holton,1968j  Cherewick,1958).  patho-  Here v a r i a t i o n  abounds i n both morphology and p h y s i o l o g y . In many cases where sexual r e p r o d u c t i o n i s absent, the d i v e r s i t y of progeny  types cannot be e x p l a i n e d on the  b a s i s of gene mutation a l o n e .  F o r t h i s reason, para-  s e x u a l i t y (Pontecorvo, 1956), otherwise known as somatic recombination, has been suggested as the mechanism by which these pathogenic f u n g i maintain t h e i r P a r a s e x u a l i t y may  variability.  be thought o f as g e n e t i c recombination^  where no f i n e c o o r d i n a t i o n between recombination, segregat i o n and r e d u c t i o n e x i s t s .  In f a c t , the o p e r a t i o n of  p a r a s e x u a l i t y has been i m p l i c a t e d i n a number of p l a n t pathogens,  i n c l u d i n g s e v e r a l r u s t s (Bartos et a l . , 1 9 6 9 ;  Bridgmon,1959? E l l i n g b o e , 1 9 6 l j  Sharma and Prasada,1969J  V a k i l i and Caldwell,19575 Waterhouse,1952i Watson,19575 Watson and L u i g , 1 9 5 8 , 1959• 1 9 6 2 ; ) , smuts (Rowell,1955t Kozar,1969; Day and Anagnostakis,1971) and  Verticillium  (Ingram,1968j H a s t i e , 1 9 6 2 ) , Cephalosporium  (Tuveson  and  2. Coy,1961), Phytophthora and Fusarium  (Wilde,1961;  Leach and Rich,1969)  (Buxton,1956) s p e c i e s .  I m p l i c i t and perhaps more important than v a r i a b i l i t y i n a d i s c u s s i o n of p l a n t pathogenic f u n g i i s the means by which new  r a c e s are o r i g i n a t e d .  There are many  cases i n the r u s t s , f o r example, where new  r a c e s have been  observed to a r i s e a s e x u a l l y (Bartos et a l . , 1 9 6 9 ; 1959J  Bridgmon and Wilcoxson,1959;  Ellingboe,196l}  Flor,1957»  Bugbee e t  Bridgmon,  al.,1968|  I 9 6 0 ; L i t t l e and Manners,19675  Nelson e t al.,1955; Nelson,1966? Sharma and Prasada, V a k i l i and C a l d w e l l , 1957; Watson and L u i g , 1958, for  Waterhouse, 1952;  1959,  many y e a r s t h a t new  1962).  Watson,  1969; 1957;  I t has been known  r a c e s and b i o t y p e s of p a r a s i t i c  f u n g i a r i s e through h y b r i d i s a t i o n , mutation, and heterok a r y o s i s but i t seems not unreasonable  to suspect t h a t  somatic recombination i s a l s o r e s p o n s i b l e i n l a r g e p a r t for  the o r i g i n of new  r a c e s (Person, 1 9 5 8 ) .  The p a r a s e x u a l c y c l e has been shown to operate i n a v a r i e t y of fungi including Aspergillus nidulans, (Pontecorvo and Raper, 1952), A. n i g e r , and  Penicillium  chrysogenum, (Buxton, 1 9 5 6 ) , Coprinus s p e c i e s and S c h i z o phyllum commune ( C a s s e l t o n , 1965)1 as w e l l as i n the phytopathogenic  f u n g i mentioned above.  I t appears  therefore,  l e g i t i m a t e to conclude t h a t the p a r a s e x u a l c y c l e i s not a r a r e o d d i t y but t h a t i t s occurrence i s f a i r l y  widespread  3. i n nature and t h a t i t may w e l l p l a y a p a r t i n the v a r i a t i o n of  host-parasite relationships.  The p r e c i s e extent o f  the o p e r a t i o n o f the p a r a s e x u a l c y c l e i n nature cannot be gauged s i n c e many s p e c i e s i n which i t might occur have so f a r n o t been s u i t a b l e f o r experimentation o f t h i s kind.  A major d i f f i c u l t y  i n a s s e s s i n g the occurrence o f  p a r a s e x u a l i t y i n nature i s due t o the l a c k o f c r i t e r i a for  i t s detection.  N e v e r t h e l e s s , s u f f i c i e n t evidence i s  a v a i l a b l e to make a study o f the p r o c e s s o f somatic recombination smut,  i n a pathogen such as U s t i l a g o h o r d e i , a  worthwhile. Before d e s c r i b i n g the p r e s e n t study, a b r i e f  o u t l i n e o f the s t e p s i n the p a r a s e x u a l c y c l e as v i s u a l i s e d by Pontecorvo  ( 1 9 5 6 ) w i l l be g i v e n and the evidence f o r  them c o n s i d e r e d .  Three s t e p s appear to be e s s e n t i a l :  (a) H e t e r o k a r y o s i s , (b) f o r m a t i o n o f heterozygous  diploid  n u c l e i and t h e i r m u l t i p l i c a t i o n and (c) o c c a s s i o n a l s e g r e g a t i o n and recombination a t m i t o s i s i n c o n j u n c t i o n with haploidisation of d i p l o i d n u c l e i .  A more d e t a i l e d  e x p l a n a t i o n o f the p a r a s e x u a l c y c l e i s g i v e n i n F i g u r e 1 .  FIGURE 1 STEPS IN THE PARASEXUAL CYCLE AS THEY MAY OCCUR IN U. HORDEI. (AFTER PONTECORVO). 13b^Haploid homokaryotic strain^-(abc d)  Hyphal  anastomosis  Haploid heterokaryotic mycelium (abc d) + (ABC DT  13D H a p l o i d homokaryotic s t r a i n (ABC D)^ 3  5a  3  2X10~  6  Haploid n u c l e i sort themselves out  8  (abc d) (ABc D) (aBC d) (ABC D) 12  (abc  Heterozygous d i p l o i d homokaryotic mycelium  Heterozygous d i p l o i d n u c l e i  _2 5b 10 heterozygous or ? d i p l o i d n u c l e i undergo m i t o t i c c r o s s i n g over  11  haplo-diploid heterokaryotic myceliumi  u n l i k e n u c l e i fuse  ^10" d i p l o i d " n u c l e i undergo haploidisation 3  13a Haploid homokarytic s t r a i n s , some o f which are recombinant  d) + (ABC D)+  [Jjg  =5!  (abc d) (ABC D; 10  Haploid nuclei some o f which are recombinants  (abc:£.) 11 4  (abc (ABc (ABc (aBC etc.  D i p l o i d i s a t i o n p r i o r to t e l i o s p o r e formation  13b  1. abc/ABC r e p r e s e n t 3 l i n k e d genes and d/D another one u n l i n k e d 2. The genotypes o f the v a r i o u s kinds o f nucleus are e n c l o s e d i n parentheses. 3 . The occurrence f i g u r e s are those given f o r A s p e r g i l l u s n i d u l a n s .  dl D) d) d)  5. There i s adequate evidence each o f the major steps i n v o l v e d . a s s o c i a t i o n , s e p a r a t i o n and  documented f o r  Heterokaryosis  entails  subsequent r e g r o u p i n g of  g e n e t i c a l l y u n l i k e n u c l e i i n m u l t i n u c l e a t e c e l l s as a r e s u l t of hyphal f u s i o n s .  In the Basidiomycetes, d i k a r y o s i s ,  which i s simply h e t e r o k a r y o s i s i n v o l v i n g two  different  n u c l e i , i s a step i n the sexual p r o c e s s and as such i s a p r e r e q u i s i t e f o r i n f e c t i o n o f b a r l e y by U. h o r d e i . The  formation o f heterozygous  d i p l o i d s i n vegeta-  t i v e c e l l s , or d i p l o i d i s a t i o n , i s not so c l e a r l y demonstrable i n smuts although t h e r e i s c o n c l u s i v e evidence r i n g i n other f u n g i . T o l m s o f f , 1972) artificial,  In V e r t i c i l l i u m  f o r i t occur-  (Ingram,  1968;  v e g e t a t i v e d i p l o i d s occur i n nature w h i l s t  g e n e t i c methods have been d e v i s e d f o r the  s e l e c t i o n of spontaneously  o c c u r r i n g heterozygous  diploids  i n A s p e r g i l l u s n i d u l a n s (Roper, 1952), U s t i l a g o maydis, ( H o l l i d a y , 1 9 6 l b ) , and U. v i o l a c e a e (Day and Jones, C y t o l o g i c a l evidence Ehrlich  for diploidisation i s contradictory.  (1958), working w i t h U. maydis, observed  mycelium r e s u l t i n g from a c r o s s o f two the host was  1968).  i n i t i a l l y uninucleate.  i n c r e a s e , however, i n the percentage  t h a t the  haploid l i n e s i n  There was  a progressive  of d i k a r y o t i c  cells  u n t i l e l e v e n days a f t e r i n o c u l a t i o n a t which time the t r e n d r e v e r s e d and u n i n u c l e a t e c e l l s again predominated. changes were i n t e r p r e t e d as b e i n g due  These  to d i k a r y o t i s a t i o n  6. f o l l o w e d by n u c l e a r unions.  The l a t t e r were i n f e r r e d  from measurement o f n u c l e a r s i z e , which corresponded w i t h t h a t of chlamydospore  nuclei.  Thus i t was  well  concluded  t h a t karyogamy o c c u r r e d p r i o r to spore f o r m a t i o n . I n d i r e c t evidence f o r d i p l o i d i s a t i o n was by L e a c h and R i c h  (i960)  a l s o obtained  i n Phytophthora i n f e s t a n s where  hyphal f u s i o n s were observed c y t o l o g i c a l l y and p r o d u c t i o n o f p a r e n t a l and recombinant  types of zoospores was  The a c t u a l presence of d i p l o i d n u c l e i , however, was  obtained. never  observed. Bakerspigel  (1965)»  on the o t h e r hand, obtained  i n c o n c l u s i v e r e s u l t s upon c y t o l o g i c a l i n v e s t i g a t i o n of  19  d i f f e r e n t s p e c i e s and lk m i l l i o n n u c l e i f o r evidence of n u c l e a r f u s i o n s i n h e t e r o k a r y o t i c mycelium.  He  concluded  t h a t such f u s i o n s p o s s i b l y occur a t a r a t e lower than 1 i n 10.  Thus the evidence f o r the occurrence of h e t e r o -  zygous d i p l o i d s i n v e g e t a t i v e c e l l s o f f i l a m e n t o u s f u n g i has been almost e x c l u s i v e l y g e n e t i c . d i p l o i d i s a t i o n i s a rare  I t seems c e r t a i n  that  event.  