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Interactions of cowpea strains of southern bean mosaic virus and of tobacco mosaic virus in cowpea and… Molefe, Thandie Leagajang 1979

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INTERACTIONS OF COWPEA STRAINS OF SOUTHERN BEAN MOSAIC VIRUS AND TOBACCO MOSAIC VIRUS IN COWPEA AND  OF  PINTO BEAN  91  by THANDIE  LEAGAJANG MOLEFE  B . S c . , The U n i v e r s i t y o f B o t s w a n a , L e s o t h o and S w a z i l a n d , B.Sc, M.Sc,  The U n i v e r s i t y o f C a l i f o r n i a ,  California  Davis,  Polytechnic State University,  A T H E S I S SUBMITTED  San L u i s O b i s p o ,  THE REQUIREMENTS FOR THE DEGREE OF PHILOSOPHY  in THE FACULTY OF GRADUATE STUDIES (Department o f P l a n t  We a c c e p t t h i s to  Science)  t h e s i s as c o n f o r m i n g  the required  standard  THE UNIVERSITY OF B R I T I S H September Q  Thandie  1971  IN PARTIAL FULFILMENT OF  DOCTOR OF  Leagajang  COLUMBIA  1979 Molefe,  1970  1979  1973  In  presenting  an  advanced  the I  Library  further  for  degree shall  agree  scholarly  by  his  of  this  written  this  thesis  in  at  University  the  make  that  it  purposes  for  freely  permission may  representatives. thesis  partial  be  It  of  for  for  extensive by  the  understood  gain  shall  nf  P l a n t  not  be  Columbia  Date  K.  DE-6  B P 75-5 I  \  E  copying  that  S c i e n c e  The U n i v e r s i t y o f B r i t i s h 2075 Wesbrook P l a c e Vancouver, Canada V 6 T 1W5  the  requirements  reference  Head  permission.  Department  of  B r i t i s h Columbia,  available  granted  is  financial  fulfilment  of  agree  and  of my  I  this  or  allowed  without  that  study. thesis  Department  copying  for  or  publication my  ii  ABSTRACT Double i n f e c t i o n by cowpea s t r a i n s o f s o u t h e r n bean mosaic (CP-SBMV) and o f tobacco mosaic v i r u s (CP-TMV) caused a d d i t i v e reductions  i n C a l i f o r n i a b l a c k e y e cowpea.  virus growth  P l a n t h e i g h t , weight and  numbers o f seed and pods were s i g n i f i c a n t l y reduced by double i n f e c t i o n and by CP-TMV s i n g l e i n f e c t i o n compared t o h e a l t h y infected plants.  S i n g l y and doubly  pea p l a n t s developed symptoms  i n doubly  inoculated  and CP-SBMV-sing 1 y  C a l i f o r n i a b l a c k e y e cow-  CP-SBMV symptoms on t h e primary l e a v e s , but CP-SBMV  infected t r i f o l i a t e s  were masked by CP-TMV symptoms.  CP-TMV symptoms d i d not mask CP-SBMV symptoms i n s y s t e m i c a l l y trifoliate conditioned  leaves o f another cowpea v a r i e t y , V 4 5 B o t s . -  infected  CP-TMV i n f e c t i o n  s y s t e m i c i n f e c t i o n o f V45~Bots by CP-SBMV, as i n d i c a t e d by  i n f e c t i v i t y , s e r o l o g y and a n a l y t i c a l s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u gation.  CP-TMV a l s o induced s u s c e p t i b i l i t y o f P i n t o t o i n f e c t i o n by  CP-SBMV, as a s c e r t a i n e d microscopy.  by i n f e c t i v i t y ,  immunodiffusion and e l e c t r o n  A n a l y t i c a l sucrose density gradient  ments demonstrated  that  i n doubly  inoculated  c e n t r i f u g a t i o n measure-  p r i m a r y leaves o f C a l i f o r n i a  b l a c k e y e cowpea CP-SBMV and CP-TMV were s y n t h e s i z e d same leaves  singly inoculated.  f o l l o w i n g simultaneous times t h a t  CP-SBMV s y n t h e s i s  l e s s than  in the  in t r i f o l i a t e  leaves,  i n o c u l a t i o n s o f p r i m a r y l e a v e s , was enhanced 5  in singly infected t r i f o l i a t e  l e a v e s , whereas CP-TMV synthe-  s i s was not g r e a t l y a f f e c t e d . When CP-TMV preceded  CP-SBMV i n t h e p r i m a r y  leaves  by 2k and 7 2 hr CP-SBMV s y n t h e s i s was enhanced more i n t r i f o l i a t e  leaves  t h a t were u n d i f f e r e n t i a t e d a t t h e time o f i n o c u l a t i o n than i n  those o f p l a n t s s i m u l t a n e o u s l y i n o c u l a t e d . into preformed concentration  When CP-TMV preceded  3 r d t r i f o l i a t e leaves by 2 2 h r , the r a t i o in doubly  infected  t i s s u e t o that  2 . 7 versus 1 . 9 when both v i r u s e s a r r i v e d  was  leaves. 3rd  When e i t h e r v i r u s preceded  CP-SBMV  o f CP-SBMV  in s i n g l y  infected  tissue  s i m u l t a n e o u s l y a t these  the o t h e r by 7 2 hr i n t o  preformed  t r i f o l i a t e leaves the s y n t h e s i s o f the c h a l l e n g i n g v i r u s was g r e a t l y  retarded.  CP-SBMV s y n t h e s i s was a l s o enhanced by CP-TMV  differential  temperature  synchronous  system o f i n f e c t i o n .  r i o n s o f both v i r u s e s were d e t e c t e d i n the same c e l l was  d e t e c t e d by i n f e c t i v i t y  CP-TMV  infection  infection  neutralization  p r e d i s p o s e s the host c e l l s  thus the enhanced s y n t h e s i s o f CP-SBMV.  test.  under  Although v i -  no genomic masking  It i s t h e o r i z e d  to i n f e c t i o n  that  by CP-SBMV and  The e f f e c t o f CP-TMV  on CP-SBMV s y n t h e s i s in cowpea seems to be a p h y s i o l o g i c a l  infection  one.  CP-SBMV,  but not CP-TMV, was t r a n s m i t t e d through p l a n t e d seed and decontaminated embryos o f C a l i f o r n i a taminated  B u f f e r e x t r a c t s made from  embryos a l s o were i n f e c t i o u s f o r CP-SBMV.  both v i r u s e s . seed  blackeye cowpea.  Double i n f e c t i o n o f C a l i f o r n i a  t r a n s m i s s i o n o f CP-SBMV from  Seed c o a t s contained  blackeye cowpea decreased  1 3 - 5 to 7.6%.  Buffer extracts of  h e a l t h y seed were i n h i b i t o r y t o i n f e c t i v i t y o f both v i r u s e s . t i o n o f seed of  CP-TMV.  reduced  infectivity  It i s a l s o concluded  a r e s u l t of embryo i n f e c t i o n  of CP-SBMV that seed  decon-  Germina-  in the seed c o a t s , but not t r a n s m i s s i o n o f CP-SBMV i s  r a t h e r than c o n t a m i n a t i o n w i t h v i r u s in  the seed c o a t s .  Supervisor's signature  iv  TABLE OF CONTENTS Page ABSTRACT  i i  L I S T OF TABLES  x  L I S T OF FIGURES  x  ACKNOWLEDGEMENTS  1  4  LITERATURE REVIEW Mixed p l a n t A. E f f e c t  virus  on p l a n t  4  infections  5  growth  B. E f f e c t o n c o n c e n t r a t i o n a n d d i s t r i b u t i o n o f v i r u s e s C. E f f e c t  on t r a n s m i s s i o n  D. S t r u c t u r a l  II. III.  In v i t r o s t r u c t u r a l  2.  In v i v o  Synchronous  of viruses  interactions  1.  Seed  structural  between  viruses  interactions interactions  systems f o r p l a n t  transmission  by v e c t o r s  of plant  virus  replication  viruses  II. III.  ...  8 14 16 17 20 25 29 40  MATERIALS AND METHODS I.  v  xvii i  INTRODUCTION  I.  i  The v i r u s e s  40  H o s t s and t h e i r p r o p a g a t i o n  41  Inoculation  o f hosts f o rv i r u s and i n o c u l a t i o n  purification of hosts  43 43  A.  Propagation  B.  Purification of viruses  44  1.  P u r i f i c a t i o n o f CP-SBMV  44  2.  P u r i f i c a t i o n o f CP-TMV  46  Page C.  D. IV.  Determination 1.  Determination  2.  Estimation of concentration  Interactions in California  1. 2.  V45~Bots  concentration  of purified  ..  48  v i r u s ...  48  blackeye  50  i n cowpea and B o t s w a n a  51 local 51  cowpeas  51  Inoculations  53  Assay  Movement o f CP-SBMV and CP-TMV f r o m into 3rd t r i f o l i a t e  leaves blackeye  C.  virus  i n f e c t i o n s o f CP-SBMV and CP-TMV  variety  B.  of purified  P r e p a r a t i o n o f CP-SBMV and CP-TMV a n t i s e r a  Mixed A.  48  of the virus concentration  inoculated primary  leaves of C a l i f o r n i a  cowpea  55  1.  Trimming  55  2.  Inoculations  55  E f f e c t o f sequence formed  of a r r i v a l  3rd t r i f o l i a t e  o f each v i r u s i n the pre-  l e a f on t h e c o n c e n t r a t i o n  of the 56  other 1. 2. D.  55  I nocul a t ions  55  Assay  I n t e r a c t i o n s o f CP-SBMV and CP-TMV e y e cowpea u n d e r  in California  synchronous c o n d i t i o n s  black57  1.  Trimming  57  2.  Inoculations  57  3.  Assay  58  vi  Page E.  V.  f o rstructural interactions in California  blackeye  cowpea d o u b l y  i n f e c t e d by CP-SBMV and CP-TMV..  B.  Inocu1 a t i o n s  59  2.  Assay  59 62  in Pinto  62  Inoculations 1.  Inoculations with  intact  2.  Inoculations with  CP-SBMV-RNA a n d i n t a c t  3.  Assay  CP-SBMV a n d i n t a c t  CP-TMV.  CP-TMV ... 63  65  Extraction of ribonucleic acid 1.  E x t r a c t i o n o f RNA f r o m p u r i f i e d  2.  E x t r a c t i o n o f RNA f r o m c r u d e s a p  virus preparation..  Propagation  hosts  B.  D i s t r i b u t i o n o f CP-SBMV a n d CP-TMV blackeye  66 66 67  o f CP-SBMV and CP-TMV  A.  fornia  62  64  Seed t r a n s m i s s i o n  67  f o r CP-SBMV a n d CP-TMV  i n seed p a r t s o f C a l i -  cowpea and 3 B o t s w a n a cowpeas  67  1.  Harvesting  67  2.  Decontamination procedures  67  3.  A s s a y o f embryos and s e e d l i n g s produced  on p l a n t s  derived  from  seeds  i n f e c t e d by CP-SBMV a n d 68  CP-TMV C.  59  1.  I n t e r a c t i o n s o f CP-SBMV and CP-TMV A.  VI.  Analysis  Effect of germination  on CP-SBMV a n d CP-TMV  i n seed  coats  69  1.  G e r m i n a t i o n o f seed  69  2.  Infectivity  69  assay  v i i  Page D.  Effect  of healthy  m a t u r e s e e d e x t r a c t s on  infectivity 70  o f CP-SBMV a n d CP-TMV  VII.  1.  E x t r a c t ion  2.  I n f e c t i v i t y assay  Production  .  70 70  o f seed f o r y i e l d a n a l y s i s  71  RESULTS I.  Estimation of concentration sucrose  II.  70  Effect  density gradient of single  of purified virus  by a n a l y t i c a l 71  centrifugation  and d o u b l e  infections  on C a l i f o r n i a  black-  eye  cowpea a n d B o t s w a n a cowpea v a r i e t i e s  71  A.  C o m p a r i s o n o f symptoms  71  B.  C.  D.  1.  Symptoms i n C a l i f o r n i a  2.  Symptoms i n B o t s w a n a cowpea v a r i e t i e s  Effect  blackeye  71  cowpea  83 83  on p l a n t g r o w t h  1.  P l a n t growth  in California  2.  Plant growth  i n B o t s w a n a cowpea v a r i e t i e s  Comparison o f e f f e c t s  blackeye  83  cowpea  84  o f CP-SBMV a n d CP-TMV on y i e l d 84  and  seed c h a r a c t e r i s t i c s  1.  Yield  2.  Seed c h a r a c t e r i s t i c s  in California  3.  Seed c h a r a c t e r i s t i c s  i n B o t s w a n a cowpea v a r i e t i e s . .  of California  Distribution  and c o n c e n t r a t i o n  nucleoproteins 1.  blackeye  cowpea... 89 89  o f CP-SBMV and CP-TMV 89  and c o n c e n t r a t i o n  in t h e i n o c u l a t e d primary cowpea  blackeye  i n cowpea  Distribution  eye  84  cowpea  o f CP-SBMV and CP-TMV  leaves of C a l i f o r n i a  black89  viii  Page 2.  Distribution  and c o n c e n t r a t i o n  B o t s w a n a cowpea v a r i e t y 3.  Distribution in  h.  in primary  leaves o f  V*45-Bots  3k  and c o n c e n t r a t i o n o f CP-SBMV and CP-TMV  trifoliate  Distribution  leaves of C a l i f o r n i a blackeye  cowpea..  and c o n c e n t r a t i o n o f CP-SBMV and CP-TMV  in t r i f o l i a t e  l e a v e s o f B o t s w a n a cowpea  variety 109  V45-Bots D.  Distribution  a n d c o n c e n t r a t i o n o f CP-SBMV and CP-TMV i n  3rd  trifoliate  1.  Arrival  leaves of C a l i f o r n i a blackeye  o f CP-SBMV and CP-TMV a t 3 r d  cowpea  ...  110  T i m e - c o u r s e s y n t h e s i s o f CP-SBMV a n d CP-TMV i n preformed 3rd t r i f o l i a t e  3.  Effect  o f sequence o f a r r i v a l  trifoliate Concentration trifoliate nously 1.  virus III.  on t h e  i n t h e preformed 3rd 111  o f CP-SBMV and CP-TMV  i n the preformed 3rd  leaves of C a l i f o r n i a blackeye by d i f f e r e n t i a l  o f synchronous  cowpea  temperature  synchro-  manipulation.  i n f e c t i o n on a c c u m u l a t i o n  interactions  in C a l i f o r n i a blackeye  1.  Analytical  2.  Infectivity  sucrose  116  b e t w e e n CP-SBMV and  cowpea  density gradient centrifugation..  neutralization  116  of  i n 3rd t r i f o l i a t e leaves  Analysis for structural CP-TMV  o f each v i r u s  leaves  infected  Effect  111  leaves  concentration of the other  E.  110  trifoliate  1 eaves 2.  98  123 123 126  ix  Page IV.  I n t e r a c t i o n o f CP-SBMV a n d CP-TMV A.  128  of Pinto  Inoculation with  intact viruses  128  2.  Inoculation with  CP-SBMV-RNA and i n t a c t CP-TMV  130  3.  Starch  lesions  130  Time-course synthesis with  o f CP-SBMV  in tissue also  inocu133  CP-TMV  Confirmation primary  D.  ino-  1.  lated C.  128  bean  I n t e r a c t i o n o f CP-SBMV and CP-TMV a f t e r s e q u e n t i a l culation  B.  in Pinto  o f CP-SBMV  in local  lesions of Pinto ^38  leaves  Serology  1 3  2.  Electron microscopy  138  Influence  of Cornell  i s o l a t e o f CP-TMV o n CP-SBMV i n f e c -  tion of Pinto V.  Seed t r a n s m i s s i o n A.  1 41 o f CP-SBMV and CP-TMV  -\ LL]  i n cowpea  D i r e c t p l a n t i n g o f s e e d p r o d u c e d on C a l i f o r n i a  blackeye  cowpea p . l a n t s . . 1.  Freshly  2.  E f f e c t o f seed  ^1  harvested  vine-dry  storage  mature seed  duration  141  on v i r u s  transmis-  sion 3.  145  E f f e c t o f seed m o t t l e and  B.  8  1.  on t r a n s m i s s i o n  CP-TMV t h r o u g h p l a n t e d  o f CP-SBMV  D i r e c t p l a n t i n g o f s e e d s f r o m s i n g l y and d o u b l y B o t s w a n a cowpea v a r i e t i e s  1 45  seed infected  145  Page C.  Distribution 1.  of CP-SBMV and CP-TMV  D i s t r i b u t i o n o f CP-SBMV and CP-TMV d i f f e r e n t developmental  1^9  in seed p a r t s in seed p a r t s o f  stages o f seed o f C a l i f o r -  n i a blackeye cowpea 2.  149  D i s t r i b u t i o n o f CP-SBMV and CP-TMV  in seed p a r t s o f 158  Botswana cowpea v a r i e t i e s 3.  D i s t r i b u t i o n o f CP-SBMV and CP-TMV  in p l a n t e d embryos 158  of C a l i f o r n i a blackeye cowpea seed k.  E f f e c t of age o f C a l i f o r n i a blackeye cowpea on  5.  CP-SBMV or CP-TMV  E f f e c t of h e a l t h y seed e x t r a c t and  D.  s u s c e p t i b i l i t y to e i t h e r  II. III.  165  and i n f e c t i v i t y o f v i r u s  seed o f C a l i f o r n i a blackeye cowpea  167 170  DISCUSSION I.  163  on i n f e c t i o n o f CP-SBMV  CP-TMV on GA 21 and N_. g l u t i n o s a  E f f e c t o f germination on s u r v i v a l on  seedling  Interaction A.  E f f e c t s on p l a n t  B.  Distribution  C.  In v i v o  Interaction  170  in cowpea growth  and c o n c e n t r a t i o n o f v i r u s e s  structural in P i n t o  Seed t r a n s m i s s i o n  interactions  170 171 177 180 182  SUMMARY  189  LITERATURE CITED  190  xi  LIST OF TABLES  TABLE I. II.  III.  Page Composition Relationship  and p r o p e r t i e s  o f CP-SBMV and CP-TMV  3  between CP-SBMV and CP-TMV c o n c e n t r a t i o n s and 80  t h e i r absorbance  areas  E f f e c t of single  (SI) and simultaneous double  (Dl) i n f e c t i o n s  of CP-SBMV and CP-TMV on growth o f C a l i f o r n i a blackeye 86  cowpea IV.  E f f e c t o f s i n g l e and simultaneous double  i n f e c t i o n s by  CP-SBMV and CP-TMV on growth o f Botswana v a r i e t y  V45"Bots 87  cowpea V.  E f f e c t of s i n g l e and simultaneous double  i n f e c t i o n s o f CP-  SBMV and CP-TMV on pod and seed y i e l d o f C a l i f o r n i a blackeye 88  cowpea VI.  Relative  amounts o f CP-SBMV and CP-TMV n u c l e o p r o t e i n i n s i n g l e  (Si) and double  (Dl) i n f e c t i o n s o f primary  leaves o f C a l i f o r 95  n i a blackeye cowpea VII.  