F i n a l l y , the evidence f o r s e g r e g a t i o n and recomb i n a t i o n a t m i t o s i s and f o r h a p l o i d i s a t i o n i s e x t e n s i v e . These phenomena have been f o l l o w e d i n d i p l o i d d u r i n g both the sexual and p a r a s e x u a l c y c l e s  1956).  strains (Pontecorvo,  M i t o t i c h a p l o i d i s a t i o n has been induced i n  U. v i o l a c e a e by p - f l u o r o p h e n y l a l a n i n e (Day and Jones,  1968)  7.  and the r e s u l t i n g h a p l o i d l i n e s show a wide range of genotypes,  both recombinant  the o r i g i n a l d i p l o i d . at are  and p a r e n t a l f o r markers i n  I t a l s o occurs spontaneously but  a much lower frequency, i n d i c a t i n g t h a t d i p l o i d  nuclei  r e l a t i v e l y s t a b l e through many g e n e r a t i o n s o f somatic  cells.  T h i s o b v i o u s l y would enhance the o p p o r t u n i t y f o r  m i t o t i c c r o s s i n g - o v e r and s e g r e g a t i o n to o c c u r .  The  first  demonstration o f m i t o t i c recombination i n f u n g i f o l l o w e d the o b s e r v a t i o n of homozygous d i p l o i d segregants from heterozygous d i p l o i d s i n A . n i d u l a n s .  T h i s was f o l l o w e d  by a s i m i l a r demonstration i n U. maydis, where a u x o t r o p h i c segregants, s t i l l d i p l o i d , were o b t a i n e d from p r o t o t r o p h i c heterozygous d i p l o i d s ( H o l l i d a y , 1961)  and another i n  U. v i o l a c e a e (Day and Jones, 1968). It  seems c e r t a i n then t h a t somatic recombination,  an i n t e g r a l p a r t o f the p a r a s e x u a l c y c l e , i s an important f e a t u r e of the l i f e c y c l e o f c e r t a i n f u n g i .  Indeed, Raper  (1959) has i m p l i c a t e d i t s s i g n i f i c a n c e i n a l l Basidiomycetes, which o f course i n c l u d e s the smuts.  8. INTRODUCTION The knowledge t h a t somatic recombination occurs i n c u l t u r e both spontaneously and a f t e r i n d u c t i o n i n  U s t i l a g o maydis (Rowell, 1955? H o l l i d a y , 1 9 6 l b , 1965) and U. v i o l a c e a e (Day and Jones, 1968, 1969) has s t i m u l a t e d s i m i l a r i n v e s t i g a t i o n s o f the process i n another smut, namely U s t i l a g o h o r d e i .  Dinoor and Person  (1969) obtained  growth o f the d i k a r y o t i c phase o f U. h o r d e i i n c u l t u r e . The authors i n d i c a t e t h a t h a p l o i d recombinant  s p o r i d i a have  been o b t a i n e d from " f o r c e d d i k a r y o n s " and i n view o f the known p a r a s e x u a l i t y i n the two s p e c i e s mentioned  above,  conclude t h a t somatic recombination can occur i n v i t r o i n U. h o r d e i a l s o . F u r t h e r evidence t h a t the p a r a s e x u a l c y c l e , as w e l l as the normal o b t a i n e d by Kozar  s e x u a l c y c l e , occurs i n U, h o r d e i was  (196?, 1969a).  Compatible  combinations  o f mutant s p o r i d i a were used t o i n f e c t the b a r l e y host. A f t e r some time the p a r a s i t e was r e c o v e r e d as s p o r i d i a o r as t e l i o s p o r e s .  Evidence i n d i c a t e s t h a t the n u c l e i w i t h i n  the i n f e c t i v e d i k a r y o n undergo f u s i o n and produce  diploid  s p o r i d i a c h a r a c t e r i s e d by an i n d e f i n i t e Bauch t e s t (see M a t e r i a l s and Methods) and b i o c h e m i c a l "wild type"  appearance.  I f these d i p l o i d i n f e c t i o n s p o r i d i a were t o undergo h a p l o i d i s a t i o n , thereby p r o v i d i n g somatic recombination o f p a r e n t a l  9.  b i o c h e m i c a l markers,  a l l the requirements o f the p a r a s e x u a l  c y c l e would have been f u l f i l l e d In f a c t recombinant  (see L i t e r a t u r e r e v i e w ) .  types were observed amongst s p o r i d i a  e x t r a c t e d from the host p r i o r to t e l i o s p o r e f o r m a t i o n .  The  d i f f i c u l t y i n i n f e r r i n g somatic recombination o c c u r r i n g i n v i v o , however, l i e s i n the f a c t t h a t recombination c o u l d p o s s i b l y have occurred i n the inoculum mixture to p e n e t r a t i o n of the h o s t .  A l t e r n a t i v e l y , i t may  prior  have  o c c u r r e d i n the i n v i t r o grown c u l t u r e s a f t e r they had been e x t r a c t e d from the h o s t . In an e f f o r t to c l a r i f y the p r o c e s s , Dinoor (unpublished) u t i l i s e d a "double i n o c u l a t i o n technique". In t h i s technique some c u l t u r e s were used on the seed (e.g. ad-arg-leuc+ad~)  and o t h e r s were i n j e c t e d i n t o the  s e e d l i n g s grown from these i n o c u l a t e d seed (e.g. ad~arg-leuc""+leuc~). Recovery  of w i l d type mutants, which  c o u l d o n l y have a r i s e n by a t l e a s t two c o n s e c u t i v e recombina t i o n a l events, one of which would n e c e s s a r i l y be a s e x u a l , t a k i n g p l a c e i n the host p r i o r to t e l i o s p o r e f o r m a t i o n , would demonstrate  the o p e r a t i o n of somatic recombination  i n s i d e the p l a n t .  P r e l i m i n a r y r e s u l t s were encouraging  but the work was  d i s c o n t i n u e d owing to the l a c k o f r e p e a t -  a b i l i t y i n the experiment.  In a d d i t i o n , the numbers of  p l a n t s used were u n r e a l i s t i c a l l y s m a l l f o r the d e t e c t i o n of such a r a r e phenomenon (see L i t e r a t u r e r e v i e w ) .  10.  The  o b j e c t i v e o f the present study was  p r o v i d e an unequivocal demonstration  of somatic  to recombina-  t i o n , i f indeed i t does occur, i n o p e r a t i o n d u r i n g the development of U. h o r d e i i n s i d e the host p l a n t . two  As i n the  p r e v i o u s l y d e s c r i b e d s t u d i e s , b a r l e y seeds were  inoculated a r t i f i c i a l l y d e f i c i e n t mutants.  w i t h mixtures of n u t r i t i o n a l l y  In t h i s study, however, the  matings were arranged  i n such a way  gametic  as to p r e c l u d e the  occurrence o f a r e c o m b i n a t i o n a l event p r i o r to p e n e t r a t i o n of  the h o s t .  Furthermore,  the p o s s i b i l i t y o f a recombin-  a t i o n a l event o c c u r r i n g a f t e r e x t r a c t i o n of s p o r i d i a the host was i n t h i s study  from  e l i m i n a t e d by the s e l e c t i o n process employed (see Methods).  1 1 .  MATERIALS AND METHODS.  A.  STRAINS 1,  U s t i l a g o h o r d e i (Pers.) Lagerh.  The Hood  mutants used i n t h i s study were induced by  u s i n g UV i r r a d i a t i o n and were d e r i v e d from  ( 1 9 6 6 )  the w i l d type s t r a i n s I ^  +  and  the m a j o r i t y o f these mutations  .  I t was assumed t h a t  were p o i n t mutations  rather  than chromosomal a b e r r a t i o n s , s i n c e the former a r e known commonly to r e s u l t a f t e r UV  irradiation.  The p a r t i c u l a r auxotrophic s t r a i n s used were chosen ( 1 )  f o r ease of d e t e c t i o n i . e . n o n - l e a k i n e s s , ( 2 ) f o r  the absence o f any evidence o f c l o s e l i n k a g e w i t h other markers such as the mating type l o c u s or other n u t r i t i o n a l mutations  and ( 3 )  f o r t h e i r n o n - p a t h o g e n i c i t y on Hannchen  and Vantage b a r l e y c u l t i v a r s when present i n the homozygous state. noted  Table 1 shows the s t r a i n s chosen.  I t should be  t h a t the double mutant s t r a i n s used i n each case were  d e r i v e d from the c o r r e s p o n d i n g s i n g l e mutants.  2.  Hordeum v u l g a r e L,  F o r t h i s study, two v a r i e t i e s o f b a r l e y , Hannchen and Vantage, were chosen.  These v a r i e t i e s are known to be  h i g h l y s u s c e p t i b l e to i n f e c t i o n from c r o s s e s between the w i l d type s t r a i n s of the fungus from which the auxotrophic mutants (see Table  1 )  were d e r i v e d  (Thomas,  1 9 6 5 ) .  12. I n a d d i t i o n to the l a b o r a t o r y stock o f Vantage seed, some Vantage was obtained from B u c k e r f i e l d s L t d . , New Westminster.  Care was taken to d i s t i n g u i s h between  these two types o f Vantage s i n c e t h e i r g e n e t i c a n c e s t r y was l i k e l y to be d i f f e r e n t .  B. CULTURE OF THE FUNGUS. 1.  Media.  A m o d i f i e d Vogel's  (1956) medium was used.  Complete medium contained: 5g« D i f c o y e a s t e x t r a c t , 5gt s a l t - f r e e casein  h y d r o l y s a t e (N.B. Co.), l O g . dextrose,  50mg. tryptophane, 20ml, Vogel's s a l t s o l u t i o n  (ibid),  10ml. v i t a m i n s o l u t i o n ( H o l l i d a y , 1961), 1 l i t r e water.  distilled  F o r s o l i d medium 2% D i f c o bacto agar was added to  make agar p l a t e s and 2.5$ to make s l a n t s . Minimal  medium contained 20ml. Vogel's  s o l u t i o n and l O g . dextrose per l i t r e  salt  of d i s t i l l e d  Supplemented minimal medium was prepared to  H o l l i d a y (1961).  