N u c l e o p r o t e i n y i e l d o f CP-SBMV and CP-TMV doubly  (Dl) i n o c u l a t e d  systemically  infected  in s i n g l y  ( s i m u l t a n e o u s l y ) primary 3rd and 4 t h t r i f o l i a t e  (SI) and  leaves and  leaves o f 99  Botswana cowpea v a r i e t y V 4 5 B o t s _  VIII.  I n f e c t i v i t y o f CP-SBMV cowpea v a r . V 4 5 B o t s -  in e x t r a c t s  at d i f f e r e n t  with CP-SBMV alone o r w i t h CP-TMV  from primary  leaves o f  intervals after  inoculation 100  xi i  TABLE IX.  Page C o n c e n t r a t i o n o f CP-SBMV and o f CP-TMV (undifferentiated blackeye  in 3 r d  trifoliate  a t time o f i n o c u l a t i o n ) o f C a l i f o r n i a  cowpea a f t e r s i n g l e and s e q u e n t i a l i n o c u l a t i o n s 105  of primary X.  leaves  Movement o f CP-SBMV and o f CP-TMV of C a l i f o r n i a  XI.  blackeye  leaves  cowpea  112 before  (-) CP-SBMV and o f CP-TMV when CP-SBMV a r r i v e d  (+) and a f t e r  (+) and before  (-) CP-TMV  117  R e l a t i v e amounts o f CP-SBMV and CP-TMV on GA 21 and 1^. g l u t i nosa r e s p e c t i v e l y  recovered  v i t y of a r t i f i c i a l XIII.  i n t o 3rd t r i f o l i a t e  C o n c e n t r a t i o n o f CP-SBMV when CP-TMV a r r i v e d after  XII.  ."  a f t e r n e u t r a l i z a t i o n of i n f e c t i -  and n a t u r a l mixtures  R e l a t i o n s h i p between number o f l o c a l  127  l e s i o n s and v a r y i n g con-  c e n t r a t i o n s o f e i t h e r CP-SBMV or CP-TMV cha11enge-inocu1ated on P i n t o primary XIV.  ( P u r i f i e d ) on P i n t o primary  Transmission  XVII. XVIII.  leaves  132  of CP-SBMV and CP-TMV from seed  s i n g l y or doubly XVI.  129  E f f e c t o f p r e - i n o c u l a t ion w i t h CP-TMV on i n f e c t i v i t y o f CPSBMV-RNA  XV.  leaves  infected  California  produced by  blackeye  E f f e c t o f s t o r a g e d u r a t i o n o f seed  on seed  CP-SBMV t o s e e d l i n g s o f C a l i f o r n i a  blackeye  cowpea  transmission of cowpea  E f f e c t o f seed m o t t l e on t r a n s m i s s i o n o f CP-SBMV and CP-TMV. Seed t r a n s m i s s i o n of CP-SBMV and CP-TMV t h r e e Botswana l o c a l v a r i e t i e s  1 44  146 147  in p l a n t e d seed of 148  xiii  TABLE XIX.  Page Distribution California  o f CP-SBMV and CP-TMV  in pooled seed p a r t s o f  blackeye cowpea o f d i f f e r e n t  developmental 1 50  stages XX.  Efficacy cially  XXI.  contaminated  Distribution ted of  singly  inoculated  152  contaminated California  in 15-20 day green  old infec-  immature seed  parts  blackeye cowpea which were 153  individually  Distribution ted  artifi-  embryos  o f CP-SBMV and CP-TMV  and a r t i f i c i a l l y  assayed XXII.  o f decontaminating CP-SBMV and CP-TMV from  in 3 0 - 3 5 day; o l d i n f e c -  o f CP-SBMV and CP-TMV  and a r t i f i c i a l l y  contaminated  seed p a r t s o f C a l i f o r n i a  immature (dough stage)  blackeye cowpea d i r e c t l y assayed 1 55  i nd i v idua11y XXIII.  Distribution  of CP-SBMV and CP-TMV  and a r t i f i c i a l l y  contaminated  mature seed o f C a l i f o r n i a  Distribution infected  parts 156  individually  o f CP-SBMV and CP-TMV  in seed p a r t s o f s i n g l y  ( S i ) v i n e d r y mature seed of C a l i f o r n i a  Distribution  o f CP-SBMV and CP-TMV  blackeye  individually...  T r a n s m i s s i o n o f CP-SBMV seeds and a r t i f i c i a l l y  159  in seed p a r t s o f Botswana 160  cowpea v a r i e t i e s XXVI.  (N)  (A) seed p a r t s o f v i n e d r y  cowpea whose e x t r a c t s were d i r e c t l y assayed XXV.  infected  blackeye cowpea whose seed  e x t r a c t s were d i r e c t l y assayed XXIV.  in n a t u r a l l y  in embryos o f n a t u r a l l y contaminated  embryos  infected 161  xiv  TABLE XXVII.  Page T r a n s m i s s i o n o f CP-SBMV and CP-TMV  in embryos o f n a t u r a l l y  infected  contaminated  seeds and in a r t i f i c i a l l y  California in  XXVIII.  5% N a P 0 3  blackeye cowpea planted a f t e r  embryos o f  decontamination 162  4  T r a n s m i s s i o n o f CP-SBMV and CP-TMV when i n o c u l a t e d  to h e a l t h y  c o t y l e d o n s and or r a d i c l e - p l u m u l e shoots XXIX.  E f f e c t o f an e x t r a c t o f h e a l t h y seed o f C a l i f o r n i a  164 blackeye  cowpea on i n f e c t i v i t y o f CP-SBMV and CP-TMV XXX.  E f f e c t o f germination on i n f e c t i v i t y and s u r v i v a l and  CP-TMV  in the seed  coats  166 o f CP-SBMV 169  XV  LIST OF FIGURES  FIGURE 1.  Page Absorbance p r o f i l e s f o l l o w i n g c e n t r i f u g a t i o n o f 0 . 2 ml o f purified  2.  CP-TMV through sucrose  Third t r i f o l i a t e and  6.  in sucrose  columns  doubly  California  in sucrose  columns  columns  78  blackeye cowpea s i n g l y 82  i n f e c t e d by CP-SBMV and CP-TMV blackeye cowpea p l a n t s s i n g l y and doubly i n f e c t e d  density gradient  t r a c t s o f primary  85  columns, o f 0 . 2 ml o f c l a r i f i e d ex-  leaves o f C a l i f o r n i a  QQ  blackeye cowpea  R e l a t i v e absorbance p r o f i l e s f o l l o w i n g c e n t r i f u g a t i o n , through sucrose  density gradient  t r a c t s o f 3rd t r i f o l i a t e 9.  76  R e l a t i v e absorbance p r o f i l e s f o l l o w i n g c e n t r i f u g a t i o n , through sucrose  8.  74  (mg/ml) and absor-  density gradient  leaves o f C a l i f o r n i a  columns  by CP-SBMV and CP-TMV 7.  72  (mg/ml) and absor-  density gradient  R e l a t i o n s h i p between CP-TMV c o n c e n t r a t i o n bance area o f CP-TMV  5.  density gradient  R e l a t i o n s h i p between CP-SBMV c o n c e n t r a t i o n bance area o f CP-SBMV  k.  density gradient  Absorbance p r o f i l e s f o l l o w i n g c e n t r i f u g a t i o n o f 0 . 2 ml o f purified  3.  CP-SBMV through sucrose  columns, o f 0 . 2 ml o f c l a r i f i e d ex-  R e l a t i v e amounts o f CP-SBMV and CP-TMV n u c l e o p r o t e i n s wt.)  in primary  92  leaves  leaves o f C a l i f o r n i a blackeye cowpea  (mg/g f r . afc  xv i  FIGURE 10.  Page R e l a t i v e amounts o f CP-SBMV and CP-TMV n u c l e o p r o t e i n s (mg/g f r . wt.) in the 1 s t , 2 n d , 3 r d and 4 t h t r i f o l i a t e of  11.  and  13.  California  102  blackeye cowpea  E l e c t r o n micrographs of  12.  California  leaves  o f c e l l s o f primary and t r i f o l i a t e  blackeye cowpea doubly  infected  leaves  by CP-SBMV  CP-TMV  106  Time-course r e p l i c a t i o n  o f CP-SBMV and CP-TMV as  by t h e i r  respective nucleoprotein contents  Relative  i n f e c t i v i t y o f CP-SBMV  California  indicated 114  in 3 r d t r i f o l i a t e  blackeye cowpea, i n f e c t e d  leaves o f  synchronously s i n g l y by 119  CP-SBMV and doubly by CP-SBMV and CP-TMV 14.  Relative  i n f e c t i v i t y o f CP-TMV  California  in 3 r d t r i f o l i a t e  blackeye cowpea, i n f e c t e d  leaves o f  synchronously  singly 121  by CP-TMV and doubly by CP-SBMV and CP-TMV 15.  16.  N u c l e o p r o t e i n accumulation o f CP-SBMV and CP-TMV blackeye cowpea 3 r d t r i f o l i a t e  leaves synchronously  Primary  and doubly  intact 17.  in C a l i f o r n i a  leaves o f P i n t o s i n g l y CP-SBMV and i n t a c t  Time-course r e p l i c a t i o n singly  and doubly  inoculated  infected with  431  CP-TMV  o f CP-SBMV  inoculated with  in P i n t o primary intact  leaves  CP-SBMV and i n t a c t 134  CP-TMV 18.  Time-course r e p l i c a t i o n o f CP-SBMV singly CP-TMV  . .-124  and doubly  in P i n t o primary  i n o c u l a t e d w i t h CP-SBMV-RNA  leaves  and i n t a c t 136  xv i i FIGURE 19-  P  Immunodiffusion  E l e c t r o n micrographs rified  21.  of CP-SBMV-like p a r t i c l e s  P i n t o primary leaves i n o c u l a t e d  CP-TMV 22.  i n o c u l a t e d w i t h CP-SBMV and CP-TMV..  from P i n t o primary l e a f  (Cornell  139  (arrows) pu-  lesions  142  singly with  intact  CP-TMV  i s o l a t e ) and doubly w i t h i n t a c t CP-SBMV and i n t a c t  (Cornell  Relative  g  r e a c t i o n s o f crude sap from P i n t o primary  leaves s i n g l y and doubly 20.  a  isolate)  143  i n f e c t i v i t y o f CP-SBMV  germinated  and ungerminated  blackeye cowea p l a n t s s i n g l y  in seed coat e x t r a c t s o f  seeds d e r i v e d infected  from  California  by CP-SBMV  168  e  ACKNOWLEDGEMENTS  My g r a t i t u d e  i s expressed  t o the f o l l o w i n g members of the s t a f f  of A g r i c u l t u r e Canada Research S t a t i o n , Vancouver.  Dr. Marvin Weintraub,  D i r e c t o r , f o r use o f the e x c e l l e n t f a c i l i t i e s a t h i s s t a t i o n ; Dr. H.W.J. Ragetl i f o r a l l o w i n g  me extended use o f h i s growth chamber f o r e x p e r i -  ments on synchronous  i n f e c t i o n ; Bea Schroeder, f o r a s s i s t a n c e with e l e c -  t r o n microscopy o f s e c t i o n s ; Connie N i c h o l s microscopy and s e r o l o g y ;  Wes McDiarmid f o r the e x c e l l e n t  work; Al Mosher f o r s u p p l y i n g cal  fittings  suggestions; lable  styrofoam m a t e r i a l  in e l e c t r o n  photographic  and f o r making  in the styrofoam chamber; Dr. J.H. Tremaine f o r the L i b r a r y s t a f f  locally;  tistical  for assistance  for obtaining  reference  electri-  invaluable  materials  and Drs.Bryan D. F r a z e r and J.W. H a l l f o r a d v i c e  unavaion s t a -  ana l y s i s .  I am g r a t e f u l l y indebted  t o Drs.  B.B. B r a n t l e y  and CW.  Kuhn,  Georgia Experiment S t a t i o n and Department o f P l a n t Pathology and Plant Genetics, 21  r e s p e c t i v e l y , U n i v e r s i t y o f G e o r g i a , Athens, f o r seed o f Georgia  cowpea; Dr. M i l t o n Z a i t l i n ,  University,  Ithaca,  f o r the C o r n e l l  mosaic v i r u s ; Nat M a h l a t j i e , Agricultural  Department o f P l a n t  Pathology, C o r n e l l  i s o l a t e o f cowpea s t r a i n o f tobacco  Seed M u l t i p l i c a t i o n U n i t , Department o f  Research, Gaborone, Botswana, f o r seeds o f Botswana cowpea  var i e t i e s . I am g r a t e f u l to Drs. who,  R.J. Copeman, J.B. Hudson and R. Stace-Smith  as members o f my s u p e r v i s o r y  committee, gave s u g g e s t i o n s  in the p r e -  p a r a t i o n of the t h e s i s manuscript. Hamilton, my and  for  thesis  indebted to Dr. R  s u p e r v i s o r , f o r h i s help in choosing  his u n f a i l i n g  couragement and  I am e s p e c i a l l y  guidance,  the p r o j e c t  p a t i e n c e , i n v a l u a b l e s u g g e s t i o n s , en-  support d u r i n g the t h r e e years  I spent under h i s super  vi sion. I g r a t e f u l l y acknowledge the s c h o l a r s h i p granted Canadian  Commonwealth  generous study me  S c h o l a r s h i p and  leave granted  to do these s t u d i e s and L a s t , though not  first  d r a f t of t h i s  I am  least,  thesis.  to me  to me  by  F e l l o w s h i p Committee.  by the Botswana Government  the The enabled  grateful.  I thank my w i f e A l i c e  f o r t y p i n g the  J  INTRODUCTION Cowpea (Vigna u n g u i c u l a t a L. Walp) i s i n f e c t e d viruses  in i t s natural  bean mosaic was  first  virus  (Shepherd  and F u l t o n ,  1962)-  (Shepherd  s t r a i n o f southern bean mosaic  1974).  lar  and F u l t o n ,  1962),  (Tremaine and Wright,  virus  lated  by Capoor  (1962).  CP-TMV  1975)-  infects  serologically  CP-TMV  the French  i t is serologi1967) t o the bean  both bean and  related,  is also distantly  virus  s e r o l o g i c a l l y re-  (Bawden and K a s s a n i s , 1968; Kassanis  The chemical compositions and p r o p e r t i e s o f CP-SBMV I for interpretations  and e s p e c i a l l y  d e a l i n g with s t r u c t u r a l  in the s e c t i o n  Both CP-SBMV and CP-TMV are very contagious with  of t h e i r  behaviour,  interactions.  infectivity  sap o f 20 t o 265 days and over 8 y e a r s , r e s p e c t i v e l y . also  but not  i s c o n s i d e r e d to be s i m i -  and CP-TMV a r e g i v e n in T a b l e  viruses  America  (Lamptey, 1972; Lamptey and Hamilton,  is also  t o the bean s t r a i n of SBMV.  to the type s t r a i n o f TMV  and Varma,  in  (Lister  (Kassanis and Varma, 1975) t o the southern sunn-hemp mosaic  described  CP-TMV  (SBMV) d e s c r i b e d by Zaumeyer and  from Ghana, A f r i c a  The Ghana s t r a i n o f SBMV  identical  Africa  from cowpea i n North  R e c e n t l y a s t r a i n o f SBMV that  cowpea was i s o l a t e d  (CP-TMV).  Although CP-SBMV does not i n f e c t  but not i d e n t i c a l  (1943).  virus  in cowpea in N i g e r i a ,  1955) and CP-SBMV was i s o l a t e d  related,  Harter  the cowpea s t r a i n s o f southern  (CP-SBMV) and o f tobacco mosaic  (Phaseolus v u l g a r i s )  cally  including  reported and i s o l a t e d  and Thresh,  bean  habitat,  by a number of  i n f e c t many cowpea v a r i e t i e s and c u l t i v a r s ,  survival  These  2  especially  the C a l i f o r n i a  t h i s host,  thus  blackeye  type.  They a l s o m u l t i p l y well in  i t was a p p r o p r i a t e to study  Obj ect i v e s :  The o b j e c t i v e s o f t h i s  e f f e c t s o f CP-SBMV and CP-TMV  their effects  in  it.  t h e s i s were to compare the  in s i n g l e and double  i n f e c t i o n s o f Vigna  ungu i c u l a t a L. Walp var. E a r l y Ramshorn and in t h r e e Botswana cowpea v a r i e t i e s with  respect t o symptom e x p r e s s i o n , d i s t r i b u t i o n and concen-  t r a t i o n o f each o f the v i r u s e s in v a r i o u s and  transmission  actions  through seed;  and t o determine  (e.g., e n c a p s i d a t i o n o f v i r a l  p r o t e i n ) occur  l e a v e s , and d i s t r i b u t i o n in i f structural  RNA by heterologous  as a consequence o f double  inter-  v i r u s coat  infection.  S t u d i e s o f mixed v i r u s i n f e c t i o n s cannot only o f f e r a b e t t e r understanding  o f the l i f e c y c l e s o f v i r u s e s , but can a l s o o f f e r an  understanding  o f how v i r u s e s i n t e r a c t w i t h the host c e l l s .  tical  aspect.of  a mixture of  such  i n t e r a c t i o n s stems from the f a c t  may suppress  o r accentuate  that v i r u s e s in  symptoms o r a f f e c t  one of the v i r u s e s by v e c t o r s or through  seed.  The prac-  transmission  3  T a b l e |.  Composition arid p r o p e r t i e s o f CP-SBMV and CP-TMV  CP-SBMV  CP-TMV  3  b  Properties of p a r t i c l e s V i r i o n m o l e c u l a r weight (M.W.)  6.6 x 1 0 d  39-4 x 1 0 d  I s o e l e c t r i c point  3.9 - 6.0  3-5  5.85  3.2  1,60  1.2  115 S  187 S  Vector  leaf beetles  unknown  Seed t r a n s m i s s i o n  e q u i v o c a l (?)  none  i sometr i c  r i g i d rod  Optical density 0 D  (E°^*  2 6 o  6  n m )  260/ 280 0 D  Sedimentation c o e f f i c i e n t  (S2Q ) W  6  c  c  Structure of p a r t i c l e s Shape  40 nm; 300 nm  Length o f p a r t i c l e s 25 nm  18 nm  Number o f p a r t i c l e components  one  two  Protein subunit  2.9 x lO^d  1.65 x 1 0 ; 1.81 x 1 0 d  P r o t e i n amino a c i d r e s i d u e s  270  161  RNA  1.4 x 10 d  0.3 x 1 0 ; 2.0 x 1 0 d  21*  5%  Diameter Composition o f p a r t i c l e s  M.W.  M.W.  6  RNA percentage o f v i r i o n  a  Data  from Shepherd  4  6  4  6  (1971)  A l l data from K a s s a n i s and Varma (1975) and Gibbs (1977) except f o r (c) where data a r e from Z a i t l i n and I s r a e l (1975) f o r t h e type s t r a i n o f TMV; v a l u e s a r e assumed t o be ca t h e same f o r CP-TMV.  k  LITERATURE REVIEW I. a  Mixed p l a n t  virus  h o s t by two v i r u s e s  interacting affect yield and  are often  in single  of the infection of  d i f f e r e n t from those  infections.  