water. according  The i n d i v i d u a l growth f a c t o r s were  added to the minimal medium as r e q u i r e d : amino a c i d s lOOmg., p u r i n e s and p y r i m i d i n e s lOmg., and v i t a m i n s 1 mg. per litre. All  media were s t e r i l i s e d by a u t o c l a v i n g f o r 15  minutes a t 151b. use.  p r e s s u r e and 121  degrees Centigrade  before  13. TABLE 1 M u t a n t s o f U. h o r d e i  Experiment  Somatic Recombination  Genotype  AdU40  NiaX95  2-15  Ad"Nia Ad^Nia" Ad~Nia~  MetV364 SerV138 2-34  Met~Ser Met s e r " Met"Ser" +  a a A  LeuV4-17 ArgV240  Leu"Arg* Leu_Arg~ Leu"Arg"  a a A  Leucine Arginine Leucine +  Arg~Leu* Arg_Leu~ Arg~Leu~  a a A  Arginine Leucine Arginine + Leucine  * +  A a  ArgV242 LeuV4-17  2-55  I4  *  study.  Strains  2-53  Multiple Infection  used i n t h i s  Mating type a a A  +  +  E/C ArgV35 ProV324 PanV271 PdxV26 MetV375 LeuV4 IlvU26  Arg" Pro Pan" Pdx" Met" Leu" Ilv"  A A A A A A A  & & & & & & &  Nutritional requirement Adenine Niacin Adenine +  Niacin  Methionine Serine . .Methionine + Serine  Arginine  a a a a a a a  Arginine Proline Pantothenic A c i d Pyridoxine Methionine Leucine I s o l e u c i n e and Valine  Denotes w i l d type.  Each c u l t u r e was r o u t i n e l y t e s t e d f o r i t s n u t r i t i o n a l requirements and i t s mating type p r i o r to use.  14  2. G r o w t h o f t h e f u n g u s . Shake c u l t u r e s were m a i n t a i n e d i n D e l o n g flasks in  fitted  a New  flasks their  w i t h Morton  contained total  liquid  capacity.  was  3.  A l l incubation  placed These  of cultures,  of liquid  made a t 22 d e g r e e s C e n t i g r a d e ( - 2 d e g r e e s ) .  Techniques. tests.  fungus e x h i b i t s b i p o l a r  incompatibility  and  therefore  i s important to e s t a b l i s h the mating types of a l l  cultures was  closures  medium t o a p p r o x i m a t e l y 40$  a. M a t i n g type  it  steel  Brunswick Psychrotherm i n c u b a t o r - s h a k e r .  or s o l i d ,  This  stainless  culture  involved.  Compatibility  o f the s p o r i d i a l  d e t e r m i n e d by t h e B a u c h t e s t r e f e r r e d  Hoiton  (1957).  Here,  however, t h e t e s t s  o u t on c o m p l e t e medium o w i n g c a r r y i n g more t h a n one suchfaden  (infection  t o by F i s c h e r had  t o be  to the r e l u c t a n c e  a u x o t r o p h i c marker  to  approached  carried strains  hyphae) on m i n i m a l medium.  S p o r i d i a r e c o v e r e d from smutted  ft  of  and  produce  b . Random s p o r e a n a l y s i s and r e p l i c a  f r o m t h e shake  lines  heads  plating.  o f b a r l e y were t a k e n  c u l t u r e s when t h e c o n c e n t r a t i o n o f  sporidia  7  10 -10'  per ml.  (lOmg/ml.) were added  One  o r two  t o e a c h shake  bacterial proliferation.  Counts  drops of achromycin  culture  to prevent  o f t h e s p o r i d i a were made  u s i n g a S p e n c e r haemocytometer w i t h improved Neubauer  ruling  15.  and  appropriate  d i l u t i o n s were made t o g i v e a p p r o x i m a t e l y  100  c o l o n i e s per p l a t e .  S t e r i l e c o n d i t i o n s were ensured  throughout. When t h e s e c o l o n i e s became v i s i b l e (about d a y s ) , c o l o n i e s were p i c k e d o f f w i t h s t e r i l e and p l a n t e d  3-4  toothpicks  t o "master" p l a t e s o f complete medium on  o f 25 s e c t i o n s .  grids  For each a n a l y s i s made, a p p r o x i m a t e l y  200 c o l o n i e s were t e s t e d .  A f t e r 3-4  days i n c u b a t i o n ,  the  c o l o n i e s were r e a d y f o r r e p l i c a t i o n . R e p l i c a p l a t i n g was  performed a c c o r d i n g  L e d e r b e r g and L e d e r b e r g ' s t e c h n i q u e ( 1 9 5 ) ,  otherwise  2  known as the " V e l v e t Pad Method".  C o l o n i e s were r e p l i c a t e d  t o m i n i m a l medium, to each o f the r e q u i r e d media and  t o lawns o f  and  supplemented  on complete medium i n  o r d e r to determine the m a t i n g type (see B3a. A f t e r 4-5  to  above).  days the p l a t e s were examined t o determine the  biochemical  c h a r a c t e r i s t i c s of the c o l o n i e s .  c. T e t r a d  analysis.  I s o l a t i o n of the f o u r p r o d u c t s o f m e i o s i s promycelium and  t h e i r subsequent t r a n s f e r t o new  s e t up s e p a r a t e c u l t u r e s was ( D i c k i n s o n , 1926) and m i c r o f o r g e .  from the budding  done by s t a n d a r d  media t o  procedure  u s i n g a De Fonbrune m i c r o m a n i p u l a t o r A s l i g h t m o d i f i c a t i o n o f the method  was  employed to e l i m i n a t e the d i f f i c u l t and hazardous p r o c e s s of s i n g l e t e l i o s p o r e i s o l a t i o n .  Instead,  a s m a l l drop of  16. the  teliospore  having f i r s t way  5-10  and  after  chosen in  s u s p e n s i o n was p l a c e d on t h e a g a r b l o c k ,  greatly  diluted  the suspension.  In this  t e l i o s p o r e s were p l a n t e d t o e a c h a g a r b l o c k g e r m i n a t i o n , t h e most s u i t a b l e  one c o u l d  f o r micromanipulation, l e a v i n g the r e s t  the centre of the b l o c k .  analysed  The t e t r a d s c o u l d  be  untouched t h e n be  f o r t h e i r r e q u i r e m e n t s on s u p p l e m e n t e d  minimal  media.  C. HOST INOCULATION 1.  AND  PLANTING.  Techniques.  a . B a s i c method o f s e e d The to  barley  s e e d s were i n o c u l a t e d w i t h t h e f u n g u s a c c o r d i n g  t h e method o u t l i n e d  t r e a t e d w i t h 0.12$ 1:320  diluted the  seed  formalin  ( i . e . 40$  i n order to s t e r i l i s e  which  i s thought  The wet s e e d s were s p r e a d  t o w e l s t o a i r d r y f o r 24-48  to a i d  thinly  hours.  c u l t u r e s were i n o c u l a t e d  s l a n t s and a l l o w e d t o grow f o r 3-4  days.  to fresh  Fresh  agar  inoculum  s l a n t s was p l a c e d i n t h e a p p r o p r i a t e q u a n t i t y o f  liquid for  were  The s e e d s were t h e n washed i n r u n n i n g t a p  Sporidial  from  A l l seeds  formaldehyde  w i t h w a t e r ) f o r one h o u r  f o r 30 m i n u t e s .  on p a p e r  by T a p k e ( 1 9 4 2 ) .  s u r f a c e and l o o s e n h u l l s ,  infection. water  inoculation.  complete  48-60  medium  hours u n t i l  and m u l t i p l i e d  i n shake  the c u l t u r e s appeared  culture  thick.  17.  The b a r l e y seeds to be i n o c u l a t e d were counted i n t o batches of  about 100 and p l a c e d i n f l a t - b o t t o m e d #7  crystalite vials.  snap-cap  S p o r i d i a l inoculum was poured over the  seeds u s i n g equal q u a n t i t i e s o f the two mating types i n each v i a l . jar 20  The v i a l s were p l a c e d uncovered i n a d e s s i c a t o r  (without d e s s i c a n t ) , and evacuated f o r 20 minutes a t l b . vacuum, i n order to draw the inoculum under the seed  hulls.  Excess inoculum was poured o f f and the seeds dumped  i n t o l a b e l l e d #2 M a n i l l a c o i n envelopes.  These envelopes  were l e f t unsealed so t h a t the seeds c o u l d a i r dry a t room temperature.  A f t e r a minimum o f three days, the seeds  were sown. b. E x p e r i m e n t a l p l a n . The f i r s t and main experiment was designed to d e t e c t somatic recombination o c c u r r i n g i n the fungus d u r i n g i t s development i n s i d e the h o s t .  To t h i s end, seeds were i n o c u l a t e d  i n the manner shown i n T a b l e 2 . the  field  i n May, 1 9 7 2 .  Each row was 10 f e e t l o n g and  c o n t a i n e d about 100 seeds. apart.  The seeds were then sown i n  The rows were spaced one f o o t  A f t e r about three months the b a r l e y p l a n t s were  examined c a r e f u l l y f o r the presence o f smut and any smutted heads c o l l e c t e d . The second experiment was c a r r i e d out as a s u p p o r t i v e study t o the f i r s t experiment. to  I t was designed  demonstrate t h a t m u l t i p l e i n f e c t i o n , a p r e r e q u i s i t e f o r  18. TABLE Plan  f o r seed  Test number  i n o c u l a t i o n i n somatic experiment. Cross  Combination 1 2  l  2  C  A  recombination  3 ,  a  Variety  2-15  AdU4o + NiaX95  2-15  AdU4o  H  10  H  10  >  6 4  b H  y  3°  2-15  4  AdU40  1  C  NiaX95 NiaX95  d  2-34  2  2-34  MetV364  V H  3  2-34  SerV138  H  4  MetV364  SerV138  V H V  1  2-53  2  2-53  3  L e u V 4 l 7 + ArgV240 LeuV4l7 ArgV240  10 10  h b  h b  b  H  > H  v 2-53  6 4  > v  a  10  >  MetV364 + SerV138 H v  2  3  6 4  V.  e  b  H  h V H V  b  4  LeuV4l7  ArgV240  Number o f rows  20 10 10 20 10 10 20 10 10 20 10 10  20 10 10 20 10 10 20 10 10 20 8 12  19. TABLE 2 (CONT.) Cross Test  Combination 1  a  A 2-56  a  Variety  ArgV242+ LeuV4l7  H  V. V H V V H b b  4  2  2-56  ArgV242  D  3  2-56  LeuV4l7  V  Number o f rows 20 10 10 20 10 10 20 1  0  r  ArgV242  a  LeuV4l7  H  20  * V v  D  8  12  H = Hannchen V = Vantage a . A s s t a t e d a b o v e , e q u a l q u a n t i t i e s o f t h e two o p p o s i t e m a t i n g t y p e s were u s e d i n a l l c o m b i n a t i o n s i . e . when two c u l t u r e s o f t h e same m a t i n g t y p e , a^. and a » a r e u s e d i n one c r o s s , Volume o f A = Volume a + a andV olume a ^ * Volume a, 2  r  1  2  b . The V a n t a g e s e e d d e s i g n a t e d V was B u c k e r f i e l d ' s L t d . ( s e e A2 a b o v e ) .  