induced  Mixed v i r u s  and g r o w t h o f t h e h o s t p l a n t ,  by t h e  infections  i n f e c t i o n s may a l s o  a l t e r the transmission  v e c t o r s o r t h r o u g h s e e d and i n r a r e  susceptibility virus.  may o c c u r .  virus or the protein made o f m i x e d p r o t e i n  reviewed viruses  interactions  Such s t r u c t u r a l  capsid  were reviewed  has  been r e v i e w e d  has  induce  i n mixed  capsid  bacterial  and p l a n t  been made (Dodds and H a m i l t o n ,  by t h e r e l e v a n t  i n f e c t i o n h a v e been  in transmission  and  will  both  unrelated by Ross  of viruses  (1974).  by a p h i d s interactions  i n v i t r o and i n v i v o ,  1976).  of this thesis  adjective  genome may be  viruses.  between r e l a t e d  viruses,  t o encap-  of the other  ( 1 9 6 3 ) and more r e c e n t l y  infections  structural  lead  (Rochow, 1972) a n d a r e v i e w o f s t r u c t u r a l  Throughout the text fied  from both  Interactions  by K a s s a n i s  r o l e o f mixed v i r u s  i n t e r a c t i o n s may  s u r r o u n d i n g one o f t h e v i r u s e s  between v i r u s e s  The  i n t h e same c e l l  by t h e p r o t e i n  s u b u n i t s donated  in recent years.  between a n i m a l ,  o f one o f t h e v i r u s e s  i n s t a n c e s o n e v i r u s may  replicate  s i d a t i o n o f t h e genome o f o n e v i r u s  The  Such  o f a h o s t o t h e r w i s e r e g a r d e d a s immune t o t h e o t h e r  If the interacting viruses  interactions  may  symptom t y p e o r s e v e r i t y  c o n c e n t r a t i o n o r d i s t r i b u t i o n o f one o r both o f t h e v i r u s e s .  double by  viruses  infections. Characteristics  t h e w o r d " v i r u s " when n o t q u a l i -  mean"plant v i r u s " a n d  foreither  animal  5  or  bacterial  virus  the appropriate adjectjye  is concerned wholly with between a n i m a l  viruses  plant  viruses  will  be u s e d .  and t h e r e f o r e  This  thesis  interactions  and between b a c t e r i o p h a g e s w i l l  n o t be d e s c r i b e d  e x c e p t w h e r e an e x a m p l e may be u s e f u l . Effects  o f double v i r u s  in  several  of  the doubly  infected 1957;  ways.  There could  infected  plants  i n f e c t i o n s on a p l a n t  plants  (Bennett,  and  Dawson, 1 9 7 3 ; S i e v e r t ,  A.  Effects The  1949),  by d o d d e r  H o l m e s , 1956; 1968;  was f o u n d  l a t e n t mosaic v i r u s  Y (PVY) and TMV  fruit  o r seed y i e l d  plants. virus  doubly  infected  a n d p o t a t o m o t t l e v i r u s o r TMV  (Sievert,  by v i r u s e s  a n d CCMV ( H a r r i s o n  plants.  (Holmes, 1 9 5 6 ) ,  1973) and CCMV a n d t o b a c c o (Demski and J e l l u m , may a l s o  i n cowpea d o u b l y  yield  ringspot  1975).  show a r e d u c t i o n  of healthy or singly  infected  and G u d a u s k a s , 1 9 6 8 )  Synergistic  (Bennett,  (CCMV) (Kuhn a n d Dawson, 1 9 7 3 ) ,  a s compared t o t h e y i e l d  Thus seed y i e l d  infected  Kuhn  1975).  and t o b a c c o e t c h v i r u s  (Kuhn and Dawson, 1973) was s i g n i f i c a n t l y singly  1969;  t o be r e d u c e d a s a r e s u l t o f d o u b l e  and CCMV and s o y b e a n m o s a i c v i r u s Plants  G a r c e s - O r e j u e l a and P o u n d ,  S c h m i t t h e n n e r and Gordon,  CP-SBMV and cowpea c h l o r o t i c m o t t l e v i r u s  virus  of healthy or singly  1 9 7 3 ; Demski and J e l l u m ,  tomato aspermy v i r u s  potato virus  height or weight  growth.  height of plants  infections  in yield,  as compared t o t h a t  1949;  H a r r i s o n and G u d a u s k a s ,  on p l a n t  be a r e d u c t i o n  c a n be m a n i f e s t e d  in infected  by c u c u m b e r m o s a i c  a n d by CP-SBMV a n d CCMV  reduced as compared  t o that o f  r e d u c t i o n s were observed i n  6  seed o f soybean d o u b l y pod  mottle virus  ringspot  virus  The results  infected  (Ross,  1968)  soybean mosaic v i r u s  most s t u d i e d i n a change  e f f e c t of double v i r u s  infections  i n symptom s e v e r i t y o r t y p e . inoculated  with  separately.  in mixed v i r u s  One o f t h e e a r l y  and t o b a c c o  1969) i s that  When p l a n t s  a pair of viruses  symptoms c a n be more s e v e r e t h a n t h e symptoms i n d u c e d  viruses  a n d bean  o r by s o y b e a n m o s a i c v i r u s  ( S c h m i t t h e n n e r and G o r d o n ,  taneously or sequentially of  1963,  by e i t h e r  studies  that  are simul-  the complexion  by e a c h o f t h e  (Vanterpool,  i n f e c t i o n was t h e o b s e r v a t i o n  which  1926)  the so-called  streak  d i s e a s e o f t o m a t o was d u e t o s y n e r g i s t i c e f f e c t s o f TMV a n d p o t a t o virus  X (PVX).  Subsequent s t u d i e s  such as t h e d o u b l e (Bennett,  1949)  interactions  i n f e c t i o n o f t o b a c c o by TMV and t o b a c c o e t c h  by TMV and c u c u m b e r m o s a i c v i r u s  virus  ( G a r c e s - O r e j u e l a and  1957).  Pound,  Interaction extensively Double  o f PVX and PVY i n t o b a c c o  studied  i s one system t h a t  by R o s s and h i s s t u d e n t s a t C o r n e l l  i n f e c t i o n o f t o b a c c o by PVX and PVY i n s y s t e m i c a l l y  results and  revealed other s y n e r g i s t i c  in extensive severe veinal  necrosis  R o s s , 196lb) a n d i n s e v e r e n e c r o s i s  PVY-inocu1ated  half  t h e two v i r u s e s  Using  t h e same v i r u s  p a i r a n d h o s t Thomson  found  increased  symptom s e v e r i t y  infected 1955;  leaves Stouffer  the midvein adjacent t o  l e a v e s when l e a v e s a r e i n o c u l a t e d  opposite halves with  University.  (Rochow and R o s s ,  along  has been  separately  on  ( D a m i r d a g h a n d R o s s , 1967)-  following  (1961) a n d C l o s e (1964) double  i n f e c t i o n o f tobacco.  7  Double i n f e c t i o n of  pea  by  a l f a l f a mosaic v i r u s and  mosaic v i r u s  results  mosaic v i r u s  in combination w i t h bean pod  necrosis,, and  d i s t o r t i o n and  Ross, 1 9 7 2 a ) .  virus  in the most severe symptoms  mottling  C i t r u s doubly  showed o n l y p s o r o s i s  yellow vein  vein  196l)  Tobacco mosaic v i r u s on  infected  by  1972; by TMV  and  caused by  BMV  alone  barley  BMV  and  by  in p l a n t s  (Peterson and  leaves  (Hamilton and  (BSMV) and  CP-SBMV developed  v i r u s and  psorosis  infected  infected  but  by  singly  not  plants  TMV,  in such doubly  by  vein  infected plants  the  all  were doubly latter  infected by  plants  plants  BSMV  doubly  was  (Dodds, infected  developed c h l o r o t i c m o t t l e to the general  Nichols,  1977).  mosaic  Double  lower than  in  pattern  i n f e c t i o n of  in severe symptoms, even though  of e i t h e r v i r u s was  Brakke,  Cowpea p l a n t s  (BMV)  in a d d i t i o n  BSMV r e s u l t e d  nucleoprotein y i e l d  Lee  alone.  Hami1 t o n , 1 9 7 2 ) . However, b a r l e y  invaded  severe  (Ross, 1 9 6 8 ;  when doubly  infects barley  symptoms  bromegrass mosaic v i r u s  systemically  Soybean  induced  yellow vein  However, when b a r l e y  but  d i f f e r e n t from those  Dodds and  virus  s t r i p e mosaic v i r u s  a b l e to move s y s t e m i c a l l y , were no  by  than when s i n g l y  i t s own  infected.  barley  soybean f o l i a g e  symptoms and  v i r u s a l o n e or y e l l o w v e i n  become s y s t e m i c a l l y  mottle virus  infected  1967).  (Ford,  e n a t i o n v i r u s more severe symptoms were  (Weathers, I 9 6 0 ,  expressed enation  v i r u s and  virus  of  bean y e l l o w  in s i n g l e  the  infections  1973).  doubly  inoculated  s i m u l t a n e o u s l y w i t h CCMV  systemic symptoms of  both v i r u s e s  on  the  and  inoculated  8  primary  leaves.  CCMV sometimes caused n e c r o t i c e t c h i n g s ,  s i n g l e or double i n f e c t i o n s .  !t was  i n t e n s i f i e d and  also noticed  had  mottled seed  B.  E f f e c t on  leaf abscision  that  occurred  a l l seed produced on  infected  concentration  and  d i s t r i b u t i o n of v i r u s e s .  above there may  or d i s t r i b u t i o n of v i r u s e s  change may  be e i t h e r enhancement or s u p p r e s s i o n of one  interacting viruses.  An  not  observed  concentration  1949)  infected in s i n g l y of PVY  by PVX  and  or TMV  PVY  in the  or PVX  in s i n g l y  and  of PVX,  TMV,  was  PVX  l e s i o n s caused by PVX the  r a t i o of PVX  i n f e c t e d ones was  32°C, a temperature not  or  optimal  11.5  f o r PVX  :  distri-  symptoms. was  either and  in tobacco, doubly  2 to 11  Ross, 1954,  inoculum c o n t a i n i n g  local  both of the  or dodder l a t e n t mosaic v i r u s  (Rochow and  Ross ( I 9 & l a ) found that  to that  or  con-  of dodder l a t e n t mosaic v i r u s  concentration  infected plants  in the number of and  The  TMV  addition  The  when tomato p l a n t s were i n f e c t e d by  dodder l a t e n t mosaic v i r u s and virus.  In  infection.  Such a change in v i r u s c o n c e n t r a t i o n  in the  (Bennett,  tobacco etch  in a mixed  always be c o r r e l a t e d with s e v e r i t y of  increase  by  plants  a l s o be a change in the  centration  may  induced  Dawson, 1 9 7 3 ) .  (Kuhn and  doubly  with  coats.  to the e f f e c t s d i s c u s s e d  bution  infected  c h a l l e n g e d k to 6 days l a t e r w i t h CCMV, n e c r o s i s  CP-SBMV was CCMV was  However when cowpea, s i n g l y  whether in  also  times as much as 1955).  The  presence  an  increase  caused  (Thomson, I 9 6 0 in doubly  that  Stouffer  infected  plants  when p l a n t s were growing at synthesis  in s i n g l y  inoculated  9  plants. of PVX PVX  The c o n c e n t r a t i o n of PVX in s i n g l y  synthesis.  the i n o c u l a t e d recently,  leaves was  may  restricted  be enhanced  at a temperature  in double  i s enhanced most by PVY  that when PVX  l e s s than when PVY when PVX  arrived  Ross  after  they have been  preceded  60 hours  PVX  l a t e r than PVY.  Another  PVY  suggested  enhancement  by PVX.  In another study  i t was  that enhancement o f PVX  occurs  They was  minimal  s t r a i n o f PVX,  not n e c e s s a r i l y need the p h y s i c a l  in the same c e l l .  and  (1974b)  invaded by PVY.  by 6 hours.  to i n f e c t i o n  helper v i r u s  PVX  More  Ross  (19&7) and  by 12 hours; enhancement was  mechanism r e s p o n s i b l e f o r the enhancement could be a  found  above 30°C.  i n f e c t i o n s Goodman and  invaded the leaves b e f o r e PVY,  p r e d i s p o s e s the c e l l s  f a c t o r ( s ) and may  out of  when leaves in which g r e a t e s t i n t e r a c t i o n  enhanced most o n l y i f i t preceded  that PVY  (19°C) optimal f o r  to g a i n a b e t t e r understanding of the mechanism  takes p l a c e are invaded by PVX observed  twice that  that movement of PVX  the c l o s e - t i m i n g concept of Damirdagh and  that PVX  was  i n f e c t i o n s was  p l a n t s at a temperature  C l o s e (1964) a l s o observed  in an attempt  by which PVX used  infected  in double  It would  however, appear  The nature o f the translocatible presence of the  (Goodman and  in c e l l s doubly  Ross,  197^a)  infected  by  PVY.  The degree of i n t e r a c t i o n may by Lee and  Ross  (1972a) who virus  be  observed  an  mosaic  leaves a l s o  virus.  However, in bean, bean pod m o t t l e v i r u s l e s i o n s by soybean  by the host as shown  i n c r e a s e in the c o n c e n t r a t i o n of  soybean  d u c t i o n of l o c a l  in soybean  influenced  mosaic  infected  virus  by bean pod  mottle  i n t e r f e r e d with the pro(Lee and  Ross,  1972b).  10  Apart from enhancement there a l s o could  in the c o n c e n t r a t i o n  be a n t a g o n i s t i c  e f f e c t s on the c o n c e n t r a t i o n  or both o f the i n t e r a c t i n g v i r u s e s ,  when e i t h e r v i r u s and severe e t c h (Bawden and K a s s a n i s ,  and  PVY have s i n c e  and  Gooding, 1 9 7 0 ) and t h i s  related viruses. concentration  even when they a r e c o n s i d e r e d  been found  v i r u s were i n o c u l a t e d 1945).  inoculated  the c o n c e n t r a t i o n  However, severe etch  to be s e r o l o g i c a l l y r e l a t e d  type  of interaction  A c y c l i c pattern  of reduction  infections  (Purcifull  i s common between and i n c r e a s e  of a l f a l f a mosaic v i r u s was reduced  In pea  mosaic v i r u s  plants  even  t u r n i p mosaic v i r u s and  independently; however t h e c o n c e n t r a i f i t was introduced  days a f t e r  mosaic v i r u s  i n o c u l a t i o n with c a u l i f l o w e r  Double i n f e c t i o n s o f b a r l e y  virus  induced by bean y e l l o w mosaic  inoculated  replicated  (Ross, 1 9 6 7 ) ,  t i o n o f t u r n i p mosaic v i r u s was reduced  and  i n the  w i t h a l f a l f a mosaic v i r u s and bean y e l l o w mosaic  In simu1taneousy doubly  cauliflower  virus  ( G a r c e s - O r e j u e l a and Pound, 1 9 5 7 ) .  though the l a t t e r u s u a l l y masked symptoms virus.  simultaneously  o f e i t h e r v i r u s was observed when cucumber mosaic v i r u s and  TMV were in double doubly  o f one  M u l t i p l i c a t i o n o f PVY or Hyocyamus v i r u s 3 was prevented  unrelated.  to tobacco  of one o f the v i r u s e s  by BSMV and BMV  15 to kO .  (Kamei et a l , 1 9 6 9 ) .  (Peterson and Brakke, 1 9 7 3 )  of cowpea by CCMV and CP-SBMV (Kuhn and Dawson, 1 9 7 3 ) r e s u l t e d in  s y n e r g i s t i c e f f e c t s on symptomology, but the c o n c e n t r a t i o n s o f both BSMV and BMV and o f CP-SBMV were reduced centration  by double  o f CP-SBMV in cowpea s i m u l t a n e o u s l y  reduced by 50% of that  in s i n g l y  inoculated  i n f e c t i o n s . The con-  inoculated  plants  with CCMV was  (Kuhn and Dawson,  1973)-  11  The  c o n c e n t r a t i o n o f CCMV f o l l o w i n g simultaneous  affected.  When p l a n t s s u p p o r t i n g  c h a l l e n g e - i n o c u l a t e d with v i r u s was c o n s i d e r a b l y inoculated a f t e r  r a p i d m u l t i p l i c a t i o n o f one v i r u s were  the o t h e r ,  reduced.  the f i r s t  i n o c u l a t i o n s was not  the c o n c e n t r a t i o n o f the c h a l l e n g i n g  However, when the c h a l l e n g i n g v i r u s was  v i r u s had passed the r a p i d s y n t h e s i s phase, the  s y n t h e s i s o f the c h a l l e n g i n g v i r u s was the same as in h e a l t h y p l a n t s  inocu-. •  l a t e d a t the same time. When two v i r u s e s of such  interact  i n t e r a c t i o n may be an e f f e c t on the d i s t r i b u t i o n o f one o f the  viruses.  In c o t t o n p l a n t s s i n g l y  s t r e a k v i r u s the v i r u s could and  in the same host, one o f the consequences  not from non-inocu1ated  were doubly  inoculated with  be recovered leaves  (Costa,  1969).  is similar  tobacco s t r e a k v i r u s .  inefficiently  In s i n g l y  Hamilton,  doubly  1972) or TMV and BMV  I n f e c t i o n o f b a r l e y by  (Hamilton  and N i c h o l s ,  from n o n - i n o c u l a t e d  and N i c h o l s , 1977) or could only  concentrated  more than 10 times  Dodds and Hamilton,  1972).  streak replicated  leaves.  and Dodds, 1 9 7 0 ; Dodds, 1 9 7 2 ; Dodds  (Hamilton  1977) were  in doubly  p l a n t s was very much enhanced in the 3 r d and 4 t h l e a v e s .  (Hamilton  tobacco  i n o c u l a t e d b a r l e y p l a n t s TMV  i n o c u l a t e d t o b a r l e y , the c o n c e n t r a t i o n o f TMV  TMV c o u l d be not be: recovered  leaves,  i n v a s i o n of c o t t o n  in both the i n o c u l a t e d and non-inocu1ated  However, when TMV and BSMV (Hamilton and  systemic  t o that o f c o t t o n p l a n t s by the B r a z i l i a n  v i r u s , but not i d e n t i c a l .  tobacco  from the non-inocu1ated  Anthocyanosis v i r u s c o n d i t i o n e d  p l a n t s by the B r a z i l i a n  leaves  However, when the p l a n t s  i n o c u l a t e d w i t h Anthocyanosis v i r u s and the B r a z i l i a n  thus suggesting  very  tobacco  o n l y from the i n o c u l a t e d  s t r e a k v i r u s , the l a t t e r c o u l d be recovered  TMV  the B r a z i l i a n  In some cases  leaves when  be d e t e c t e d  inoculated  alone  i f e x t r a c t s were  and Dodds, 1 9 7 0 ; Dodds, 1 9 7 2 ;  12  I n t e r a c t i o n o f TMV w i t h BSMV o r w i t h BMV but not i d e n t i c a l Ross,  in baeley  is similar,  to that between PVX and PVY in tobacco  (Rochow and  1 9 5 4 , 1 9 5 5 ) in that  participating because  viruses.  i t results  However, the two types o f i n t e r a c t i o n . d i f f e r  PVX i s r e a d i l y d e t e c t e d  whereas TMV c o u l d be d e t e c t e d after spread  in e x t r a c t s o f  o f TMV  infected  infected  10-fold.  ( 3 0 ° C ) seems t o be n e c e s s a r y For t h i s  reason  Dodds, 1 9 7 0 ) t h a t under temperature  s u s c e p t i b l e t o TMV.  and Dodds, 1 9 7 0 ;  stress,  that o f PVX by PVY. by i n t e r a c t i o n  presence cally be  of a  s y s t e m i c a l l y when  1 9 5 1 ; Finlay,  interpreted with c a u t i o n ,  mixed  in s i n g l e  s t r a i n o f the same v i r u s  (Norris,  infections  between  Yellow aucuba mosaic v i r u s d i d not become  S t r a i n s o f tomato s p o t t e d w i l t v i r u s  the host  (Hamilton  b a r l e y may become  s y s t e m i c when i n o c u l a t e d a l o n e , but d i d s o . i n . t h e presence  invade  s i n c e a high  i t was suggested  o f v i r u s may a l s o be a f f e c t e d  other v i r u s e s and s t r a i n s .  (Benda, 1 9 5 7 ) .  systemic  T h e r e f o r e t h e enhancement o f TMV by BSMV o r by  BMV seems t o be much .higher than Distribution  (Hamilton  tissue,  plants only  Also  in b a r l e y seems t o be temperature-dependent  and N i c h o l s , 1 9 7 7 ) .  Hamilton  singly  in t i s s u e o f s i n g l y  e x t r a c t s had been c o n c e n t r a t e d more than  temperature  and  i n t h e enhancement o f one o f t h e  that o c c u r r e d  have t r a n s m i t t e d the s t r a i n s .  1952).  that  o f TMV  t h a t d i d not normal  i n f e c t i o n s d i d so in the infected  the host  However, these r e s u l t s  systemi-  should  because i t appears they were based on in the f i e l d , where a v e c t o r c o u l d  13  Double i n f e c t i o n by one  of the  v i r u s e s may  i n t e r a c t i n g v i r u s e s . The  considered  v e i n enation  v i r u s were g r a f t e d  i n f e c t e d the  t h i s type o f  i n t e r a c t i o n has  other s i m i l a r instances  found that on  N_. tabacum  are  reported.  i t s own  More r e c e n t l y  (Pound et a l , 1962)  i t was  reported  (Tochihara,  Nicotiana  interaction 1959).  glutinosa  When two  or 1959)  (Tochihara,  Koike, 1973)  is believed  to be  that  Johnsongrass  immune to sugarcane  i n f e c t i o n of Johnsongrass by the  two  viruses  are  influence  sugarcane mosaic believed  to  be  same v i r u s . viruses  in the same c e l l  r e p l i c a t e together or  of the  requisites for structural  recent  o b s e r v a t i o n . (Goodman and occurred  in the  same host they may  in a d j a c e n t c e l l s . T h i s  is one  in tobacco  It  in the presence of cucumber mosaic v i r u s .  i n t e r e s t i n g phenomenon s i n c e  s t r a i n s of the  sized  i t seems  in a " s t a b i l i z e d " mixture with maize dwarf mosaic v i r u s . The  is an  vein  alone d i d become i n f e c t e d by t h i s v i r u s when  of maize dwarf mosaic v i r u s on virus  is  plants  is the  i n f e c t N_. g l u t i n o s a  ( G i l l a s p i e and  (Sorghum halepense L. Pers.) which mosaic v i r u s when i n o c u l a t e d  (Ross, 197^)  Another example  infect  L.  I960).  (Weathers,  P_. t r i f ol i a t a  cucumber mosaic v i r u s  P v i r u s d i d not  to  (Weathers, 1 9 6 1 ) . Although  been termed e x c e p t i o n a l being  tr i foliata  i n f e c t e d by y e l l o w  to healthy  (cv. B r i g h t Yel low) , but. i t d i d  B r i g h t Yellow  i t was  Ponci rus  l a t t e r host s y s t e m i c a l l y  between P v i r u s , a tymovirus, and  and  c i t r u s species  immunity of the host  taken from lime p l a n t s doubly  both v i r u s e s  was  also alter  immune to y e l l o w v e i n v i r u s when i t is alone  However, when s c i o n s v i r u s and  two  is of  i n t e r a c t i o n s and  be  i n t e r e s t because i t a l s o because of  Ross, 197^a) that enhancement of PVX  in c e l l s doubly  infected  by  PVX  synthe-  and  PVY.  .  the  <  14  Crystalline the  i n c l u s i o n s o f t o b a c c o e t c h v i r u s and TMV  same c e l l s  of doubly infected plants  e t a l , 1967)•  Fujisawa  (1949) t h a t  have  a DNA v i r u s ,  e t a l , 1969)-  occurred  together  Ross, 1972a). and  and TMV  individual  be s e e n  i n t h e same  m o s a i c v i r u s a n d b e a n pod m o t t l e  These v i r u s - h o s t systems a l s o r e s u l t  ment o f o n e o f t h e v i r u s e s  i n t h e m i x t u r e and i t h a s been  and  Ross,  C.  E f f e c t o n t r a n s m i s s i o n o f v i r u s e s by v e c t o r s in the transmission  shown (Goodman  i s another consequence  by v i r u s e s .  The b e s t e x a m p l e o f t h i s phenomenon i s t h a t b e t w e e n  ticed  different  that  t h e MAV  Rhopalosiphum padi  doubly  , normally  the aphid-transmitted  The same o b s e r v a t i o n infected barley  transmitted  not transmitted  serolo-  I t had been n o -  f r o m b a r l e y by  L. i n a s i n g l e : i n f e c t i o n , ' was t r a n s m i t t e d when  in a double i n f e c t i o n w i t h 1970).  of double i n f e c t i o n of a plant  i s o l a t e s o f b a r l e y y e l l o w dwarf v i r u s . isolate  cells.  b e h a v i o u r o f a v i r u s by a v e c t o r o r  through seed  gically  infection  i n t h e enhance-  1974a) t h a t PVX e n h a n c e m e n t o c c u r s i n d o u b l y i n f e c t e d  Alterations  (Goodman  ( D o d d s , 1974)  ( H a m i l t o n and N i c h o l s , 1977) r e s u l t e d i n d o u b l e  cells.  RPV  isolate  was a l s o made when MAV a n d RMV  (Rochow, 1 9 7 5 ) .  by R_. ma i d i s o n l y when  virus  ( L e e and  i n f e c t i o n o f t o b a c c o by PVX a n d PVY o r TMV  R o s s , 1974a) a n d o f b a r l e y by e i t h e r BSMV and TMV  o r BMV of  Soybean  with  w i t h c a u l i f l o w e r mosaic v i r u s  i n t h e same c e l l s o f d o u b l y i n f e c t e d p l a n t s  Double  Cauli-  i s able to co-infect turnip plants  p a r t i c l e s and p a r t i c l e s o f t u r n i p m o s a i c v i r u s c o u l d (Kamei  and P r i c e  r e p l i c a t e d i n t h e same c e l l s .  t u r n i p m o s a i c v i r u s and X - b o d i e s t o g e t h e r  cell  found i n  and P r i c e , 1949;  I t was a l s o s u g g e s t e d by M c W h o r t e r  the viruses could  flower mosaic v i r u s ,  (McWhorter  h a v e been  In t h i s  i toccurred  (Rochow, isolates  i n s t a n c e MAV c o u l d i n mixed  i t was  be  infections with  15  i s o l a t e RMV  which  is normally  ma id i s, however, d i d not i n j e c t e d with  transmitted  transmit  The  to  on  RMV  i n s t a b i l i t y of t h i s  dependence of the MAV  explained  and  e x t r a c t s made from doubly  f a i l u r e of R_. maid is to t r a n s m i t attributed  RMV  1972,  MAV  isolate  in v i t r o  i s o l a t e on e i t h e r RPV  1975)  and  (Rochow,  or RMV  has  was  1975). been  i n t e r a c t i o n s (genomic masking)  which w i l l  v i r u s C and  transmitted  ions with  The  be d i s c u s s e d  later.  t r a n s m i s s i o n which  i n f e c t i o n s , but does not seem to i n v o l v e s t r u c t u r a l  t i o n s . Potato and  fed or  in v i t r o c o n d i t i o n s  There i s yet another type of dependent from mixed  when i t was  infected plants.  under  the b a s i s of s t r u c t u r a l  (Rochow, 1970,  by R. ma i d i s. Rhopalos i phum-  PVY  then on  by aphid  or  potato vectors  i f aphids  plants  i f each v i r u s o c c u r r e d  fed f i r s t  and  interac-  aucuba mosaic v i r u s were o n l y  on  plants  Govier,  between p a r t i c l e s of the h e l p e r and  acquired  in mixed  infect-  i n f e c t e d s i n g l y by  i n f e c t e d by e i t h e r potato v i r u s C or potato  mosaic v i r u s (Kassanis  results  PVY  aucuba  1971). Formation of aggregates the dependent v i r u s e s as well  as  m o d i f i c a t i o n of s u r f a c e s t r u c t u r e s or s u r f a c e charge of the  stylets  by PVY  to e x p l a i n  are some of the mechanisms which have been suggested  t h i s phenomenon (Kassanis The aphid was of the  frequency  Govier,  of t r a n s m i s s i o n  increased  in mixed  l a t t e r v i r u s was  From t h i s  and  of  a l f a l f a mosaic v i r u s may  pea  streak v i r u s ,  of a l f a l f a mosaic v i r u s by the  i n f e c t i o n with  decreased  observation  1971).  (Hampton and  i t was be  pea  inferred  increased  thus the high frequency  streak v i r u s while Sylvester,  that the in mixed  of  pea that  1969)•  concentration i n f e c t i o n s with  i t s transmission  by the  pea  16  aphid  (Hampton a n d S y l v e s t e r ,  nation that  i s n o t t e n a b l e and indeed  virus  concentration,  in dependent D.  Structural  interactions  between  other prerequisites  that of  reviewed  i n which they  does n o t g u a r a n t e e  s t r u c t u r a l components.  which favour o r prevent s t r u c t u r a l ,  interactions  capsid  Some h a v e been  1976).  t h e t e r m i n o l o g y o f Dodds a n d H a m i l t o n  interactions will  several  between two v i r u s e s  followed.  (1976) f o r  Thus,genomic masking w i l l  t o mean e n t i r e e n c a p s i d a t i o n o f a genome o f o n e v i r u s  protein  role  replicate or  However,  interactions  p e r s e , i n t h e same c e l l  be a n e x c h a n g e o f v i r u s  In t h i s t h e s i s  used  1972)  any s i g n i f i c a n t  between t h e two v i r u s e s .  (Dodds a n d H a m i l t o n  structural  (Rochow,  i n t h e same c e l l , t h e r e may be a n e x c h a n g e  preclude structural  synthesis,  there w i l l  thefactors  expla-  viruses  i n f e c t t h e same h o s t  s t r u c t u r a l components  therefore  i t h a s been s u g g e s t e d  p e r s e , seems n o t t o p l a y  t h e i r assembly s i t e s c o i n c i d e  and  | t a p p e a r s , however, t h i s  transmission.  When t w o v i r u s e s  of  1969).  o f the other v i r u s , as adopted  be  by h e t e r o l o g o u s  f r o m Yamamoto a n d A n d e r s o n  (1961) by Dodds a n d H a m i l t o n (1976). T h e t e r m " p h e n o t y p i c m i x i n g " similarly will  be used i n t h e c o n t e x t o f S t r e i s i n g e r  by Dodds a n d H a m i l t o n of the  (1976), t o mean a p r o t e i n  heterologous protein  made o f a m i x t u r e  s u b u n i t s from a t l e a s t two v i r u s e s  genome o f o n e o f t h e v i r u s e s . W h e r e  phenotype  capsid  (1956), a n d a d o p t e d  (coat protein), there could  also  both parental  surrounding  viruses  have a  be a v i r i o n made o f b o t h  17  p a r e n t a l p r o t e i n s and/or t h e i r n u c l e i c a c i d s .  O b v i o u s l y where.one o f t h e  p a r e n t s e x i s t s o n l y as a f r e e n u c l e i c a c i d , as i n some TMV p r o t e i n - d e f e c t i v e mutants, no second  parental v i r i o n w i l l  be formed nor w i 1 1 p h e n o t y p i c  mixing occur. 1-  In v i t r o s t r u c t u r a l Studies of s t r u c t u r a l  interactions i n t e r a c t i o n s between p l a n t v i r u s e s were  f i r s t done i n v i t r o and o n l y r e c e n t l y have attempts been made t o d e t e c t the phenomenon o f s t r u c t u r a l  interactions in vivo.  Perhaps s t i m u l a t e d  by t h e p i o n e e r work o f F r a e n k e l - C o n r a t and W i l l i a m s t r a t e d t h a t TMV coat p r o t e i n c o u l d be reassembled to  form  (1955)  which demons-  i n v ? t r o w i t h i t s RNA  i n f e c t i o u s r o d s , s e v e r a l l a b o r a t o r i e s have s i n c e r e p o r t e d t h e r e -  c o n s t i t u t i o n o f a number o f v i r u s e s (Matthews and H a r d i e , 1 9 6 6 ; H i e b e r t et  al,  1968;  Wagner and B a n c r o f t ,  1968;  Atabekov e t a l ,  1970a;  Breck and  Gordon, 1 9 7 0 ; Kado and K n i g h t , 1 9 7 0 ; Jonard e t a l , 1 9 7 2 ; F r i t s c h e t a l , 1973).  An important o b s e r v a t i o n o f these i n v t t r o s t u d i e s i s t h a t t h e r e  i s a h i g h degree o f s p e c i f i c i t y between t h e n u c l e i c a c i d o f a v i r u s and i t s homologous coat p r o t e i n . D e s p i t e t h e s p e c i f i c i t y o f r e c o n s t i t u t i o n between homologous components success has been a c h i e v e d h e t e r o l o g o u s components. reassembled  in vitro  i n assembling v i r i o n s  from  Thus, p r o t e i n s o f s m a l l s p h e r i c a l v i r u s e s were  i n t o v i r i o n s w i t h RNAs from h e t e r o l o g o u s v i r u s e s ( H i e b e r t  et  al,  of  BMV, broadbean m o t t l e v i r u s and BMV-RNA by t h e coat p r o t e i n s o f :CCMV  1968).  In t h i s study CCMV-RNA was e n c a p s i d a t e d by coat p r o t e i n s  18  or broadbean m o t t l e v i r u s . virus  (TYMV)-RNA  w i t h TMV  In v i t r o protein  of s h o r t rods than normal TMV  assembly of t u r n i p y e l l o w mosaic  resulted  rods  the i n f e c t i v i t y o f the assembled  in a much higher p r o p o r t i o n  (Matthews and  product was  i n c u b a t i o n w i t h r i b o n u c l e a s e (RNase);  Hardie,  very much reduced  thus s u g g e s t i n g the  of such p a r t i c l e s . Assembly, of PVX-RNA w i t h TMV product that was  highly  resistant  1970), but no  in v i v o  these v i r u s e s  (Goodman and Ross, 1974c).  products and  Other  and  (Breck and  et a l , 1970a).  have s p e c i a l  i t was  sites,  p r o t e i n assembled  suggested  that  w i t h the two  RNAs.  r e c o n s t i t u t ion ( B u t l e r and reassembly  of TMV  may  Klug, 1971)  i n t e r n a l l y . n e a r the 3' OH end..In another  excess  Because both  that TYMV-RNA  similar  a s s i g n e d t o be the  be b i d i r e c t i o n a l  reassembly  p r o t e i n , under some  ( F r i t s c h et a l , 1973)  end was  protein  in which TMV-RNA and TYMV-RNA  p o s s i b l y a t the 5' OH end,  However, s i n c e the 5' OH  that  (Kado  in the presence of  protein.  initiation  recent evidence  site indicates  (Otsuki et a l , 1977)  study  may  to those on  TMV-RNA, which are a l s o recognized e q u a l l y by h e t e r o l o g o u s  in TMV  assembly  coat p r o t e i n  TMV-RNA and TYMV-RNA were e q u a l l y recognized by TMV pH c o n d i t i o n s ,  Gordon,  between  heterologous  F r i t s c h et a l (1973) observed  However, in c o m p e t i t i v e experiments  were both present TMV  in a  BSMV-RNA w i t h cucumber v i r u s - 4 p r o t e i n , PVX-RNA  of TMV-RNA w i t h homologous p r o t e i n o c c u r r e d host RNA.  a f t e r ".  resulted  coat p r o t e i n and TMV-RNA w i t h cucumber v i r u s - 4 coat  (Atabekov  Further,  instability  i n t e r a c t i o n s were observed  i n c l u d e RNA o f cucumber v i r u s - 4 with TMV  Knight, 1970)  w i t h TMV  protein  to RNase treatment  structural  1966).  starting  (Jonard e t a l , 1972)  TMV-RNA  19  or a l f a l f a  mosaic v i r u s RNA d i d not form n u c l e o p r o t e i n complexes with  TYMV p r o t e i n c a p s i d s . affinity  T h i s suggests  that TYMV p r o t e i n may have g r e a t e r  f o r i t s own RNA than f o r h e t e r o l o g o u s RNAs.  Recently SBMV was s u c c e s s f u l l y assembled and  sowbane mosaic v i r u s RNA (Tremaine  with  and Ronald,  i t s homolgous RNA 1977).  Both  assembly  products were i n f e c t i o u s on P i n t o and Chenopodium a r m a r a n t i c o l o r , respectively.  The assembly  dodecyl  products were a l s o r e s i s t a n t  t o 1% SDS  (sodium  s u l p h a t e ) to which n a t i v e SBMV v i r i o n s a r e r e s i s t a n t .  a l s o as s t a b l e as and. i d e n t i c a l  t o SBMV v i r i o n s ,  absorbance  patterns, electrophoretic mobility  microscopy  and i n f e c t i v i t y . with  They were  in r e s p e c t to u l t r a v i o l e t  in agarose  gels,  electron  However, when sowbane mosaic v i r u s  was  assembled  new  products were a l s o u n s t a b l e in ] % SDS..  protein  i t s homologous RNA or w i t h h e t e r o l o g o u s SBMV-RNA the  In some c a s e s , the h e t e r o l o g o u s l y r e c o n s t i t u t e d product i s infectious 1970;  ( H i e b e r t et a l , 1968; Atabekov et a l , 1970a; Breck and Gordon,  Kado anf Knight, 1970; F r i t s c h e t a l , 1973) although  infectivity product. was  is usually  l e s s than that o f the homologous  its specific  reassembly  In o t h e r cases the i n f e c t i v i t y o f the heterologous  drastically  reduced  by RNase treatment  product  (Matthews and Hardie,  1966).  The above examples a r e o f in v i t r o genomic masking. Phenotypic mixing related  has been demonstrated  and u n r e l a t e d v i r u s e s .  m o t t l e v i r u s were r e c o n s t i t u t e d  in v i t r o  between  P r o t e i n s u b u n i t s o f CCMV, BMV and broadbean i n v i t r o t o form a ; p r o t e i n c o n t a i n i n g  20  mixed s u b u n i t s of these v i r u s e s tituted was  product c o n t a i n i n g a mixture  resistant  s t r a i n s was  product.  demonstrated  s t r a i n s were assembled 2.  The  In v i t r o  indicated  phenotypic mixing  of the common s t r a i n  Structural  down.  common as those between animal Hamilton,  1976).  that mixed  were mixedly encapsidated particle. mixed  In v i v o s t r u c t u r a l  (1951) observed  homologous  and  in v i v o  bacteriophages  i n t e r a c t i o n s between  Bailey,  19^6)  when i t was  phages gave new  types,  that when b a c t e r i o p h a g e s T2 and  bacterial  host, the DNA  of T2  Jk  was  in the coat p r o t e i n o f Th to form a g e n o m i c a l l y masked  In another  study  i n f e c t i o n of T2 and Tk  observed  (Delbruck and  i n o c u l a t e d to t h e i r  c a l l y mixed and was  viruses  i n f e c t i o n s with T2, Tk and T6  Szilard  to occur  In a d d i t i o n , o t h e r s u b t l e b a r r i e r s must  i n t e r a c t i o n s between p l a n t v i ruses  b a c t e r i o p h a g e s were suspected  Novick and  tomato  i n t e r a c t i o n s are one o f the consequences of mixed  s p e c i f i c i t y must be broken  found  between  In order f o r genomic masking or phenotypic mixing  are not as  residual  (Otsuki et a l , 1 9 7 7 ) .  the v i r u s e s must have common or adjacent assembly s i t e s , and  (Dodds and  by  interactions.  In v i v o s t r u c t u r a l  be removed.  recons-  o f two or more d i f f e r e n t p r o t e i n s  when p r o t e i n s of the common and  with RNA  In v i v o s t r u c t u r a l  infections.  Bancroft, 1968).  to snake venom p h o s p h o d i e s t e r a s e , as  i n f e c t i v i t y of the new TMV  (Wagner and  (Streisinger, resulted  1956)  i t was  found  that a  in the f o r m a t i o n of both  g e n o m i c a l l y masked p a r t i c l e s .  (Yamamoto and Anderson, 1961)  phenotypi-  R e c i p r o c a l genomic masking  in v i v o between P221  and  P22h  21  phages.  Among the many examples between animal  v i r u s e s , the i n t e r -  a c t i o n between the genome o f foot-and-mouth d i s e a s e v i r u s and the coat p r o t e i n o f bovine of  interest  enterovirus  because o f the economic  d i s e a s e as a , l e t h a l Apparently  s i m i l a r v i r u s e s (Sarkar,  The  i n the southern  d e t e c t i o n o f in v i v o observed  1970a, 1970b;  1969;  Kassanis  in v i v o s t r u c t u r a l  1971)  is  importance o f foot-and-mouth  disease of c a t t l e  p l a n t v i r u s e s was f i r s t  Bastow,  (Trautman and S u t m o l l e r ,  structural  hemisphere.  i n t e r a c t i o n s between  between r e l a t e d and m o r p h o l o g i c a l l y  Atabekov e t a l ,  1970b;  Kassanis and  and C o n t i , 1971; Atabekova e t a l ,  i n t e r a c t i o n s observed  in these  e n c a p s i d a t i o n o f the RNAs o f t e m p e r a t u r e - s e n s i t i v e  1975).  studies involved  coat p r o t e i n o r  p r o t e i n - d e f e c t i v e mutants o f s t r a i n s of TMV by the coat p r o t e i n of the protein-competent with  strains.  the t e m p e r a t u r e - r e s i s t a n t  l e s i o n s than when alone.  The TMV mutant Ni 118, i n mixed v u l g a r e s t r a i n o f TMV,  From these  and  strain et and  (Kassanis and Bastow,  no s t r u c t u r a l  RNAs of the Ni 118 and  components;  however,  who reported  in the  and o f the T u r i n  In other s t u d i e s  i n t e r a c t i o n s occurred i f they had access encapsidated  masked  that  the genomes o f Ni 118  1970a, 1970b)  (Kassanis and C o n t i , 1971) o f TMV.  a l , 1975)  These  A14 and d o l i c h o s e n a t i o n mosaic  The CP-TMV p r o t e i n a l s o e n c a p s i d a t e d  PM2 mutants  1969).  mutants were g e n o m i c a l l y  coat p r o t e i n s o f TMV s t r a i n s of v u l g a r e , virus.  (Sarkar,  (1970b)  by Atabekov e t a l  the RNAs o f Ni 118 and flavum  induced more  r e s u l t s genomic masking o f Ni 118-RNA  by the coat p r o t e i n o f v u l g a r e was i n f e r r e d r e s u l t s were confirmed  infections  (Atabekov  between coat p r o t e i n s to their  homologous  the RNA o f Ni 118 a t  22  a temperature which was non-permissive f o r the p r o d u c t i o n  o f Ni 118 coat  prote i n. Genomic masking can a l s o occur s i m i l a r , but u n r e l a t e d  viruses.  in v i v o between two s t r u c t u r a l l y  Isolates of barley yellow  structurally  s i m i l a r but s e r o l o g i c a l l y  by d i f f e r e n t  aphid  species.  of a non-aphid t r a n s m i t t e d mitting doubly  strain  resulted  infected plants  Recently  and they a r e t r a n s m i t t e d  Heterologous e n c a p s i d a t i o n strain  by the coat  in t h e aphid  of t h e RNA genome  p r o t e i n o f an aphid  transmission  o f the former from  i n v i v o s t r u c t u r a l i n t e r a c t i o n s have been d e t e c t e d In t i s s u e e x t r a c t e d  from  i n f e c t e d by BSMV and TMV t h e genome o f TMV was found  in the coat  trans-  1970, 1972, 1975).  (Rochow,  structurally dissimilar viruses. doubly  unrelated  dwarf v i r u s a r e  between  barley encapsidated  p r o t e i n o f BSMV (Dodds and Hamilton, 1971; Dodds, 1972,  Dodds and Hamilton,  197*0.  p l a n t s were t r e a t e d with  1974;  When e x t r a c t s prepared from doubly i n f e c t e d  antiserum to TMV^not a l l TMV i n f e c t i v i t y was  a b o l i s h e d whereas the i n f e c t i v i t y was removed by antiserum treatment of a mixture o f e x t r a c t s from p l a n t s s i n g l y i n f e c t e d by each v i r u s . of doubly  infected plants  lost  BSMV i n f e c t i v i t y a f t e r treatment with  homologous antiserum. The a s s o c i a t i o n of TMV-RNA w i t h about 8* o f the RNA w i t h i n masking was detected In such doubly the same c e l l .  only  i n leaves  that s y n t h e s i z e d  its  BSMV p r o t e i n was  BSMV v i r i o n s (Dodds, 1974).  i n f e c t e d leaves  Extracts.,  T h i s genomic  more TMV than BSMV.  p a r t i c l e s of both v i r u s e s appeared i n  Genomic masking o f BSMV-RNA by BMV p r o t e i n was  suspected  23  in doubly  infected  barley  (Peterson and Brakke, 1973),  but u n f o r t u n a t e l y  this  i n t e r a c t i o n was not confirmed by a n t i b o d y n e u t r a l i z a t i o n o f i n f e c t i -  vity  test.  ected and  Genomic masking o f BMV-RNA by BSMV coat p r o t e i n  in another study  1970) nor  (Morris,  Although phenotypic mixing  ence  similar viruses,  in v i v o  reported  was reported  their protein  et a l , 1977)-  and  1978).  pH  inoculated  had been doubly  r e s u l t s of s t r u c t u r a l  i n d i c a t e that  studies  The phenomenon was  e t a l , 1977).  P r o t e i n sub-  in v i t r o and when  t o tobacco p l a n t s ,  phenotypic mixing  (Atabekova e t a l , 1975;  Taliansky  common and tomato s t r a i n s o f TMV when TMV was i s o l a t e d from that  in v i v o  i t s occurr-  s u b u n i t s o f sunh-hemp mosaic v i r u s  s u b u n i t s was observed  protoplasts Takebe,  viruses.  that  Mixed coat p r o t e i n was a l s o observed between p r o t e i n sub-  u n i t s o f the  for  between p l a n t  recently  s t r a i n s o f TMV form aggregates  these s t r a i n s were doubly  The  i t was not u n t i l  s t r a i n s o f TMV ( S k o t n i c k i  2  u n i t s o f aucuba and  of  i s l i m i t e d by geometric b a r r i e r s t o  t o occur between p r o t e i n  (= CP-TMV) and U  between TMV .  1977).  BMV (Hamilton and N i c h o l s ,  structurally  was i t d e t e c t e d  was not det-  inoculated  i n t e r a c t i o n s , discussed  conditions optimai for  interactions.  between v i r a l  or  the  approach  conditions,  use o f a d d i t i v e s ,  in v i t ro  above, seem t o  reconstitution  be they b u f f e r  that may favour  RNA and h e t e r o l o g o u s coat p r o t e i n .  in v i t r o ^ may not be those which are  (Otsuki  in v i t r o may not always be the same  The general  i s t o search f o r optimal  gradients  w i t h the two s t r a i n s  suitable for  molarities,  interactions  Such optimal  conditions  interaction  in the host.  24  Although the success o f in v i t r o found  in v i v o , the new product  than a product d e r i v e d there and  r e c o n s t i t u t ion seems ; to surpass  i s e i t h e r lower i n y i e l d or l e s s i n f e c t i v e  from homologous reassembly.  In some experiements  seems t o be a c o r r e l a t i o n between the success o f i n v i t r o  In v i v o s t r u c t u r a l  occurred  interactions.  p r o t e i n aggregates  and aucuba s t r a i n s o f TMV  in v i t r o  (Taliansky  (Atabekova et a l , 1 9 7 5 ) -  of PVX-RNA with TMV p r o t e i n was achieved and  Gordon, 1 9 7 0 ) i t was not detected Where i n v i v o s t r u c t u r a l  unrelated  viruses  and  Brakke, 1 9 7 3 )  ted  (Dodds and Hamilton, 1 9 7 4 )  t i s s u e leads  the e n c a p s i d a t i o n  It has been  specula-  o f TMV-RNA by BSMV  o f l a r g e amounts o f TMV-RNA  i n doubly  to great  enhancement o f TMV i n  I n t e r a c t i o n between BMV and BSMV in doubly  i n s i n g l y i n f e c t e d p l a n t s and i t has been suggested  Brakke, 1 9 7 3 )  possibly  Peterson  bar 1 e y r r e s i i 1 t s i n decreased y i e l d o f e i t h e r v i r u s compared t o  their yield and  1974c).  to excess RNA which becomes a v a i l a b l e t o BSMV p r o t e i n .  3 and 4 o f b a r l e y .  infected  Breck  between  Dodds and Hamilton 1 9 7 * * ;  I n t e r a c t i o n between TMV and BSMV leads leaves  assembly  (Goodman and R o s s j .  have been g i v e n .  that  both in  (Atabekov et a l , 1 9 7 0 a ;  in vivo  1974;  two e x p l a n a t i o n s  p r o t e i n occurs because s y n t h e s i s infected  Although ?n v i t r o  i n t e r a c t i o n s have o c c u r r e d  (Dodds, 1 9 7 2 ,  1977).  et a l ,  Genomic masking o f Ni 118-RNA by U2 TMV p r o t e i n a l s o occurred v i v o and i n v i t r o  assembly  Thus i n v i v o phenotypic mixing  between the p r o t e i n s u b u n i t s o f  which a l s o formed  that  that e n c a p s i d a t i o n  be due to a great  o f BSMV-RNA by BMV p r o t e i n  (Peterson could  excess o f BMV p r o t e i n over that of BSMV p r o t e i n .  25  The  r e s u l t s obtained  1977)  with TMV and-BMV i n b a r l e y  (Hamilton and N i c h o l s ,  where no s t r u c t u r a l i n t e r a c t i o n s were observed between the two  v i r u s e s , suggest that one has t o c o n s i d e r  other  reasons than excess o f  e i t h e r RNA o r coat  p r o t e i n o f one o f the v i r u s e s , because TMV was a l s o  enhanced  3 and 4 a l t h o u g h BMV was suppressed  in leaves  Indeed excess o f e i t h e r components may p l a y a very leading dified. was,  al,  t o s t r u c t u r a l i n t e r a c t i o n s unless  Indeed, found t o be very  homologous s p e c i f i c i t y  can be mo-  high  between TMV s t r a i n s (Atabekova e t  replication.  study o f p l a n t v i r u s r e p l i c a t i o n i s l i m i t e d t o a great  the l a c k o f a s u i t a b l e system which a f f o r d s synchronous  r e p l i c a t i o n of virus plast  system  i n the c e l l s .  in plant v i r o l o g y  (Takebe and O t s u k i ,  of TMV.  proto-  1969) introduced a  Inoculation o f protoplasts  i n f e c t i o n and an a c c e l a r a t e d  synthesis  Between 8 and 22 hours a f t e r i n o c u l a t i o n o f p r o t o p l a s t s w i t h TMV,  synthesis  to i n c r e a s e  plant v i r u s  increased  r a p i d l y (Takebe and O t s u k i ,  linearly until  et a l , 1975).  1975).  rapid  extent  i n f e c t i o n and  However the advent o f the  approach t o the study o f p l a n t v i r u s e s .  with TMV r e s u l t s i n t h e i r  TMV  minors r o l e i n  1975).  The  new  leaves.  S p e c i f i c i t y o f RNA-protein and p r o t e i n - p r o t e i n i n t e r a c t i o n  I I Synchronous systems f o r p l a n t v i r u s  by  i n these  72 hours  The u s e f u l n e s s  1969) and continued  (Takebe e t a l , 1971,  o f the p r o t o p l a s t  system  cited  i n Dawson  i n t h e study o f  i n f e c t i o n and r e p l i c a t ion has r e c e n t l y been reviewed  The main l i m i t a t i o n s o f the p r o t o p l a s t  (Takebe  system are that the  26  i s o l a t e d mesophyll c e l l s cannot u s u a l l y be maintained f o r long after  infection. At about the same time as the development  a n e a r l y synchronous system TMV  times  synthesis  of the p r o t o p l a s t technique  in i n t a c t c e l l s was  utilized  in the study of  in tobacco (Ni1sson-Ti11gren et a l , 1969).  T h i s system  took advantage o f r a p i d l y d e v e l o p i n g young leaves which were in a n e a r l y synchronous stage of i n f e c t i o n k or 5 days a f t e r - i n o c u l a t i o n w i t h The obvious d i s a d v a n t a g e w i t h t h i s system infection starts  i s that the exact time at which  i s not known.  A system that a f f o r d e d both synchronous of the c e l l s  (1973).  in t h e i r n a t i v e s t a t e was  i n f e c t i o n and  r e p l i c a t i o n of v i r u s e s  preservation  developed by Dawson and  T h i s system has s i n c e proven to a f f o r d  of c e l l s and  TMV.  synchronous  (Dawson and S c h l e g e l ,  Schlegel  infection  1973; 1976a  1976b, 1976b, 1976c; Dawson et a l , 1975) s i m i l a r to that achieved in the p r o t o p l a s t system.  