o b t a i n e d from  c . C o m b i n a t i o n s 2, 3 and 4 i n e a c h t e s t c o n s t i t u t e t h e necessary c o n t r o l s f o r t h i s experiment i . e . the p a i r - w i s e c o m b i n a t i o n s o f e a c h o f t h e c u l t u r e s u s e d i n one t e s t . d. A l t h o u g h t h e c u l t u r e s used i n Combination 4 o f each t e s t were o f t h e one m a t i n g t y p e and t h e r e f o r e i n c o m b a t i b l e , i t was s t i l l n e c e s s a r y t o make t h i s c o n t r o l ( s e e C2 b e l o w ) . e. E a c h t e s t was d i v i d e d i n t o two i d e n t i c a l p a r t s i n o r d e r to provide r e p l i c a t e s . R e p l i c a t e s were t r e a t e d s e p a r a t e l y i n d i f f e r e n t b a t c h e s a n d sown i n d i f f e r e n t p a r t s o f t h e field.  TABLE 3 P l a n f o r seed i n o c u l a t i o n s i n m u l t i p l e i n f e c t i o n Test  experiment  Combination A a a a a a a a a 1^ E~ ArgV35 ProV324 PanV271 Pdxv26 MetV375 LeuV4 I l v U 2 6 1  +  +  2  +  +  3  +  +  +  4-  +  +  +  +  5  +  +  +  +  +  6  +  +  +  +  +  7  +  +  +  +  8  +  +  +  +  + +  + +  + +  +  +  TABLE 3 (CONT.) Test  Combination A ArgV35  B  A A A ProV32i+PanV271 PdxV26  -  -  2  +  +  3  +  +  +  4  +  +  +  +  5  +  +  +  +  6  +  +  7  +  +  A  A  MetV375  LueV4  A  IlvU26  a ~  T e s t B formed a r e c i p r o c a l  +  +  +  + + .  +  +  +  +  +  +  +  +  +  +  +  (with r e s p e c t to mating types) r e p l i c a t i o n  Once a g a i n , equal q u a n t i t i e s o f the two mating types were used i n each as e x p l a i n e d i n Table 2,  o f T e s t A. combination  22.  the success of the f i r s t  experiment,  can a c t u a l l y occur.  Crosses were made a c c o r d i n g to the schedule shown i n Table 3.  A l l the i n o c u l a t i o n s were made to the Hannchen c u l t i v a r  and  i n each case one row  of 50 seeds was  planted.  Each  row was  4-g- f e e t l o n g and the rows were spaced 6 inches  apart.  T h i s experiment was  October, (-5  1972.  i n the greenhouse i n  Seeds were grown a t 75 degrees F a h r e n h e i t  degrees) u s i n g a 15  et a l . (1962).  performed  hour day as suggested by S c h a f e r  During i t s growth, the crop had to be  t r e a t e d w i t h i n s e c t i c i d e f o r aphids and s p i d e r mites w i t h sulphur dust f o r powdery mildew.  and  These problems and  the crowding of the seeds i n the bench caused a number of p l a n t s to d i e but s u f f i c i e n t data was  still  to the p r o v i s i o n of a r e p l i c a t e b e i n g made.  obtained owing Smutted heads  were c o l l e c t e d a f t e r about 4 months. 2. R a t i o n a l e . The  somatic recombination experiment  was  designed to d e t e c t the occurrence of t h i s phenomenon i n the fungus w i t h i n the i n f e c t e d host p l a n t p r i o r to t e l i o spore f o r m a t i o n .  The method depends on the occurrence  o f double i n f e c t i o n .  The mutant s t r a i n s chosen  f o r each  t e s t were known not to permit s p o r u l a t i o n when brought homozygosity.  to  F o r example, i n one t e s t , the markers were  arranged i n the f o l l o w i n g  way:  23.  S t r a i n 1 : A Leu"Arg" Strain 2 » a Leu~Arg  +  S t r a i n 3 '> a L e u A r g ~ +  where A and  a are the two  mating  types.  When these s t r a i n s are mixed together  and  poured over  the s u s c e p t i b l e b a r l e y seeds f o r i n o c u l a t i o n , two are expected, both e q u a l l y l i k e l y  ( p r o v i d i n g t h a t there i s  no d i f f e r e n t i a l i n tendency to mate and s t r a i n s were present 'A'  t h a t the two  i n equal q u a n t i t i e s and  mating type e q u a l l e d  t h a t of  matings  'a'  the q u a n t i t y  'a* mating  of  type)t  #1  X #2  to g i v e dikaryon  Leu~Arg" + L e u ~ A r g  and #1  X #3  to give dikaryon  Leu Arg  +  + Leu Arg  .  Thus, those p l a n t s which have been i n f e c t e d s i n g l y , or c a r r y a s i n g l e i n f e c t i n g genotype, should s i n c e n e i t h e r dikaryon alone;  show no  smut a t  i s capable of c a u s i n g i n f e c t i o n  both dikaryons are homozygous f o r one  o f the two  maturity,  n u t r i t i o n a l markers.  Consequently, any  which does have smut would be of i n t e r e s t and o f the t e l i o s p o r e s would be  or the  other plant  investigation  informative.  C o n t r o l s were necessary i n t h i s experiment to ensure t h a t the i n d i v i d u a l p a i r - w i s e  matings ( i . e . #1X#2,  #1X#3, #2X#3) on the s u s c e p t i b l e host gave no r e s u l t . c o n t r o l s would a l s o provide  a check f o r mutation o f  mutant c u l t u r e s back to w i l d type.  Otherwise the  and #3,  l o o k i n g f o r smutted heads and  the  experiment  c o n s i s t e d simply o f i n o c u l a t i n g w i t h the mixtures of #2  These  #1,  checking these  24. for  markers p r e s e n t i n s i n g l e The  entirely event  exclude  on  time  this rationale  the p o s s i b i l i t y  o f an  asexual  whilst  the s u r f a c e o f the seed p r i o r  span  the purposes ated  by  occurring during inoculation,  resting The  method d i c t a t e d  teliospores.  involved, of t h i s  the fungus  i s actually Since  inside  o f the event  the  are  to p e n e t r a t i o n . that, for  such a l i k e l i h o o d  to the g r e a t e r p o s s i b i l i t y  recombination  the s p o r i d i a  however, i s so b r i e f  study,  cannot  may  be  subordin-  occurring  whilst  plant.  the success o f the somatic  recombination  experiment  w h o l l y depended upon t h e o c c u r r e n c e o f  double  infection,  i t was  experi-  c o n s i d e r e d n e c e s s a r y t o show by  ment t h a t more t h a n one The  experiment  described  was  either The to  1^  guished  Cherewick  matings  the  occur.  manner t o  (1964).  The  that  m u t a n t s were  on Hannchen when c r o s s e d w i t h  (depending  different from  and  could i n fact  i n a similar  infection  or  sporidial seven  s e t up  by P e r s o n  known t o p r o d u c e  infection  on t h e i r  were a r r a n g e d  genotypes,  each  o t h e r s by v i r t u e  mating  type).  i n s u c h a way  that  capable of being of t h e i r  w h e t h e r any two,  i t c a n be d e d u c e d , by random s p o r e smut on a p l a n t  three, four,  five,  has  been p r o d u c e d  s i x or seven  distin-  nutritional  r e q u i r e m e n t s , were p r e s e n t t o g e t h e r i n t h e i n o c u l u m I n t h i s way,  up  by  infections.  mixture.  analysis, either  one,  25.  RESULTS A. SOMATIC RECOMBINATION EXPERIMENT. 1.  Harvesting. TEST 1.  I t was n o t i c e d  (see Table 2)  t h a t many of the 8000 p l a n t s i n t h i s t e s t  were smutted, which was not expected.  Therefore,  the  o r i g i n a l c u l t u r e s used were r e t e s t e d and i t was found t h a t the Ad U4-0 c u l t u r e had r e v e r t e d was consequently  to w i l d type.  This test  disregarded.  TEST 2 . of 16000 p l a n t s , none were smutted.  Out  TEST J3. One p l a n t out of 16000 was smutted.  I t occurred  i n combina-  t i o n 1 of the t e s t and was t h e r e f o r e of g r e a t i n t e r e s t . It  had two smutted t i l l e r s  and was designated  501 s i n c e  t h i s was the number of the row i n which i t was found. TEST 4 . o f 16000 p l a n t s , none were smutted.  Out  N.B.  None of the c o n t r o l combinations i n t e s t s 2 , 3 or  4 showed smut.  2.  Random Spore A n a l y s i s . Table 4 shows the r e s u l t s of the f i r s t  spore a n a l y s i s t h a t was made.  random  26.  TABLE  Preliminary  random s p o r e a n a l y s i s  4 - 4 -  4.  Leu A r g Number o f coloniesi %  It  2  3.8  c a n be s e e n  recovered i.e.  —  Leu Arg  30  Total1  4  from  —  _  Leu A r g  21  126  19.1  19.9  20.6  16.6  100  the t a b l e  process of elimination  that  it  was p o s s i b l e  further, as was  then bases,  colonies  requirements  medium o r on medium or leucine  +  tested  described  then v i t a m i n s . was  by a s i m i l a r p r o c e s s , t o n i c o t i n i c  when r e t e s t e d ,  by  Holliday  Once i t was  f o r a vitamin,  t o n a r r o w t h e i n v e s t i g a t i o n down  I t was  by a  on m e d i a l a c k i n g  t h e unknown r e q u i r e m e n t  t h e r e s u l t o f a spontaneous  arginine.  