The advantages of the system a r e : a l a r g e amount  of t i s s u e can be accomodated; c e l l s are synchronous w i t h r e s p e c t to t h e i r developmental s t a g e and stage o f i n f e c t i o n ; and to b u i l d and use.  i t i s easy and i n e x p e n s i v e  Its main d i s a d v a n t a g e i s t h a t , u n l i k e p r o t o p l a s t s ,  is spread of v i r u s between i n t a c t c e l l s .  T h i s system was  there  used as a working  model f o r the i n t e r a c t i o n o f CP-TMV and CP-SBM in some s e c t i o n s of t h i s t h e s i s and thus i t w i l l The d i f f e r e n t i a l  be d i s c u s s e d  in d e t a i l s .  temperature synchronous system o f Dawson and  Schlegel  (1973) i s based on m a n i p u l a t i o n of the d i f f e r e n t p a r t s of the p l a n t a f t e r i n o c u l a t i o n of the lower leaves w i t h a v i r u s .  Essentially  the lower  27  (leaves ace', i n o c u l a t e d placed  i n a p o l y s t y r o f o a m chamber m a i n t a i n e d  (high) f o r rapid lated  virus  synthesis.  the plant i s  at a permissive  temperature  A t t h e same t i m e t h e u p p e r  non-inocu-  l e a v e s a r e a l l o w e d t o p r o t r u d e o u t s i d e t h e s t y r o f o a m chamber  they a r e maintained sis  i n a c o n v e n t i o n a l manner a f t e r w h i c h  at a non-permissive  but not f o r t r a n s l o c a t i o n .  achieved.by growth  placing  chamber m a i n t a i n e d  s y n t h e s i s in the inoculated  system  chamber m a i n t a i n e d  synthesis in the e n t i r e  Since  replication  applicable  A rapid  (Dawson and S c h l e g e l ,  whereby t h e degree o f synchrony, p r o t e i n c u r v e s o f TMV, et a l , 1975).  s y n t h e s i s o f TMV  ss-RNA and  (Dawson and S c h l e g e l , between p a r e n t a l v i r a l  applied  a  permissive  synchronous  virus  s y n t h e s i s was a c h i e v e d  by  infectivity  t o t h a t f o r TMV  and  nucleo-  i n p r o t o p l a s t s (Dawson  to study  the k i n e t i c s of  i t sattendant double-stranded  structures  a r e b e l i e v e d t o be i n t e r m e d i a t e s  and p r o g e n y v i r u s  ( B i s h o p and L e v i n t o w ,  with  1973, 1976b, 1 9 7 6 c ; Dawson e t a l , 1975)  K i 1 l a n d - B r a n d t and N i 1 s s o n - T i 1 1 g r e n , bacterial  •  leaves the e n t i r e  temperature  has a l s o been u s e d  1976b) w h i c h RNA  '. :  After  at a temperature  as i n d i c a t e d  is similar  T h i s system  /.  to the study of v a r i o u s aspects of the  of plant viruses.  CCMV and TMV  is  synthe-  plant.  i t s development the d i f f e r e n t i a l  has p r o v e n  temperature  a t t h e d e s i r e d low temperature.  i s moved t o a g r o w t h  for virus  The n o n - p e r m i s s i v e  (low) f o r v i r u s  t h e e n t i r e s t y r o f o a m chamber'. i n a c o n t r o l l e d  s u i t a b l e period of v i r u s plant  temperature  where  i n p l a n t ( J a c k s o n e t a l , 1972;  1973; H a m i l t o n ,  1971) v i r u s e s .  in the study of i n h i b i t o r s which  1974) and a n i m a l  The s y s t e m  block different  has a l s o  steps  and  been  in the  28  r e p l i c a t i o n of CCMV a n d TMV In s t u d y i n g to know i f the by the order action that  plant viruses  is being  studied.  investigate  Goodman and  The virus  in double and and  mosaic v i r u s than that  in doubly  infected tissue  inoculated  doubly  which o t h e r s  (Goodman and  ment of PVX,  but  Infection. In  the study of mixed  Barker and  in doubly that  inoculated  i s , an equal  protoplasts  no mention was  but  p r o t o p l a s t s was  protoplasts.  i n o c u l a t e d with PVX Ross,  Harrison,  1974b)  protoplasts  number of  of TMV  was  the  that of cucumber  c o n s i s t e n t l y lower  In the same study and  1977a,  i n f e c t e d by e i t h e r  A l s o the c o n c e n t r a t i o n  inoculated  to  d i f f e r e n t v i r u s e s with  been used  TMV  inter-  in the r e p l i c a t i o n  in s i n g l e i n o c u l a t i o n s was  in s i n g l y i n o c u l a t e d  p l a s t s were a l s o  in doubly  stages  Takebe, 1 9 7 6 ;  Takebe, 1 9 7 6 ) .  same in s i n g l y or doubly  was  and  already  independently of each o t h e r ,  (Otsuki  t i s s u e in which the  (1974b) showed evidence  i n t e r a c t i o n s of two  Cucumber mosaic v i r u s and  protoplasts virus  of c r i t i c a l  system has  i n f e c t i o n s (Otsuki  multiplied  Ross  r e p l i c a t i o n in a mixed  protoplast  1977b).  in the  is a f f e c t e d  A synchronous system o f f e r s an o p p o r t u n i t y  intracellular  to t h e i r  1976c).  i t synergism or antagonism,  to be maximally enhanced by PVY  c y c l e of the v i r u s e s .  1976a,  Schlegel,  i n f e c t i o n s i t is d e s i r a b l e  of the v i r u s e s  there must be a c o i n c i d e n c e  respect  in mixed  i n t e r a c t i o n , be  of a r r i v a l  f o r PVX  (Dawson and  TMV,  proto-  a combination  found to r e s u l t in the enhance-  made whether the c o n c e n t r a t i o n  enhanced or o t h e r w i s e a f f e c t e d under c o n d i t i o n s  of  PVX  of synchronous i n f e c t i o n .  29  When p r o t o p l a s t s were doubly  Inoculated  with r a s p b e r f y  ringspot  v i r u s and the CAM s t r a i n o f tobacco r a t t l e v i r u s numerous aggregates o f raspberry',  ringspot  inoculated  protoplasts  and that ted  Harrison,  v i r u s were found throughout the cytoplasm o f doubly but not in s i n g l y i n o c u l a t e d  1977a)-  raspberry  It was l a t e r found  t h e i r formation  by mixing t h e two v i r u s e s . centration of either virus. in the p r o t o p l a s t s  (Barker  I I I Seed t r a n s m i s s i o n  and H a r r i s o n ,  of plant  detected  1977b).  in the mixture.  in seed d e r i v e d  by CCMV and CP-SBMV than  i n double  Thus seed  trans-  i n f e c t i o n s with  On the other  hand CP-SBMV was  from cowpea p l a n t s  in s i n g l y i n o c u l a t e d  characteris-  doubly  plants  (Kuhn and  In s t u d i e s where s t r u c t u r a l i n t e r a c t i o n s between v i r u s e s  in double  i n f e c t i o n s were reported  suspected  (Peterson  in p l a n t s  (Dodds and Hamilton, 1974) and  and Brakke, 1973) no seed t r a n s m i s s i o n  (Dodds and Hamilton, occurred  in v i t r o  viruses.  (Ross, 1963, 1968).  more r e a d i l y t r a n s m i t t e d  Dawson, 1973).  be induced  No s t r u c t u r a l i n t e r a c t i o n s were  o f soybean mosaic v i r u s was reduced  infected  in doubly i n f e c -  i n f e c t i o n s may a l s o a l t e r seed t r a n s m i s s i o n  bean pod m o t t l e v i r u s  1977b)  Double i n f e c t i o n d i d not a f f e c t the con-  t i c s o f the seed t r a n s m i s s i b l e v i r u s mission  could  (Barker  and H a r r i s o n ,  r i n g s p o t v i r u s aggregates a l s o occurred  l e a f c e l l s and that  Mixed v i r u s  (Barker  protoplasts  1974) o r o f BSMV  inoculated with  (Peterson  and Brakke, 1973)  inoculum that contained  e i t h e r o f these v i r u s e s e n c a p s i d a t e d  in t h e coat  o f TMV  the RNA o f  p r o t e i n o f heterologous  30  virus.  It i s i n t e r e s t i n g  that BSMV was not seed t r a n s m i t t e d when i t s  RNA was presumed t o be e n c a p s i d a t e d  i n BMV p r o t e i n (Peterson  1973)  because BSMV i s known to be seed t r a n s m i t t e d  1955;  McKinney and Greeley,  is, in  however, not f u l l y single  1965).  and Brakke,  ( E s l i c k and A f a n a s i e v ,  The mechanism o f seed  transmission  understood even where the seed-borne v i r u s i s  infections.  I n v e s t i g a t i o n s on seed t r a n s m i s s i o n  i n mixed v i r u s i n f e c t i o n s must,  of n e c e s s i t y , be preceded by an understanding  o f the d i s t r i b u t i o n and  c h a r a c t e r i s t i c s o f each o f the v i r u s e s i n the seed when i t i s in a •.; single  infection.  Where these  have been r e s o l v e d  the seed t r a n s m i s s i o n o f the v i r u s e s mind, the l i t e r a t u r e d e a l i n g with infections will  be reviewed with  CP-TMV a l l e g e d seed The  infections.  With t h i s in  seed t r a n s m i s s i o n o f v i r u s e s special  in s i n g l e  emphasis on the CP-SBMV and  transmission.  phenomenon o f seed t r a n s m i s s i o n  few  viruses are transmitted  not  been reported  is puzzling  in that  through seed and a m a j o r i t y  t o be so t r a n s m i t t e d .  that f o r a v i r u s to be t r a n s m i t t e d to  in mixed  then one can study  relatively  have  The bulk o f evidence  to the next progeny  indicates  i t must be a b l e  I n f e c t the embryo i n the e a r l y stages o f I t s development and there<-  after  persist  transmission Even  in the embryo u n t i l has been c a l l e d  seed g e r m i n a t i o n .  embryonic t r a n s m i s s i o n  T h i s type o f seed (Bennett,  1969).  i n t h i s case some v i r u s e s a r e a b l e to i n f e c t embryos o f some host  p l a n t s and not o f o t h e r s . embryos o f any host  species  Why some v i r u s e s v i r t u a l l y i s not c l e a r l y understood.  fail  to i n f e c t  Inactivation of  the v i r u s in the embryo has been suggested by some (Duggar, 1930;  31  Cheo, and  1955)-  The l a c k of d i r e c t v a s c u l a r  the embryo has been i m p l i c a t e d  transmission vascular  (Bennett and Esau,  1936).  only  to the  Immunity o f the gametophytic  (Bennett, 1 9 4 0 ) i s g i v e n as another reason f o r the l a c k o f  seed t r a n s m i s s i o n  by some v i r u s e s .  the embryo has been e x p l a i n e d phosphorylated m a t e r i a l s (Caldwell,  as a reason f o r the f a i l u r e of seed  in the case of v i r u s e s that are c o n f i n e d  systems  generation  c o n n e c t i o n s between the p l a n t  1962).  on the b a s i s of i n s u f f i c i e n t  required  f o r virus synthesis  high-energy  in the embryo  However, what appears c l e a r i s t h a t seed  s s i o n o f v i r u s through embryo between host  The f a i l u r e of some v i r u s e s to invade  and v i r u s  transmi-  i n f e c t i o n depends on an i n t e r a c t i o n  1972).  (Shepherd,  This  i n t e r a c t i o n could  be  dependent on the composition o f s p e c i f i c host and v i r u s p r o t e i n s . Indeed, g l y c o p r o t e i n s were found and  in two seed t r a n s m i t t e d  cowpea mosaic v i r u s , but not in three other  seed t r a n s m i t t e d  ( P a r t r i d g e et a l ,  1974).  v i r u s e s that were not  From t h i s c o r r e l a t i o n  i t was suggested by these workers that c o v a l e n t l y - 1 i n k e d residues for  on the v i r a l  coat p r o t e i n  in nepoviruses  Harrison,  1 9 7 7 ) that  RNA-1 and  carbohydrate  may a c t as r e c o g n i t i o n  the attachment of the v i r u s to the gametophytic c e l l  However,  v i r u s e s , BSMV  i t has r e c e n l y been reported  seed t r a n s m i s s i b i 1 i t y was  sites  surfaces.  (Hanadarand  i n f l u e n c e d more by  than by RNA-2. RNA-2 of these v i r u s e s determines coat  protein  serological specificity. As f a r as i t i s known TMV  i s not seed t r a n s m i s s i b l e through the  32  embryo except f o r the the f a c t  report of Gilmer  1961;  ( T a y l o r et a l , been r e p o r t e d fruit,  Milbrath of  and  these  Bennett,  1969).  Seed t r a n s m i s s i o n of TMV  seed t r a n s m i s s i o n of TMV  Canner which had  no evidence  was  from  of TMV  to  transmitted  intact  seed.  contamination  not  been handled.  (Crowley, 1957)  of the germinating  removed from the seed coats and Broadbent,  1965)  clearly  in tomato i f s e e d l i n g s are not  virus.  in tomato s e e d l i n g s  Despite  (1957)  could  handled  unlikely  that TMV  pungent  t h i s he found  this  that  derived  transmission  embryos from v i r u s in the seed occurred  coats.  when embryos were  Later studies  showed that TMV  been surface-decontaminated  i s not  ( T a y l o r et a l ,  seed  ( i . e . transplanted)  transmitted or when  before p l a n t i n g and  thus  i t was  in the endosperm would become absorbed  into  the  germinating  embryo.  fruit  (Gilmer and  this virus.  by the  Crowley  attributed  planted.  seed had  trees  i n s i d e a f l e s h y or  to 15 to 301 of pungent pepper s e e d l i n g s He  has  in the endosperm or embryo of tomato and  Indeed, he found that no t r a n s m i s s i o n of TMV  1961;  reviewed  thus a f f o r d i n g easy s u r f a c e contamination reported  In s p i t e of  known, e a r l i e r  have been thoroughly  pepper a f t e r decontamination of seed p a r t s . TMV  (1967).  is not  in tomato seed, which i s c o n t a i n e d  (1937)  Indiana  find  Wilks  that embryonic t r a n s m i s s i o n of TMV  r e p o r t s were c o n f l i c t i n g  pulpy  and  The  embryonic t r a n s m i s s i o n of TMV Wilks,  In t h i s study TMV  1967) was  is p u z z l i n g and  recovered  in seed of pome  exceptional  from 16% of  coat-endosperm t i s s u e s of Malus p l a t y c a r p a , but not  from  for  inner seed corresponding  33  seeds whose inner seed coat-endosperm t i s s u e s had assayed p o s i t i v e l y for  TMV.  From t h i s  i t would  seed coats and endosperm. lings derived had been  appear the v i r u s was  However, TMV  to 16% of seedtissue  removed.  Kassanis and Varma, 1 9 7 5 )  i s o l a t e o f sunn-hemp mosaic v i r u s  (Kassanis and Varma, 1 9 7 5 )  not seed-borne (Capoor, 1 9 6 2 ) .  the cowpea c h l o r o t i c spot  contamination s i m i l a r  (=CP-TMV;  seed-borne in k to 20% of seed of Vigna  was  s i n e n s i s cv. Pusa Dophasli  of  transmitted  with  from p l a n t e d seeds whose inner seed coat-endosperm  The cowpea c h l o r o t i c spot  s t r a i n was  was  associated  i s o l a t e was  to that  w h i l e the type  Perhaps the seed t r a n s m i s s i o n a consequence of s u r f a c e  in pungent pepper  (Crowley, 1 9 5 7 )  s e e d l i n g s were not handled and, as suggested (Shepherd,  1972),  in which is a rare  example. Tobacco mosaic v i r u s  i s very s t a b l e and  that where seed t r a n s m i s s i o n  has been reported  contamination by the v i r u s , e s p e c i a l l y were not employed.  i n f e c t i o u s and this  i t may  resulted  be  from s u r f a c e  i f decontamination procedures  Evidence so f a r a v a i l a b l e suggests that  embryonic  t r a n s m i s s i o n of TMV. does not o c c u r . The bean s t r a i n of southern bean mosaic v i r u s for  the f i r s t  months (Zaumeyer  and  isolated  time from Fr.ench.'bean (Phaseol us vu Igar 1 s L.) and  found to be seed t r a n s m i t t e d  7  (SBMV) was  infectious.  was  to 5% of seeds that had been s t o r e d f o r  and H a r t e r ,  19^3).  This virus  Southern bean mosaic v i r u s c o u l d  i s a l s o very  stable  be recovered from a l l  3h  bean seed p a r t s u n t i l  maturity, by which time o n l y seed c o a t s c o n t a i n e d  i n f e c t i o u s v i r u s . ( C h e o , 1955)embryos was a t t r i b u t e d  The absence  o f i n f e c t i v e SBMV i n mature  by Cheo to i n a c t i v a t i o n o f the v i r u s  in mature  seed. Although Cheo (1955) o b t a i n e d 2 t o 5% seed t r a n s m i s s i o n o f SBMV w i t h 3 v a r i e t i e s o f bean, he concluded, on the b a s i s o f 50 o t h e r p l a n t e d mature seeds seed  that f a i l e d  transmitted.  t o t r a n s m i t the v i r u s , that the v i r u s  Because he f a i l e d  i s not  t o recover the v i r u s from  buffer  e x t r a c t s o f embryos o f mature seed that were assayed d i r e c t l y on a l o c a l l e s i o n host, a method o t h e r s ( S c h i p p e r s , 1963) than s e e d l i n g assay, Cheo a t t r i b u t e d to a c c i d e n t a l  found  less  sensitive  the 2 t o 5% seed t r a n s m i s s i o n  i n c l u s i o n o f " n o t - w e l l matured" seed  in the mature seed  lot. The apparent d i s a p p e a r a n c e o f SBMV in the embryos o f mature seed was  a l s o r e p o r t e d by Crowley  (1959)  (1959)-  It i s s i g n i f i c a n t t h a t -  c o u l d not recover SBMV from the embryos o f two soybean  Crowley varieties  at any stage o f development, but the v i r u s was recovered from seed c o a t s and  perisperm t i s s u e s o f more than  was  no mention McDonald  o f attempts  t o recover SBMV from p l a n t e d soybean  (1971) and McDonald and Hamilton  i n f e c t i o u s v i r u s from e i t h e r been decontaminated  (1972)  seed.  failed--.to recover  immature or mature bean embryos that had  by running tap water wash f o r 30 minutes,although  i n f e c t i o u s v i r u s could be r e a d i l y coats treated  100 seeds t e s t e d ; however, t h e r e  like-wise.  recovered from c o r r e s p o n d i n g seed  T h i s evidence l e d these workers  (McDonald  35  and Hamilton,  1972)  to p o s t u l a t e - t h a t  the mechanism o f SBMV seed  trans-  m i s s i o n , where t h i s has been r e p o r t e d (Zaumeyer and H a r t e r , 1 9 4 3 ; Cheo, 1955),  was due t o s u r f a c e c o n t a m i n a t i o n o f the embryo at g e r m i n a t i o n  with v i r u s from the seed TMV in tomato seed which  indicated  attributed  coat  in a s i m i l a r manner t o that  (Taylor et a l , 1 9 6 1 ) .  Thus the r e s u l t s o f Cheo  that SBMV c o u l d be recovered from  (McDonald,  of the e a r l i e r workers  1971;  McDonald and Hamilton,  to decontaminate  has been found to g i v e r e l i a b l e  results  1972;  in the seed floral  Lamptey and Hamilton,  l o t was 1 . 3 % .  (1955)  immature embryos was 1972)  to the f a i l u r e  the embryos, a procedure  that  (Taylor e t a l , 1 9 6 1 ) .  The Ghana s t r a i n o f SBMV was t r a n s m i t t e d (Lamptey,  reported f o r  1974);  through seed o f cowpea the r a t e o f seed t r a n s m i s s i o n  The Ghana s t r a i n could be recovered from a l l  p a r t s , some embryos and seed c o a t s o f immature and "maturing" .  cowpea seeds which p l u s a 3 0 minute  had been p r e v i o u s l y decontaminated  running t a p water wash.  However t h e v i r u s could not  be recovered from embryos o f d r y mature seed even transmitted  through p l a n t e d seed.  with detergent  though  i t was  T h i s agrees w i t h the r e s u l t s o f Cheo  ( 1 9 5 5 ) who c o u l d not recover t h e bean s t r a i n o f SBMV from embryos o f mature seed.  Although the 1.3% seed t r a n s m i s s i o n of the Ghana  through cowpea seed was a t t r i b u t e d c o n t a m i n a t i o n o f t h e emerging  (Lamptey and Hami1 ton, 1974)  s e e d l i n g s by v i r u s from the seed  strain to possible coat,no  f u r t h e r e x p l a n a t i o n was g i v e n f o r the a c t i v i t y o f the v i r u s recovered, from e x t r a c t s o f decontaminated  immature embryos.  If seed t r a n s m i s s i o n  36  of t h i s v i r u s through p l a n t e d seeH was :due to c o n t a m i n a t i o n of the seedl i n g s w i t h v i r u s from the seed c o a t , then decontamination abolished  the  infectivity  from a l l embryos.  During the course of these s t u d i e s a manuscript, p u b l i c a t i o n p r e s e n t i n g evidence f o r embryonic  1977)  laboratory.  e x t r a c t s of decontaminated  were i n f e c t i v e . taminated  submitted f o r  t r a n s m i s s i o n of the bean  (Uyemoto and Grogan, 1 9 7 7 ) ,  s t r a i n of SBMV in bean seed made a v a i l a b l e to t h i s  should have  The v i r u s was  In t h i s study  was  kindly  (Uyemoto and  Grogan,  immature embryos of t h r e e bean v a r i e t i e s  t r a n s m i t t e d at a higher r a t e when  immature embryos were p l a n t e d than  in s i m i l a r l y  decon-  t r e a t e d mature  embryos. The e q u i v o c a l evidence f o r embryonic Other  f a c t o r s may  have been r e s p o n s i b l e f o r the d i s c r e p a n c i e s reported  in the recent s t u d i e s Lamptey, 1 9 7 2 ; since It  t r a n s m i s s i o n of SBMV i s c o n f u s i n g .  (McDonald, 1 9 7 1 ;  Lamptey and  Hamilton,  McDonald and 1974;  Uyemoto and  in a l l of these cases decontamination  i s not c l e a r  i f the d i f f e r e n c e s  of buffer:embryo many more seeds  Hamilton, Grogan,  likely  1977)  procedures were f o l l o w e d .  in the r e s u l t s are due t o a high  ratio  in the e x t r a c t i o n of v i r u s or whether some workers used than o t h e r s .  Indeed  absence  of seed t r a n s m i s s i o n in a  few seeds does not mean i t does not occur at a l l (Shepherd, is a l s o  1972;  1972).  that the n e g a t i v e r e s u l t s o f some workers c o u l d be  to the presence of  inhibitors  It due  in mature embryo e x t r a c t s which c o u l d have  i n t e r f e r e d w i t h the i n f e c t i v i t y of the v i r u s which may  have been  in the  37  embryo.  Such i n h i b i t o r s have been r e p o r t e d (Cheo,  although  i t was thought  (Cheo,  1955) unlikely  1 9 5 5 ; Crowley,  they would have been  p o n s i b l e f o r the l a c k o f t r a n s m i s s i o n o f SBMV In mature seed the  i n h i b i t o r y substance was present in embryos o f both  mature bean seed.  Perhaps  immature and  assay o f embryo e x t r a c t s on i n d i c a t o r p l a n t s has been found  As f a r as CP-SBMV its  seed-borne  These workers derived this to  report  is available  and F u l t o n ,  that CP-SBMV could  1962).  in seedlings  However, s i n c e  v e r y l i t t l e work has been done i n the seed.  However, the l i t t l e  i n d i c a t e s that CP-SBMV can be c o n s i s t e n t l y  (Gay, 1 9 7 3 ; Kuhn and Dawson, 1973).  i n f e c t i o n has not been  Although the source o f  thoroughly i n v e s t i g a t e d , Gay ( 1 9 7 3 )  be recovered from a l l f l o r a l  p l a n t s o f each o f two cowpea v a r i e t i e s .  attempt  (Shepherd  on i n f e c t e d cowpea p l a n t s .  study the d i s t r i b u t i o n o f the v i r u s  seed-borne  ted  its first  r e p o r t o f CP-SBMV being seed-borne  i n f o r m a t i o n that  t o be l e s s  t h e r e seems to be c o n s i s t e n c y in  r e p o r t e d 3 t o k% seed t r a n s m i s s i o n o f CP-SBMV  from seed produced  direct  (Schippers, 1 9 6 3 ) .  i s concerned  nature s i n c e  res-  because  because o f i n h i b i t o r s o f i n f e c t i o n ,  s e n s i t i v e than s e e d l i n g assay  1955),  demonstrated  and seed p a r t s o f i n f e c -  A p p a r e n t l y Gay d i d not  recovery."-• o f CP-SBMV from dry mature seed,and  although a s c e p t i c  methods were employed t o s e p a r a t e seed p a r t s , p o s t - s e p a r a t i o n decontam i n a t i o n procedure  i s not mentioned,  However, CP-SBMV was t r a n s m i t t e d of  thus making h i s r e s u l t s  meaningless.  to an average o f 2 3 and 2 6 % when seeds  the two v a r i e t i e s were p l a n t e d (Gay, 1 9 7 3 ) .  In mixed v i r u s  infection  38  s t u d i e s CP-SBMV was t r a n s m i t t e d of  12.5% o f s e e d l i n g s d e r i v e d  through p l a n t e d  seed t o an average  from seeds from p l a n t s s i n g l y i n f e c t e d  by CP-SBMV and to 20% o f s e e d l i n g s d e r i v e d from seeds from p l a n t s doubly The  i n f e c t e d by CP-SBMV. and CCMV few s t u d i e s  (Gay, 1973;  Kuhn and Dawson, 1973;  Hamilton,  1974)  that t h i s  s t r a i n o f the v i r u s can be t r a n s m i t t e d  on seed t r a n s m i s s i o n  seed of cowpea. frequency  1973)•  (Kuhn and Dawson,  Lamptey and  o f CP-SBMV through seeds i n d i c a t e d through p l a n t e d  A l s o t h e r e seems to be no c o n f l i c t with  o f seed t r a n s m i s s i o n  (Lamptey and Hamilton, 1974)  r e s p e c t to the  o f CP-SBMV, except o f c o u r s e ,  r e s u l t s by s u r f a c e decontamination, no v i r u s was recovered  d i c a t o r host It  even though the v i r u s was t r a n s m i t t e d  i s not c l e a r i f the n e g a t i v e  embryos of d r y seed of  infection,  present  (Cheo, 1955;  from embryos  indexed  on an  inseed.  with d i r e c t assay o f a r e due t o i n h i b i t o r s  Crowley, 1955) (Cheo,  to be  1955).  i n a c t i v a t i o n was not r e s p o n s i b l e s i n c e the Ghana s t r a i n o f  SBMV was t r a n s m i t t e d  to 1.3% o f the s e e d l i n g s  The p o s s i b i l i t y o f in v i v o  the seed has been mission  by o t h e r s  spurious  through planted  in the seed e x t r a c t s , or due t o i n a c t i v a t i o n  Apparently  seed.  r e s u l t s obtained  (Lamptey and Hamilton, 1974)  reported  in s t u d i e s  where care was taken t o avoid  of dry mature seed when t h e i r e x t r a c t s were d i r e c t l y  mature  implicated  r a i s e d from p l a n t e d  i n h i b i t o r s t o manual  (Powell  mature  i n o c u l a t i o n in  and S c h l e g e l , 1970)  in the t r a n s -  o f squash mosaic v i r u s in seed o f cantaloupe.  In c o n c l u s i o n , a p o i n t that needs t o be emphasized  i s that a number  39  of v i r u s e s are present  in both t h e s e e d  v i r u s e s are not seed t r a n s m i t t e d . that seed  It has been s t r e s s e d  t r a n s m i s s i b i 1 i t y of v i r u s e s  and does not r e s u l t  from  coat and endosperm, but  in the seed c o a t s or endosperm of some seeds was as f r e q u e n t l y as that  (Shepherd,  i s a p r o p e r t y of embryo  i n f e c t i o n of o t h e r seed p a r t s .  1972)  infection  Indeed  i f virus  t r a n s m i t t e d through  in the embryo t h e r e would be abundant seed  m i s s i o n o f many more v i r u s e s .  these  seed  trans-  ko  MATERIALS AND  I.  The  METHODS  viruses.  The cowpea s t r a i n s of southern bean mosaic v i r u s tobacco mosaic v i r u s obtained  (CP-TMV) used e x c l u s i v e l y  Canada  The v i r u s e s  (henceforth  c a l l e d V.R.S. = Vancouver  had been maintained  in l y p h o l i z e d t i s s u e .  the same as that used by Weintraub  (Dr.  from Dr. R.J. Shepherd,  J.H. Tremaine,  as that  in these s t u d i e s were  from Dr. R.I. Hamilton, A g r i c u l t u r e Canada Research  Vancouver,  obtained  (CP-SBMV) and of  and R a g e t l i  Station,  Research  Station).  The CP-SBMV i s  (1970) o r i g i n a l l y  U n i v e r s i t y o f C a l i f o r n i a at Davis  personal communication).  The CP-TMV i s the same  used by M o r r i s (1974) brought from the U n i v e r s i t y o f C a l i f o r n i a  at R i v e r s i d e  (Dr. Hamilton, personal communication).  were increased t i s s u e was  separately  c a l l e d phosphate  (diamatoceous e a r t h ) . ascertained  viruses  in C a l i f o r n i a blackeye cowpea.  t r i t u r a t e d in 0.01  (henceforth  The  M potassium phosphate buffer)  P u r i t y , that  Lypholized  b u f f e r pH  7.1  in the presence of \% c e l i t e i s , freedom  from contamination,, was  by e x t r a c t i n g v i r u s from s y s t e m i c a l l y  infected  trifoliate  leaves of C a l i f o r n i a b l a c k e y e c o w p e a , d i l u t i n g i t to 1/10,000 (w/v) buffer containing hosts Single and  ] % c e l i t e and  i n o c u l a t i n g the d i l u t i o n  (Georgia 21 cowpea f o r CP-SBMV and N i c o t i a n a local  l e s i o n s were then passaged  then to C a l i f o r n i a blackeye cowpea.  serially  to l o c a l  glutinosa  in lesion  f o r CP-TMV).  through 5 t r a n s f e r s  As a f u r t h e r check, sap from  p l a n t s s i n g l y i n f e c t e d by e i t h e r CP-SBMV or CP-TMV was back-inocu1ated to the l o c a l preparation  lesion  hosts.  containing  Absence o f l o c a l  the other  l e s i o n s o f one v i r u s in a  v i r u s was taken as evidence o f f r e e -  dom from contamination o f one v i r u s by the o t h e r .  T h i s check was r o u t i -  nely done s i n c e working with mixed v i r u s e s provided  an o p p o r t u n i t y f o r  cross-contarnination. Later ded  in these s t u d i e s the C o r n e l l  by Dr. M i l t o n Z a i t l i n ,  i s o l a t e o f CP-TMV, k i n d l y p r o v i -  C o r n e l l U n i v e r s i t y , Ithaca, New York was used  in some comparison s t u d i e s on French bean I I Hosts and t h e i r The  hosts  used  (Phaseolus v u l g a r i s L . ) .  propagation. in t h i s study were C a l i f o r n i a  ungu i cu1ata L. Walp, cv. E a r l y Ramshorn), three varieties  (V. u n g u i c u l a t a ,  French bean  Botswana l o c a l  cvs. Botswana blackeye,  CP-SBMV h y p e r s e n s i t i v e cowpea (V_. u n g u i c u l a t a )  blackeye cowpea  used e x t e n s i v e l y  V26-Bots and V 4 5 B o t s ) , _  v a r i e t y Georgia  The C a l i f o r n i a  mixed  21  (GA 2 1 ) ,  blackeye cowpea was  in the s t u d i e s f o r v i r u s m u l t i p l i c a t i o n and seed  m i s s i o n . It was produced by Bunton Seed Co. L o u i s v i l l e ,  only  cowpea  (Phaseolus v u l g a r i s L. c v s . P i n t o and B o u n t i f u l ) , N i cot iana  g 1 ut i nosa and N_. tabacum cv. X a n t h i .  three  (Vigna  Botswana cowpea v a r i e t i e s in seed t r a n s m i s s i o n . infection  Department o f A g r i c u 1 t r u r a 1  The  (or l i n e s ) were used as a comparison  Only V45~Bots was used  s t u d i e s , f o r reasons given  v a r i e t i e s were k i n d l y sent  Kentucky.  trans-  later.  in v i r u s s y n t h e s i s in The seeds o f the Botswana  by Mr. N. M a h l a t j i e , Seed M u l t i p l i c a t i o n Research, Gaborone, Botswana.  Georgia  Unit, 21  42  cowpea was k i n d l y p r o v i d e d by Drs. B.B. B r a n t l e y and CW. U n i v e r s i t y of G e o r g i a , Athens, G e o r g i a .  Kuhn,  P i n t o seed was purchased  Canada Safeway S t o r e s and was r o u t i n e l y used by the greenhouse at  V.R.S.  . Bountiful  bean seed was produced  by Dominion  from  staff  Seed House,  Georgetown, O n t a r i o . N_. g l u t i n o s a and Xanthi were from seeds  produced  and used r o u t i n e l y at V.R.S. Cowpea p l a n t s f o r p r o p a g a t i o n o f v i r u s f o r p u r i f i c a t i o n t r a n s m i s s i o n were r a i s e d California raised  soil.  Cowpea  plants,  blackeye.and V 4 5 ~ B o t s , for- v i r u s n u c l e o p r o t e i n s t u d i e s were  i n the same s t e a m - s t e r i l i z e d  project; and  in a s t e a m - s t e r i l i z e d  and seed  however, t h i s s o i l  soil  d u r i n g the e a r l y p a r t o f t h i s  was o f t e n not s u i t a b l e f o r cowpea  growth  t h e r e f o r e most o f the experiments were performed w i t h p l a n t s  in Hoagland's by Johnson sown  s o l u t i o n medium  et a l  (1957)-  (Hoagland and Arnon,  When Hoagland's  1950) as m o d i f i e d  s o l u t i o n was used, seed was  in p l a s t i c f l a t s c o n t a i n i n g a seeding mix, made up o f : one p a r t  f i n e sand, one p a r t peat moss and one p a r t v e r m i c u l i t e . a f t e r seeding the s e e d l i n g s were t r a n s p l a n t e d sand  raised  in 10-cm pots. The pots were put i n small  S i x to 7 days  to s t e a m - s t e r i l i z e d bowls, f i l l e d w i t h  river Hoagland's  s o l u t i o n and the p l a n t s were maintained hydroponica11y f o r the d u r a t i o n of  the experiment  in a c o n t r o l l e d  environment  growth  room, p r o v i d e d w i t h  two banks o f S y l v a n i a f l o u r e s c e n t tubes t o g i v e a l i g h t  7,000  intensity of  lux a t the primary l e a f s u r f a c e and a temperature o f 24 t o 27°C,  18 hour p h o t o p e r i o d . in the greenhouse  A l l bean p l a n t s and i n d i c a t o r hosts r a i s e d  were maintained a t  a temperature o f  20 to  43  25°C and 14 hour" photoperiod.  