were i n v e s t i g a t e d  a f t e r t h e manner  being the missing n u t r i e n t .  cultures,  17?° o f t h e  leucine  T h e y were s u c c e s s i v e l y  that  Total  „ 26  T h e s e unknown c o l o n i e s  established  Unknown  25  with arginine,  amino a c i d s ,  501.  _  Arg  u  d i d n o t have any o f t h e e x p e c t e d  supplemented  first  Le  head  24  t h e y d i d n o t grow on m i n i m a l  (1961).  o f smut f r o m  acid  still (niacin)  assumed t h a t  mutation  showed no t r a c e  since  this  the o r i g i n a l  of a niacin  requirement. With  this  knowledge, a second  random  spore  27. a n a l y s i s was  carried  out, the r e s u l t s  o f w h i c h a r e shown  5.  i n Table  TABLE j> S e c o n d random s p o r e a n a l y s i s o f smut f r o m head  $01.  Genotype as deduced from auxonography  Mating A  Leu Arg Nia  +  23  18  41  20.2  Leu~Arg~Nia~  7  5  12  5.9  Leu~Arg Nia~  9  6  15  7.4  18  19  37  18.2  22  8  30  14.8  Leu Arg~Nia"  6  11  17  8.4  Leu Arg Nia~  7  7  14  6.9  Leu~Arg~Nia  17  20  37  18.2  109  94  203  100.0  +  +  +  Leu Arg~Nia +  +  Leu A r g N i a +  +  +  Totals  In t h i s  +  type a  Total  number  %  of total colonies  case, the requirements o f a l l the c o l o n i e s  discovered  and f i t t e d  w i t h t h e e x p e c t a t i o n s i f t h e r e had  been a m u t a t i o n t o N i a c i n cultures.  were  deficiency  i n one o f t h e p a r e n t a l  I t was o b s e r v e d , however, t h a t  the d i f f e r e n t  g e n o t y p e s d i d n o t o c c u r i n e q u a l f r e q u e n c i e s , a s w o u l d be expected  on t h e b a s i s o f random gene a s s o r t m e n t a t m e i o s i s .  28. A p o s s i b l e e x p l a n a t i o n f o r the d i s c r e p a n c y ( i . e . the severe d e f i c i e n c y of a l l N i a ~ c o n t a i n i n g c o l o n i e s ) c o u l d have been d i f f e r e n t i a l r a t e s o f m u l t i p l i c a t i o n of the v a r i o u s genotypes a f t e r spore g e r m i n a t i o n . t h i s was  In order to f i n d out i f  the case, a c o m p e t i t i o n experiment was  performed.  In t h i s experiment, a Leu A r g N i a c u l t u r e and a Leu~Arg~Nia~ c u l t u r e were each i n o c u l a t e d to 5ml. complete medium.  of l i q u i d  These were allowed to m u l t i p l y i n shake  c u l t u r e f o r 2k hours.  The c e l l s were then counted so that  the c o n c e n t r a t i o n s could be a d j u s t e d and equal numbers o f c e l l s o f each type added to 50 i n a DeLong c u l t u r e f l a s k .  ml. o f l i q u i d complete medium  The mixture was  shaken a t  c o n s t a n t temperature f o r three hours a f t e r which time the c e l l s were p l a t e d out onto complete medium to a c o n c e n t r a t i o n of about 100  c o l o n i e s per p l a t e .  Master p l a t e s were  made a f t e r three days and these were l a t e r r e p l i c a t e d to minimal medium. I t was  found that twice as many of the c o l o n i e s +  +  .  s e l e c t e d were of the Leu A r g N i a  +  —  —  type;., as o f the Leu A r g N i a  type, c o n f i r m i n g the s u s p i c i o n o f d i f f e r e n t i a l r a t e s of m u l t i p l i c a t i o n f o r the two genotypes.  E v i d e n t l y then, the  ^discrepancy was caused not by a d e f i c i e n c y of N i a ~ c a r r y i n g genotypes a f t e r spore g e r m i n a t i o n but merely by t h e i r slower r a t e of d i v i s i o n .  29. 3. T e t r a d A n a l y s i s . Great d i f f i c u l t y  was experienced i n o b t a i n i n g  complete t e t r a d s a f t e r micromanipulation o f the 501 t e l i o spores.  Out o f a t l e a s t 100 attempts, o n l y 10 complete  t e t r a d s were obtained, w i t h the help o f J.V. Groth. Table 6 shows the r e s u l t s o f the t e t r a d  analysis.  TABLE 6 T e t r a d a n a l y s i s o f 501 t e l i o s p o r e s a f t e r  T e t r a d Leu A r g N i a Number type obtained  micromanipulation.  T e t r a d Leu A r g N i a Number type obtained  + +  +  + + +  + +  +  +  + +  +  + +  + +  + + A r g = A r g i n i n e ; Leu = Leucine; N i a = N i a c i n . - i n d i c a t e s a requirement f o r the n u t r i e n t . + i n d i c a t e s no requirement f o r the n u t r i e n t .  + +  30. B. MULTIPLE INFECTION EXPERIMENT. Smutted p l a n t s were not obtained from combination  every  i n both t e s t s (see Table 3) but a t l e a s t one  smutted head was obtained f o r each type o f i n o c u l a t i o n . Where more than one smutted head was obtained f o r a p a r t i c u l a r combination,  each head was t e s t e d i n d i v i d u a l l y .  r e s u l t s of the a n a l y s i s appear i n Table 7.  The  I t can be seen  t h a t i n o n l y one head out o f the 20 examined was there any evidence o f m u l t i p l e i n f e c t i o n .  Combination  k i n Test B  showed d e f i n i t e l y t h a t two d i f f e r e n t i n f e c t i v e  dikaryons  had p a r t i c i p a t e d i n p r o d u c i n g the smut i n f e c t i o n .  TABLE 7 RANDOM SPORE ANALYSIS MADE FROM THE MULTIPLE INFECTION EXPERIMENT Test  Combination  1  2 A  3 6 7 8 1 2  B  4 5 6 7  Head  1  1 1 2 1  WT WT WT WT WT WT WT WT WT  1  2 1 2 1 1 1  2 1 1 1  2 1 2 3 4  Mating type a Number A  WT WT WT WT WT WT WT WT WT WT WT  96  40  104 104 93  44 54 57 57  175. 175 180  86  107 90 110 105 100 136  56 38 53 51 51 63  109 127  190 198 192 195  183  69  98 96  97  103 107  2  Mating type Number A a  A r g " 90 A r g " 95 A r g " 93 A r g " 105 Leu" 91 Leu" 72 89 I l v " 18 77 I l v " 15  57 50 ^5 51  A r g ^ 92 A r g 110 A r g " 84 A r g " 22 Pan" 99  47 53 34 10  56 60 50 36 52 58 84  51 52 57 54 49 73 93 95  85  94 101 98 85  --  40  25 8 2  48  33 45  48  54 51 47 10 14  45 57 50 12  51  3  Mating type Number A a  -—  Pan"  -  WT = w i l d type R e f e r to Table 3 f o r the s p o r i d i a l c r o s s e s made i n each combination.  68  26  42  32. DISCUSSION  The d e t e c t i o n of a r a r e r e c o m b i n a t i o n a l event i n a micro-organism  requires conditions that  facilitate  r e c o g n i t i o n of a n o v e l phenotype i n a v a s t p o p u l a t i o n h a v i n g the p a r e n t a l phenotype. t h i s study met  The s e l e c t i o n system employed i n  t h i s requirement! out of 12000 p l a n t s i n  which somatic recombination c o u l d have o c c u r r e d , i t was easy to p i c k out the one p l a n t i n which i t had taken p l a c e ( s u b j e c t to c o n f i r m a t i o n by the i s o l a t i o n of the o r i g i n a l markers). N u t r i t i o n a l l y d e f i c i e n t mutants were used i n t h i s study as markers f o r i n v e s t i g a t i n g the p r o c e s s of somatic recombination.  A n a l y s i s f o r t h e i r presence,  subsequent  to the p u t a t i v e somatic recombination event, p r o v i d e d c o n f i r m a t i o n t h a t t h i s one smutted  p l a n t was  indeed  produced  through somatic recombination r a t h e r than by mutation. a d d i t i o n a l safeguard a g a i n s t the l a t t e r p o s s i b i l i t y p r o v i d e d i n the c o n t r o l s of the experiment Even though  two  spontaneous  An  was  (see R e s u l t s , C 2 ) .  mutations were observed d u r i n g  the course of the experiment*  namely the adenine  reversion  to w i l d type and the mutation to n i a c i n requirement i n one o f the p a r e n t a l c u l t u r e s of 501,  none were observed i n the  p a i r - w i s e c o n t r o l combinations of T e s t 3» i n which 501 found.  was  In these there was no chance of smut b e i n g produced  u n l e s s w i l d type recombinants  had been formed as a r e s u l t of  33. some back m u t a t i o n o c c u r r i n g i n one o f t h e p a r e n t a l c u l t u r e s . From t h e r e s u l t s , t h e n , i t i s p o s s i b l e t o s a y t h a t somatic r e c o m b i n a t i o n must have o c c u r r e d i n t h e i n f e c t i v e d i k a r y o n s of  501 w h i l s t they o c c u p i e d h o s t t i s s u e . In  d e a l i n g w i t h an o b l i g a t e p a r a s i t e such as  U. h o r d e i , i t i s n o t easy t o g i v e t h e e x a c t sequence o f e v e n t s as P o n t e c o r v o  (1956) has done f o r t h e Ascomycetes.  An a t t e m p t , however, w i l l be made t o e x p l a i n t h e p o s s i b l e mechanism by which these r e s u l t s may have been o b t a i n e d . C e r t a i n d e d u c t i o n s can i m m e d i a t e l y be made from the t e t r a d a n a l y s i s (see T a b l e 6 ) .  Since non-parental  d i t y p e s a r e as numerous as p a r e n t a l d i t y p e s , no matter  which  two markers we c o n s i d e r and no matter what we assume t h e genotype o f 501 t e l i o s p o r e s t o be, i t can be c o n c l u d e d t h a t t h e r e i s no l i n k a g e between t h e markers concerned; l o c i d e t e r m i n i n g a r g i n i n e , l e u c i n e and n i a c i n  i . e . the  requirements  a r e e i t h e r a l l s i t u a t e d on d i f f e r e n t chromosomes o r s u f f i c i e n t l y f a r a p a r t on a s i n g l e chromosome t h a t recombinat i o n would appear t o be random.  T h i s i s supported by t h e  f a c t t h a t i n no t e t r a d a r e a l l t h r e e p a i r s o f markers t e t r a type.  T h i s means t h a t t h e r e can have been no t h r e e - s t r a n d  double c r o s s - o v e r s a t m e i o s i s , which i s h i g h l y u n l i k e l y i f t h e t h r e e genes were l i n k e d . As was i n t i m a t e d i n t h e l i t e r a t u r e r e v i e w ( p . 5)» s e g r e g a t i o n o f markers f o r which a d i p l o i d i s heterozygous  34.  o c c u r s d u r i n g v e g e t a t i v e m u l t i p l i c a t i o n , not o n l y as a consequence of m i t o t i c c r o s s i n g - o v e r between l i n k e d genes but a l s o as a consequence o f a process o f h a p l o i d i s a t i o n , i n which whole chromosomes, not chromosome p a r t s , a t random.  Thus somatic  "recombination" of u n l i n k e d genes,  on d i f f e r e n t chromosomes, should s t i l l  occur.  i s the form o f somatic recombination thought in this  reassort  This, to be  then,  active  study. D e t a i l s o f the process o f h a p l o i d i s a t i o n are  almost completely unknown.  H a p l o i d i s a t i o n c o u l d be a  consequence o f an a c c i d e n t a l breakdown a t m i t o s i s i n the s e p a r a t i o n of chromatids  to the two p o l e s t  i n a proportion  o f cases one daughter nucleus a r i s e s w i t h a s i n g l e s e t of chromosomes (Pontecorvo,  1956).  Breakdowns of t h i s k i n d  are well-known i n h i g h e r organisms  and r e s u l t i n a n e u p l o i d s .  A f u r t h e r deduction which can be made from the t e t r a d d a t a i s t h a t s i n c e a l l three markers are s e g r e g a t i n g i n a l l the t e t r a d s , the 501 . t e l i o s p o r e s must have been heterozygous  f o r a l l t h r e e markers.  The w i l d type  sporidia  obtained i n both the random spore a n a l y s e s and the t e t r a d a n a l y s i s bear testimony to t h i s s i n c e w i l d types can o n l y be generated i f t h e r e i s a t l e a s t one w i l d type a l l e l e a t each l o c u s i n the d i p l o i d  teliospore.  Furthermore,  the f a c t  t h a t w i l d type s p o r i d i a were obtained a t a l l i n d i c a t e s t h a t there must have been two  s u c c e s s i v e recombination  events?  35.  one taking place asexually i n the plant (somatic recombination) and the other i n the teliospores (meiotic recombination) .  This can be inferred because one member of each  possible dikaryon (the 'A' mating type) i s mutant at both the o r i g i n a l marker l o c i .  Since each of the 'a' mating type  members i s mutant at one or other of these l o c i , i t i s impossible for a wild type offspring to be generated by a single meiotic event.  The following diagrammatic represen-  t a t i o n w i l l help to make t h i s clearer* DIKARYON #lt  TELIOSPORES  Leu"~Arg~A + Leu~Arg a..... .Segregation for Arg"only, not f o r Leu . +  #2% Leu~Arg"A + Leu Arg~a.  Segregation for Leu~only, not for Argr  f  Thus, i f any teliospore segregates for both Leu" and Arg"" (thereby generating some wild type offspring) a l l three nuclei must have participated i n at least two recombinational events. With the above information and allowing for the fact that a mutation to n i a c i n requirement  occurred (see  Results A2) i n one of the parental cultures of 501, an hypothesis may be put forward which can explain how the present r e s u l t s were obtained.  O r i g i n a l l y , three s p o r i d i a l  types were present i n the inoculum mixture of 501t #1 8 Leu""Arg~Nia A #2 I Leu'Arg Nia a #3 : Leu Arg"Nia a +  +  +  +  +  36. The p o s s i b l e occurred  ways i n w h i c h  between these Now,  assuming  t y p e s when i t comes to  mating  type  somatic  types free  t h e v a r i o u s ways i n w h i c h to  produce  is  shown i n T a b l e  a r e shown i n T a b l e association  to teliospore  restrictions,  teliospores 9.  recombination  could  have  8.  of a l l nuclear  formation, subject only  i t i s possible  to  karyogamy c o u l d occur  heterozygous  determine i n order  at a l l three l o c i .  This  TABLE 8 P o s s i b l e ways i n which somatic recombination c o u l d have occurred to give r i s e to t e l i o s p o r e s 5 0 1 .  Mutation to N i a ~ in»  Diploidisation*  #1  „„  1'  Event  Dikaryons  1 Nuclear Type Leu 1 2  Haploidisation  Arg  Mating Nia Type  —  _  +  +  +  + +  _  5 6 7 2 1  Diploidisation*  1 2  Haploidisation  6 7 5 2 1  -  + +  -  + •*  -  +  +  -  —  + -  -  + +  + +  2 Nuclear Type Leu  A a  1 3  a A a A a A  8 6 9 6 9  A a  1 3  a A a A a A  6 8 6 8 6 8  _  Arg  Nia  _  _  +  +  +  + +  -  -  +  -  —  -  +  + —  -  + -  + +  -  -  +  Mating Type A a a A a A a A  + +  A a  + + + + + +  a A a A a A  TABLE 8 (CONT.) Dikaryons Mutation to Nia" i m  Event  Diploidisation* Haploidisation  #3  * T h i s event was  1  Nuclear Type Leu 1  2  ,  6  5 6 5 2 1  inferred  -  Arg  + —  + -  + +  Mating Nia Type  2  Nuclear Type Leu  + +  A a  1 3  + + + + +  a A a A a A  6 9  +•  8  + +  —  —  +  —  +  -  -  -  -  -  + -  8  Arg Nia  +  + —  -  +  Mating Type A a a A a A a A  39. TABLE 2 P o s s i b i l i t i e s f o r 501 Mutation to Nia" i n :  t e l i o s p o r e formation  Karyogamy between n u c l e a r types:  T e l i o s p o r e genotype T e l i o s p o r e Leu A r g N i a Mating Type Type  #2 + #9 +  #1  + -  a A  A  #3 + #7  +  +  + -  a A  B  #3 + #7  +  +  + -  a A  B  #2 + #8  +  +  + -  A a  C  + -  + -  a A  A  +  +  #2  #2 + #9 +  #3  + -  #3 + #5  +  (=B)  D  a  (=A)  A  With r e s p e c t to the n u t r i t i o n a l markers, i t can be seen t h a t there are o n l y two for 501,  b a s i c types of t e l i o s p o r e p o s s i b l e  A or B, r e g a r d l e s s of where we  mutation to N i a " to have o c c u r r e d . we  p o s t u l a t e the  Therefore, whilst  can say t h a t the mutation to N i a " d i d occur, i t i s  i m p o s s i b l e to say y e t e x a c t l y where. t i o n p a t t e r n s expected f u r t h e r evidence  The m e i o t i c  segrega-  from t e l i o s p o r e types A and B o f f e r  on t h i s p o i n t , as shown i n Table  10.  When the t e t r a d types g i v e n i n t h i s t a b l e are compared w i t h those a c t u a l l y obtained  i n t h i s study  (see Table 6 ) ,  it  4-0. w i l l be seen t h a t a l l the p o s s i b l e types o f t e t r a d were present  i f both types o f t e l i o s p o r e , A and B, had germinated  from the 501 head.  By i n s p e c t i o n o f Table  9,  o n l y one o f  the three p o s s i b i l i t i e s f o r t e l i o s p o r e formation  fulfills  t h i s requirement, namely that where the mutation to N i a " has occurred  i n s t r a i n 1,  the double mutant.  I t i s concluded,  t h e r e f o r e , t h a t a mutation to n i a c i n requirement  occurred  i n p a r e n t a l s t r a i n #1 and t h a t t h i s was f o l l o w e d  by the  somatic and m e i o t i c recombination events d e s c r i b e d above i n the s e r i e s o f t a b l e s 8 , 9, and 10. I t must be r e a l i s e d t h a t t h i s whole hypothesis i s based on the c o n v e n t i o n a l diploids case,  (see F i g u r e 1).  idea of parasexuality involving T h i s need n o t n e c e s s a r i l y be the  as H a r t l e y and W i l l i a m s  envisage a mechanism errors i n migration  (1971)  f o r generating  have p o i n t e d  out.  They  g e n e t i c v a r i a t i o n by  o f chromosomes a t d i k a r y o t i c m i t o s i s .  Thus an exchange o f whole chromosomes between h a p l o i d  cells  c o u l d occur without a l t e r i n g the normal chromosome complement o f the two n u c l e i .  Somatic recombination could  therefore  be e x p l a i n e d without i n v o k i n g the h y p o t h e t i c a l process o f diploidisation.  To prove t h a t somatic recombination i s a  m i t o t i c process  i n v o l v i n g d i p l o i d s r e q u i r e s the i s o l a t i o n  o f s t a b l e d i p l o i d s i n which s e g r e g a t i o n  can be s t u d i e d .  T h i s has been done s u c c e s s f u l l y i n U. maydis ( H o l l i d a y ,  196lb),  as mentioned b e l o w j but the event o f d i p l o i d i s a t i o n  41.  can o n l y be i n f e r r e d i n U. h o r d e i so f a r . of t h i s hypothesis, is  For the purposes  however, i t i s f e l t t h a t t h i s  inference  justified.  10  TABLE  T a b l e showing the m e i o t i c segregations germination of t e l i o s p o r e t y p e s .  expected from  T e l i o s p o r e Type  Tetrad Type  Leu Leu  _ A Arg_Nia_ Arg~Nia"  i  p  + +  + +  + —  —  _  + + -  +  -  + + _  3  +  - —  + +  -  -  +  + +  + +  +  Tetrad Type  L  + +  <  + +  -  -  D  +  +  6  —  + + +  —  -  + +  —  +  + +  —  —  —  +  to the d i f f i c u l t i e s experienced teliospores.  +  +  - - +  At t h i s p o i n t , c o n s i d e r a t i o n should  501  +  Leu_Arg Nia_ Leu~Arg N i a ~  —  —  -  B  be given  i n micromanipulation  o f the  I t has a l r e a d y been mentioned t h a t the  v a r i o u s s p o r i d i a l types are s u b j e c t to d i f f e r e n t i a l r a t e s of m u l t i p l i c a t i o n  (see R e s u l t s A2)  depending on the n u t r i -  t i o n a l requirements which they c a r r y .  T h i s e x p l a i n s why  the  42.  allele ratios  (the p r o p o r t i o n s of r e c o v e r y of the two homo-  logous s t r a n d s ) depart from l s l ,  b e a r i n g i n mind t h a t the  segregants are r e c o v e r e d many n u c l e a r g e n e r a t i o n s a f t e r the event which produced  them.  I t i s suggested t h a t  slower r a t e of growth and consequent  this  lowered v i a b i l i t y of  some of the s p o r i d i a l types, n o t a b l y o f those c a r r y i n g the n i a c i n requirement, may  account f o r the widespread  to r e c o v e r a l l f o u r m e i o t i c products a f t e r germination i n t h i s experiment.  failure  teliospore  Germination type i s known  t o be a f f e c t e d by the n u t r i e n t content of the medium (Holton et a l . , 1 9 6 8 )  and v a r i a t i o n s i n spore g e r m i n a t i o n amongst  the smut f u n g i are e x t e n s i v e ( i b i d . ) .  Wild type t e l i o s p o r e s ,  the o f f s p r i n g from which have no n u t r i t i o n a l requirements, are s e n s i t i v e enough to m a n i p u l a t i o n , so i t i s not  unreason-  a b l e to conclude t h a t t e l i o s p o r e s c a r r y i n g one or more n u t r i t i o n a l markers would be extremely d i f f i c u l t to handle. o n l y 1 0 complete  The  t e t r a d s obtained (Table 6 ) , however, do  p r e s e n t u n e q u i v o c a l evidence f o r somatic recombination o c c u r r i n g i n the fungus w h i l s t i t r e s i d e d w i t h i n the i n f e c t e d host p l a n t . A d d i t i o n a l evidence that t h i s was  indeed the  case comes from the m u l t i p l e i n f e c t i o n experiment. r  r e s u l t s shown i n Table 7 demonstrate two  The  c l e a r l y t h a t at l e a s t  i n f e c t i v e dikaryons can occupy an i n f e c t e d host p l a n t  a t the same time.  Furthermore, Person and Cherewick  (1964)  43.  showed t h a t i n U. k o l l e r i and do occur and may  t h a t more than one  genotype of the  be found w i t h i n a s i n g l e diseased  majority The  U. avenae, m u l t i p l e i n f e c t i o n s  of d i s e a s e d  p l a n t s had  plant.  pathogen  In f a c t ,  the  been i n f e c t e d a t l e a s t t w i c e .  dangers of e x t r a p o l a t i o n of r e s u l t s between organisms  must be r e a l i s e d but  i t seems f a i r l y c e r t a i n from these  p i e c e s o f evidence t h a t m u l t i p l e i n f e c t i o n can i n the smuts and  two  occur g e n e r a l l y  t h e r e f o r e t h a t somatic recombination  may  a l s o be a g e n e r a l  phenomenon i n t h i s group of f u n g i .  This  w i l l be d i s c u s s e d  a t g r e a t e r l e n g t h below.  The  a c t u a l event of somatic recombination i n  U. h o r d e i seems to be d i f f e r e n t from t h a t d e s c r i b e d U. maydis. U. h o r d e i  for  So f a r as i s known, none o f the c u l t u r e s of t h a t have been t e s t e d i s unstable  or r e a c t s  like  a b i s e x u a l c u l t u r e , although Kozar (1969b) d i d o b t a i n i n d i c a t i o n s t h a t some d i p l o i d s may haploid sporidia. any  have been recovered  In other words, workers f a i l e d to  t h a t any  d i p l o i d s t h a t may  This could  simply  i n most recorded  instances  described indicate  be formed i n the dikaryons  t r a n s i t o r y by v i r t u e o f extreme i n s t a b i l i t y .  immediately by  are  Although  the d i p l o i d s p e r s i s t as a  a r a r e d i p l o i d i s a t i o n event f o l l o w e d  with  find  d e f i n i t e l y d i p l o i d s p o r i d i a l c u l t u r e s l i k e those  f o r U. maydis ( H o l l i d a y , 1 9 6 l b ) .  very  along  clone, haploid-  i s a t i o n c o u l d produce much of the g e n e t i c d i v e r s i t y i m p l i c i t i n p a r a s e x u a l i t y , w i t h no d i r e c t evidence of the presence of  44.  a fusion nucleus.  So t h i s i s c e r t a i n l y not evidence a g a i n s t  the occurrence o f somatic recombination i n U. h o r d e i . The tions.  outcome o f t h i s study has important  implica-  P r i m a r i l y , i t has shown t h a t t h i s event i s not simply  a laboratory a r t i f a c t .  Secondly, i t i s the f i r s t  o f somatic recombination o c c u r r i n g i n v i v o . organism  report  S i n c e the  used here i s a p a r a s i t e , t h i s suggests t h a t a  and p r e v i o u s l y unsuspected  new  source o f v a r i a b i l i t y i s p o t e n t i a l l y  a v a i l a b l e f o r a l l e c o n o m i c a l l y damaging f u n g i . s e x u a l c y c l e i n f u n g i p r o v i d e s a complete  The  para-  e r s a t z f o r sexual  recombination i n t h a t i t p r o v i d e s f o r both recombination s h e l t e r i n g o f gene v a r i a t i o n  (Pontecorvo, 1 9 5 8 ) .  and  In the  l a t t e r r e s p e c t , i t i s more v e r s a t i l e than s e x u a l r e p r o d u c t i o n because i t i n c l u d e s two ways o f s t o r i n g gene v a r i a t i o n ( h e t e r o k a r y o s i s and h e t e r o z y g o s i s ) i n s t e a d o f o n l y one.  For  example, the formation of d i p l o i d s c o u l d a i d i n the r e t e n t i o n o f f a v o u r a b l e mutations ones.  Too  or i n the e l i m i n a t i o n of unfavourable  much emphasis, however, should not be p l a c e d on  the storage o f v a r i a t i o n i n the Basidiomycetes, s i n c e a l l d i p l o i d s reduce to h a p l o i d s i n each g e n e r a t i o n by m e i o s i s . More important here perhaps,  i s the r e a l i s a t i o n  t h a t the p a r a s e x u a l c y c l e o p e r a t i n g i n p l a n t pathogenic f u n g i c o u l d p r o v i d e an a d d i t i o n a l v e h i c l e f o r the r e a s s o r t ment o f v i r u l e n c e genes.  The use o f n u t r i t i o n a l l y  deficient  mutants here i s a t o o l i n the i n v e s t i g a t i o n o f processes of importance  i n the reassortment o f genes f o r p a t h o g e n i c i t y .  45.  S i n c e m u l t i p l e i n f e c t i o n can occur  i n b a r l e y , there i s  a l s o the p o s s i b i l i t y o f a s y n t h e s i s i n the host, by s e x u a l recombination,  para-  of b i o t y p e s p o s s e s s i n g genes f o r  v i r u l e n c e from more than two  parental biotypes.  As mentioned above (see L i t e r a t u r e Review), there are many r e p o r t s of new  races of r u s t s a r i s i n g a s e x u a l l y  but i n the m a j o r i t y of cases the authors a t t r i b u t e t h e i r occurrence F o r example, F l o r was  no  to parasexual  recombination.  (1964) s t a t e s c a t e g o r i c a l l y t h a t "there  i n d i c a t i o n t h a t parasexual  the pathogenic  are r e l u c t a n t to  processes  v a r i a t i o n o f Melampsora l i n i "  were i n v o l v e d i n even though  he d i d o b t a i n s e v e r a l a s e x u a l v a r i a n t s upon mixing u r e d i o spores  o f two  rust races.  Instead  he e x p l a i n e d these v a r i a n t s  on the b a s i s of n u c l e a r exchange f o l l o w e d by s i n g l e gene mutations or d e l e t i o n s . o r i g i n of new  The  importance o f mutations i n the  r a c e s has been mentioned by a number of  authors  ( C h r i s t e n s e n , 1959;  Holton,  1957;  Day,  i 9 6 0 , 1966? F i s c h e r and  Parmeter e t a l , 1963? Toxopeus, 1956? Zimmer  et a l . , 1963).  W h i l s t i t i s r e a l i s e d t h a t mutations are  undoubtedly a f a c t o r i n the development o f new f e l t t h a t one  other  races, i t i s  cannot r u l e out somatic recombination  another p o s s i b i l i t y , as F l o r p o i n t s out i n h i s 1971  as paper.  Other workers a t v a r i o u s times have suggested somatic h y b r i d i s a t i o n , h e t e r o k a r y o s i s , n u c l e a r p a i r i n g r e g a r d l e s s of sex and c y t o p l a s m i c  i n f l u e n c e s as b e i n g  or s e v e r a l l y r e s p o n s i b l e f o r the p r o d u c t i o n  of new  singly  asexual  46. v a r i a n t s i n the r u s t s .  More r e c e n t l y , Bartos et a l . ( 1 9 6 9 ) »  H a r t l e y and W i l l i a m s ( 1 9 7 1 ) . and Sharma and Prasada  (1969)  have e x p l a i n e d s i m i l a r r e s u l t s on the b a s i s of p a r a s e x u a l r e c o m b i n a t i o n , as i t has been d e f i n e d here.  I t seems t h a t  n e i t h e r n u c l e a r exchange nor any of the o t h e r mechanisms suggested above can account f o r both the o r i g i n and o f new  s t r a i n s among the progeny  diversity  o f mixtures of s p o r e s .  Somatic recombination, on the o t h e r hand, can accomodate both of these f a c t o r s and as such may o f o r i g i n o f new  be an important mode  s t r a i n s i n both r u s t s and  smuts.  I t i s i n t e r e s t i n g to note a t t h i s p o i n t i n biochemical studies with species of U s t i l a g i n a l e s , a n a l y s i s c o u l d have c o n s i d e r a b l e advantage meiotic analysis (Halisky, 1965).  that mitotic  over standard  M i t o t i c recombination  c o u l d become important i n the mapping o f chromosomes of organisms  l i k e U. h o r d e i , which have so f a r f r u s t r a t e d a l l  attempts a t m e i o t i c a n a l y s i s , i n the same manner as i t has been employed i n chromosome s t u d i e s o f A. n i d u l a n s (Pontecorvo and Roper, 1952) Furthermore,  and U. v i o l a c e a e (Day and Jones,  i n v i t r o p r o d u c t i o n of somatic  1969).  recombinants  c o u l d be used as a means of c i r c u m v e n t i n g passage  through  the h o s t , b a r l e y i n t h i s case, thus o b v i a t i n g the n e c e s s i t y o f a t h r e e month g e n e r a t i o n i n t e r v a l . d e s p i t e the many unknowns, may  Standard  parasexuality,  a l s o prove to be a u s e f u l  tool  f o r the e l u c i d a t i o n of g e n e t i c c o n t r o l s o f and the b i o s y n t h e t i c processes involved i n v i r u l e n c e .  In any case,  well-marked  47.  arms i n a l l the chromosomes are necessary before nature of " v i r u l e n c e " can be determined.  through  recombination. The  r e s u l t s of t h i s study suggest c e r t a i n l i n e s  for further research into this subject. we  true  There i s l i t t l e  doubt t h a t t h i s p r e r e q u i s i t e c o u l d be achieved somatic  the  With t h i s system  c o u l d study the extent o f p e n e t r a t i o n and  of a v i r u l e n t biotypes  development  i n r e s i s t a n t p l a n t s , u s i n g the method  o f s p o r i d i a l e x t r a c t i o n p r i o r to t e l i o s p o r e d e s c r i b e d by Kozar (1967).  formation  F u r t h e r , by i n o c u l a t i n g w i t h  v a r i o u s combinations of v i r u l e n c e genes, we  could i n v e s t i g a t e  (a) the p o s s i b i l i t y t h a t i n e x p r e s s i b l e genes f o r v i r u l e n c e from an a v i r u l e n t i s o l a t e w i l l be recovered other  e x p r e s s i b l e genes and  by a b i o t y p e  (b) the p o s s i b i l i t y t h a t  p h e n o t y p i c a l l y a v i r u l e n t b i o t y p e s w i l l recombine  with  two  asexually  i n the host to produce a v i r u l e n t b i o t y p e , which w i l l  thus  be able to reproduce. T h i s work a l s o has  i m p l i c a t i o n s f o r the  study  o f f u n g i whose sexual phase i s not common but i n which there i s a considerable o f v a r i a t i o n may and  d i v e r s i t y of r a c e s . be o p e r a t i o n a l and  s i n c e v a r i a t i o n i s complex  dynamic, the i d e n t i f i c a t i o n o f any  a l l are i n t e r - r e l a t e d , i s p r o b l e m a t i c . and  Since v a r i o u s mechanisms  one  mechanism, when  Mutation,  heterokaryosis  somatic recombination are a l l n u c l e a r mechanisms thought  to produce v e r s a t i l i t y i n Fungi I m p e r f e c t i ,  Versatility  48.  i s extremely important to the s u r v i v a l of a l l p l a n t pathogens, s i n c e without i t , extension  of host range to i n c l u d e  r e s i s t a n t v a r i e t i e s i s impossible.  The  f a c t that parasexual  recombination can determine v a r i a t i o n i n the host a t l e a s t i n the F u s a r i a , has (1956).  Therefore  range,  been shown adequately by Buxton  a wide f i e l d o f r e s e a r c h  both fundamental and  new  has been opened,  a p p l i e d , by work on somatic r e c o m b i n a t i o n .  Nevertheless,  i t i s only p o s s i b l e to  speculate  on the importance of p a r a s e x u a l i t y i n the s u r v i v a l and e v o l u t i o n of p l a n t pathogens i n n a t u r e , s i n c e evidence of parasexuality  i n natural populations  i s wanting.  c e r t a i n , however, t h a t the phenomenon i s not p a r t i c u l a r taxonomic u n i t or to any  I t seems  confined  to a  s p e c i f i c group of  pathogens because i t occurs i n f u n g i p r o d u c i n g disease wide range of hosts (see L i t e r a t u r e Review). which i t may Imperfecti,  The  have the g r e a t e s t s i g n i f i c a n c e i s the Fungi where i t may  be a major e v o l u t i o n a r y  o n l y i n t o the g e n e t i c s and  events per  mechanism.  cytology  research  o f recombination  se but a l s o i n t o problems of anastomosis, incom-  p a t i b i l i t y , heterokaryosis There i s no  a  group to  More complete r e a l i s a t i o n of i t s p o t e n t i a l r e q u i r e s not  on  and  extranuclear  inheritance.  doubt f o r example, t h a t the number of h a p l o i d  s t r a i n s of f u n g i g r e a t l y outweighs the number o f d i p l o i d s t r a i n s i n nature.  In f a c t , there i s o n l y one  naturally occurring stable d i p l o i d  r e p o r t of a  (Ingram, 1 9 6 8 ) .  I f the  49. parasexual why  cycle provides  such a good source  are d i p l o i d s not more common i n nature?  of v a r i a b i l i t y , What g e n e t i c  c o n t r o l s are imposed on p a r a s e x u a l i t y i n nature? On the whole, the parasexual  c y c l e seems l e s s  p e r f e c t but more f l e x i b l e than the sexual c y c l e . be  I t would  s u r p r i s i n g i f a system w i t h p o t e n t i a l i t i e s as great  those  as  of s e x u a l r e p r o d u c t i o n were merely a l a b o r a t o r y  curiousity.  Indeed, the r e s u l t s of t h i s study would i n d i c a t e  that i t i s not.  That p a r a s e x u a l i t y probably  relevant part i n natural populations  does p l a y a  i s demonstrated by  work o f L u i g and Watson (1971) i n A u s t r a l i a .  Here sexual  r e p r o d u c t i o n i n wheat r u s t i s v i r t u a l l y n o n - e x i s t e n t to the absence of the b a r b e r r y host.  the  owing  Yet the r u s t has  been  a b l e to a t t a c k s u c c e s s i v e l y a l l the r e s i s t a n t wheat v a r i e t i e s t h a t have been i n t r o d u c e d , c a u s i n g g r e a t concern.  There i s  l i t t l e doubt t h a t asexual recombinants and mutants of the fungus p r o v i d e the necessary  variability.  In c o n c l u s i o n , i t may  be s a i d t h a t v a r i a t i o n i n  smut f u n g i , i n a l l i t s r a m i f i c a t i o n s , has mental impact on p a t h o g e n i c i t y . somatic recombination,  i t s most funda-  T h i s study has  shown t h a t  a l b e i t a very r a r e event, may  to the p r o d u c t i o n o f new  and more v i r u l e n t r a c e s o f smut.  Furthermore, i t has  shown t h a t somatic recombination  occur i n pathogenic  f u n g i w h i l s t they are occupying  plant.  The  contribute  can the  host  importance o f t h i s f i n d i n g i s easy to p e r c e i v e  50.  when one c o n s i d e r s t h a t most o f the f u n g i pathogenic c u l t i v a t e d p l a n t s and,  i n c i d e n t a l l y , most o f those  to  employed  i n i n d u s t r i a l f e r m e n t a t i o n p r o c e s s e s , are a s e x u a l .  It is  r e a l i s e d , however, t h a t although c u r r e n t l y r e c o g n i s e d processes of p a r a s e x u a l i t y l a r g e l y conform to those d e s c r i b e d f o r A. n i d u l a n s (see L i t e r a t u r e Review), other v i a b l e o f recombination  are not p r e c l u d e d .  One  thing i s certain:  many more e x p l o r a t i o n s , u s i n g p a r a s e x u a l recombination p l a n t pathogenic  processes  in  f u n g i , i n t o the m o d i f i c a t i o n of host range,  i n t e r - g e n i c e f f e c t s on v i r u l e n c e and b l o c k s i n p a t h o g e n i c i t y appear warranted.  5 1 .  BIBLIOGRAPHY  1.  B a k e r s p i g e l , A. 1 9 6 5 . C y t o l o g i c a l i n v e s t i g a t i o n s of the p a r a s e x u a l c y c l e i n f u n g i . 1 . Nuclear f u s i o n . Mycopathol. Mycol. Appl. 261233-240.  2.  B a r t o s , P., F l e i s c h m a n n , G., S a m b o r s k i , D . J . and S h i p t o n , W. 1 9 6 9 . S t u d i e s on a s e x u a l v a r i a t i o n i n t h e v i r u l e n c e o f o a t crown r u s t , P u c c i n i a c o r o n a t a , and a wheat l e a f r u s t , P. r e c o n d i t a . Can. J . B o t a n y 4 7 : 1 3 8 3 - 1 3 8 7 .  3.  Bradley,  4.  S.G. 1 9 6 2 , P a r a s e x u a l phenomena i n o r g a n i s m s . Ann. Rev. M i c r o b i o l . 1 6  micro: 3 5 - 5 2 .  B r i d g m o n , G.H. 1 9 5 9 . P r o d u c t i o n o f new r a c e s o f P u c c i n i a g r a m i n i s v a r . t r i t i c i by v e g e t a t i v e f u s i o n . Phytopath_n 9:386-388. :  5.  B r i d g m o n G.H. and W i l c o x s o n , R.D. 1 9 5 9 . 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