A l l greenhouse compartments were  insect-  proof and were provided with heaters and c o o l e r s c o n t r o l l e d t h e r m o s t a t i cally.  P i n t o and B o u n t i f u l bean and GA 21 cowpea p l a n t s were used  8 to 10 days a f t e r sowing and N_. g 1 ut i nosa and Xanthi were used when 6 to 7 weeks o l d . Inoculated cowpea p l a n t s f o r v i r u s p u r i f i c a t i o n , seed transmissionand at  i n t e r a c t i o n of v i r u s e s , when r a i s e d a temperature  in the greenhouse, were maintained  of 24 to 32°C and supplemented with f l u o r e s c e n t l i g h t  to p r o v i d e a photoperiod of 18 hours. i n f e c t i o n s were r a i s e d  When cowpea p l a n t s f o r mixed v i r u s  in Hoagland's s o l u t i o n  the p l a n t s were r a i s e d in  the growth room as d e s c r i b e d above. Indicator hosts, a f t e r maintained  i n o c u l a t i o n , were r a i s e d  in a greenhouse  at 22 t o 25°C or in a growth chamber w i t h a temperature  22 to 23°C, photoperiod of 16 hours and 13,000 lux at the primary  of leaf  surface. I I I . I n o c u l a t i o n of hosts f o r v i r u s p u r i f i c a t i o n , d e t e r m i n a t i o n of v i r u s c o n c e n t r a t i o n and a n t i s e r a p r e p a r a t i o n . A.  Propagation  and i n o c u l a t i o n of h o s t s .  The C a l i f o r n i a blackeye cowpea was used e x c l u s i v e l y f o r maintanence and  propagation o f v i r u s e s f o r p u r i f i c a t i o n .  3 p l a n t s per pot. infected  The primary  Seed was sown in 10-cm p o t s ,  leaves were i n o c u l a t e d with crude sap from  source p l a n t s 8 to 10 days a f t e r sowing.  by g r i n d i n g i n f e c t e d  Crude sap was obtained  leaves in a mortar w i t h a p e s t l e  in 0.01 M potassium  kk  phosphate b u f f e r pH 7.1 plus  \% c e l i t e  (unless o t h e r w i s e s t a t e d a l l ino-  c u l a t i o n s throughout the experiments reported The  primary leaves were i n o c u l a t e d  inoculum. with  using  The i n o c u l a t e d  house at temperatures o f 2k t o 32°C. so that B.  ]% c e l i t e ) .  gauze soaked  i n the  leaves were r i n s e d  p l a n t s were maintained  in a green-  E x t r a care was taken to keep  plants  f o r t u i t o u s contamination d i d not o c c u r .  P u r i f i c a t i o n of v i r u s e s .  Fifteen  to 20 days a f t e r i n o c u l a t i o n t r i f o l i a t e  from each l o t o f p l a n t s stored  autoclaved  included  Immediately a f t e r i n o c u l a t i o n the i n o c u l a t e d  running tap water.  apart  herein  at -20°C u n t i l  leaves were  i n o c u l a t e d with e i t h e r v i r u s .  needed f o r p u r i f i c a t i o n .  harvested  The leaves were  Harvesting  and p u r i f i c a -  t i o n f o r the two v i r u s e s were never done on the same day in order  t o reduce  the o p p o r t u n i t y  procedure  of contamination.  At the end o f the p u r i f i c a t i o n  f o r each v i r u s the p u r i t y of each v i r u s p r e p a r a t i o n inoculating  i t to l o c a l  suspension was CP-SBMV  was checked by back-  l e s i o n i n d i c a t o r hosts e.g. i f p u r i f i e d i t was i n o c u l a t e d  t o N_. g l u t ! n o s a  presence o f CP-TMV and s i m i l a r l y a CP-TMV p u r i f i e d  virus  to check f o r the  suspension was  cross-checked on GA 21 f o r the presence o f CP-SBMV.  since  1.  P u r i f i c a t i o n o f CP-SBMV.  The  procedure f o r p u r i f i c a t i o n of CP-SBMV was that o f Tremaine e t j a l  i t appeared (i)  to be the most s u i t a b l e .  T i s s u e was crushed w h i l e f r o z e n diethyldithiocarbamate;  (ii)  (1976)  and s p r i n k l e d with D! ECA (sodium ;  0.004 g/g o f t i s s u e )  The t i s s u e was homogenized  in a Waring blender  in 0.1 M sodium  hS  acetate  (NaAc) b u f f e r pH 5-0 c o n t a i n i n g 0.2%  (v/v) mercaptoethanol  (1 g t i s s u e / 2 ml of b u f f e r ) , (iii)  The homogenate was  squeezed  through h l a y e r s  and the pH o f the f i l t r a t e was acetic (iv) (v)  a d j u s t e d to pH 5 w i t h g l a c i a l  acid.  The f i l t r a t e was  allowed to stand o v e r n i g h t at  T h i s f i l t r a t e was in a S o r v a l l  (vi)  of c h e e s e c l o t h  then c e n t r i f u g e d  SS-34 r o t o r .  The supernatant was  foi*  h°C.  20 minutes  The supernatant was  10,000  at  saved,  made 0. 1 M w i t h r e s p e c t to NaCl and  w i t h respect to p o l y e t h y l e n e g l y c o l  rpm  (PEG), M.W.  6,000  10%  and  s t i r r e d o v e r n i g h t to p r e c i p i t a t e the v i r u s , (vii)  The p r e c i p i t a t e was (low  (viii)  h°C  for (ix)  (x)  a f t e r which  rpm  i t was  clarified  by low speed  buffer  centrifugation  10 minutes.  hours at  28,000  The p e l l e t was b u f f e r pH 7-0 suspension  (xi)  10,000  resuspended o v e r n i g h t in 0.1 M NaAc pH 5-0  The supernatant was  3  by c e n t r i f u g a t i o n a t  speed c e n t r i f u g a t i o n ) f o r 20 minutes,  The p e l l e t was at  pelleted  centrifuged  in a Spinco no. 30 r o t o r f o r  rpm.  resuspended o v e r n i g h t in 0.1 M sodium ( t h i s b u f f e r allowed the v i r u s p e l l e t  phosphate to go  into  readily),  The v i r u s suspension was  clarified  by low speed  and the supernatant g i v e n another high speed as in ( i x ) above.  centrifugation  centrifugation  46  (xii)  The f i n a l  p e l l e t was e x t r a c t e d  overnight  phosphate b u f f e r pH 7-1, given v i r u s suspension was s t o r e d  in 0.01 M potassium  low speed c e n t r i f u g a t i o n and the  in t h i s b u f f e r c o n t a i n i n g  or 2 g r a i n s o f c h l o r o - b u t a n o l  (as a n t i m i c r o b i a l  ca one  preservative)  at 4°C. 2.  P u r i f i c a t i o n o f CP-TMV. (i)  Frozen t i s s u e was p u l v e r i z e d Waring blender containing  i n 0.1M potassium phosphate b u f f e r pH 7-5  0.1% (v/v) mercaptoethanol  nediaminetetraacetic (ii)  by hand and homogenized w i t h a  acid)  and 5-0 mM EDTA  (ethyle-  (1 g t i s s u e / 2 ml o f b u f f e r ) ,  The homogenate was s t r a i n e d  through 4 l a y e r s o f c h e e s e c l o t h or  2 l a y e r s o f ,Wiracloth. (iii)  The f i l t r a t e was c e n t r i f u g e d Sorvall  (iv)  t o PEG and s t i r r e d  10,000 rpm in a Sorval  The p e l l e t was e x t r a c t e d  to NaCl and 4% w i t h  f o r 4 hours o r o v e r n i g h t  The e x t r a c t was then c l a r i f i e d at  (vi)  SS-34 r o t o r and the supernatant was saved,  The supernatant was made 2% w i t h respect pect  (v)  f o r 30 minutes at 10,000 rpm in a  res-  a t 4°C.  by c e n t r i f u g i n g f o r 30 minutes  SS-34 r o t o r , in 0.01 M potassium phosphate  buffer  pH 7.1 overn i ght. (vii)  The v i r u s suspension was c l a r i f i e d and  (viii)  supernatant  by low speed  centrifugation  saved,  The supernatant was then c e n t r i f u g e d  i n a Spinco No. 30 r o t o r f o r  hi  3 (ix)  hours a t  28,000  The p e l l e t was taken up in the potassium phosphate b u f f e r ( i n 1/20 o f o r i g i n a l  (x)  rpm.  sap volume) o v e r n i g h t ,  The v i r u s suspension and  was c l a r i f i e d  by low speed c e n t r i f u g a t i o n  when a very pure p r e p a r a t i o n was not necessary  c a t i o n procedure was stopped a t t h i s stage.  the p u r i f i -  However when a very  c l e a n and pure p r e p a r a t i o n was r e q u i r e d such as that used f o r p r e p a r a t i o n o f antiserum by sucrose (xi)  cushioning  the v i r u s suspension  was f u r t h e r  as f o l l o w s :  Four t o 6 ml o f a v i r u s p r e p a r a t i o n  ( 1 0 mg/ml) were f l o a t e d on  top o f h to 6 ml o f kS% r i b o n u c l e a s e - f r e e sucrose  in Beckman  c e l l u l o s e n i t r a t e SW 27 tubes which were then f i l l e d oil (xii)  The v i r u s was p e l l e t e d through sucrose  mineral  5 hours  at  25,000  by c e n t r i f u g i n g in a Beckman  rpm.-  At the end o f the high speed c e n t r i f u g a t i o n the aqueous phase and  mineral  overnight (xiv)  with  to prevent c o l l a p s i n g ,  SW: 27 r o t o r f o r (xiii)  cleaned  o i l were d i s c a r d e d  and the c l e a n p e l l e t was e x t r a c t e d  in 0.01 M potassium phosphate b u f f e r pH 7.1.  The v i r u s suspension  was d i a l y s e d a g a i n s t  d i s t i l l e d water to remove sucrose  s e v e r a l changes o f  and f i n a l l y  a g a i n s t 0.01 M  potassium phosphate pH 7.1 b u f f e r . (xv)  The v i r u s p r e p a r a t i o n was s t o r e d at h°C preserved two g r a i n s o f c h l o r o - b u t a n o l .  All  c e n t r i f u g a t i o n procedures were performed a t 5°C.  with one or  48  C.  Determination  of v i r u s c o n c e n t r a t i o n .  The  c o n c e n t r a t i o n s of v i r u s in p u r i f i e d  determined by u l t r a v i o l e t virus  in c l a r i f i e d  spectrophotometry.  e x t r a c t s were e s t i m a t e d ,  g r a d i e n t c e n t r i f u g a t i o n , by comparing f i l e s with 1.  p r e p a r a t i o n s were The c o n c e n t r a t i o n s o f  a f t e r sucrose  density  the area under absorbance pro-  the area o f known v i r u s c o n c e n t r a t i o n s . Determination  of p u r i f i e d  t r a t i o n o f each p u r i f i e d ultraviolet  virus concentration.  v i r u s was determined  in  The concen-  a Beckman Model DU  spectrophotometer at an absorbance o f 0 260" U  ^  e  c  o  n  c  e  n  ~  t r a t i o n o f CP-SBMV was determined by m u l t i p l y i n g the OD^g'read i ng by the d i l u t i o n  f a c t o r and then d i v i d i n g  ( E £ ) of 5-85 (E £n 2  Q  f°  =  2  r  by the e x t i n c t i o n c o e f f i c i e n t  c o n c e n t r a t i o n o f 1 mg/ml, l i g h t  path o f 1 cm) (Shepherd, 1971); the c o n c e n t r a t i o n o f CP-TMV was~simi1ar1y determined by using an assumed E^^Q  o f 3-2 ( E ^ g = 3.2 f o r c o n c e n t r a t i o n  o f 1 mg/ml, l i g h t path o f 1 cm) (Kassanis and Varma, 1975). The  r e l a t i v e p u r i t y of each v i r u s p r e p a r a t i o n was determined by  comparing the 0 2 6 r / ^ 2 8 0 D  D  r a t  ' ° °f p u r i f i e d  v i r u s with published  values  f o r each v i r u s (Table 1) 2.  Estimation of concentration of p u r i f i e d density gradient c e n t r i f u g a t i o n .  v i r u s by sucrose  Since p a r t i a l l y  e x t r a c t s were used e x t e n s i v e l y in t h i s t h e s i s t o e s t i m a t e t i o n o f e i t h e r CP-SBMV o r CP-TMV a r a p i d method f o r doing d i f f e r appreciably  in both s i n g l e and double  so was d e s i r a b l e .  in sedimentation  clarified  the c o n c e n t r a infections,  Because CP-SBMV and CP-TMV  p r o p e r t i e s ( T a b l e ! I ) , the technique  of sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n (Brakke,  1963, 1967) would  kS  readily  separate  mixture. tions  t h e v i r u s e s and g i v e t h e i r  The s t a n d a r d  in clarified  curve,for  concentrations  rapid determination  e x t r a c t s , was p r e p a r e d  of virus  v i r u s , obtained i n 0.02  series of dilutions  pH  6 . 5 c o n t a i n i n g 0.1%  CP-TMV; t h e d e t e r g e n t CP-SBMV was d i l u t e d and  Igepon T - 7 3  g r a d i e n t m a k e r , Model  603,  Dickson  Scientific  0.1  0.5,  containing  0.02%  were always  0.25  mechanically  w i t h kO ml o f k0% s u c r o s e  The s u c r o s e  Gradient  mg/ml  a n d 0.125 with a  s o a s t o pro-^  t o kO mg/ml, f r o m t o p ;  s o l u t i o n was made by d i s s o l -  i n 0.01  obtained M  from  potassium  f o r CP-SBMV g r a d i e n t s a n d i n t h e same b u f f e r  Igepon T - 7 3 f o r CP-TMV. left  and 0.05  I n d u s t r i e s , I n c . , by m i x i n g  Co., Canada L t d , M i s s i s s a u g a ,  p h o s p h a t e b u f f e r pH 7-1  buffer  o f CP-TMV.  Schwarz/Mann u t r a p u r e r i b o n u c l e a s e - f r e e s u c r o s e ,  Becton,  way  0.25,  g r a d i e n t c o n c e n t r a t i o n o f 10 mg/ml  t o b o t t o m o f an SW k\ t u b e . ving  aggregation  columns were prepared  kO ml o f 10% (w/v) s u c r o s e duce a l i n e a r  t o prevent  t o c o n c e n t r a t i o n s o f 1.0,  Sucrose gradient  M Tris-HCl  (sodium-methy1-N-oleoy1taurate) f o r  t o concentrations o f 0.5,  CP-TMV was d i l u t e d  mg/ml.  (trishydroxymethy1-aminomethane-  f o r CP-SBMV and i n 0.02  was u s e d  virus.  a s o u t l i n e d a b o v e , was made i n t o a  M Tris-HCl  b u f f e r pH 6 . 5  hydrochloride)  concentra-  from t h e sedimentation  c h a r a c t e r i s t i c s o f known c o n c e n t r a t i o n s o f e a c h p u r i f i e d Purified  in a  overnight  Gradient  c o l u m n s made i n t h i s  before use.  c o l u m n s w e r e l a y e r e d w i t h 0.2 ml o f t h e a p p r o p r i a t e  c o n c e n t r a t i o n and c e n t r i f u g e d a t 5°C f o r 105 m i n u t e s  virus  i n an SW k\ r o t o r  50  at 39,000 rpm. in an  A f t e r c e n t r i f u g a t i o n the g r a d i e n t columns were analysed  ISCO Model 640  Model UA-5  d e n s i t y g r a d i e n t f r a c t i o n a t o r coupled  ultraviolet  absorbance monitor, and  g r a d i e n t columns were scanned at 254 was  recorded  calculated  as a peak (curve) on  by counting  small  nm  and  chart recorder.  ISCO  The  sucrose  the absorbance of each v i r u s  the c h a r t .  squares  with an  The  area under the peak  was  (each square i s e q u i v a l e n t to appro-  2 ximately can  3 mm  ) under the peak.  be r e l a t e d  curve  The  number of squares under each  to a known v i r u s c o n c e n t r a t i o n .  between area under the curve and  plotted.  T h i s standard  curve was  In t h i s way,  a  standard  each v i r u s c o n c e n t r a t i o n  used  in a l l experiments to  curve  was  estimate  the c o n c e n t r a t i o n of each v i r u s in p a r t i a l l y c l a r i f i e d e x t r a c t s . D.  P r e p a r a t i o n of CP-SBMV and  A n t i s e r a were produced by i n j e c t i o n with  CP-TMV a n t i s e r a .  injecting  two  e i t h e r v i r u s a n t i g e n , each of the r a b b i t s was  tagged as no.  1 and  each r a b b i t .  Each of the v i r u s e s p u r i f i e d  B.  1 and  potassium before  2) was  no.  2.  phosphate, 0.14,  r a b b i t (no.  mixed with an equal  emulsion  1) was  then  i n t o each hind was  repeated  (preimmune serum) was  bled from  as d e s c r i b e d above ( S e c t i o n I I I .  NaCl pH 7-0  (phosphate b u f f e r e d - s a 1 i n e ,  i n j e c t e d w i t h CP-SBMV o n l y and One  ml  M  PBS)  the mixture  injected  thigh.  the other r a b b i t  (2 mg/ml) of each d i a l y s e d v i r u s  amount of Freund's complete adjuvant  syringe until was  serum  appropriately  i n t o the r a b b i t s .  (no. 2) with CP-TMV o n l y .  in a 2-ml  Normal  Before  d i a l y s e d f o r 24 hours a g a i n s t s e v e r a l changes of 0.01  injection  One  young white r a b b i t s .  had  thickened.  and e m u l s i f i e d  One-half ml of  i n t r a m u s c u l a r l y with a 22 gauge (1  I n j e c t i o n with  f o r each r a b b i t two  inch)  the same v i r u s c o n c e n t r a t i o n  more times  at weekly  was  intervals.  the needle  51  Three weeks a f t e r the l a s t each ear at monthly  i n j e c t i o n each r a b b i t was bled a l t e r n a t e l y  intervals until  enough serum was c o l l e c t e d .  ted blood a t each b l e e d i n g was allowed  The serum was decanted and  c e n t r i f u g e d a t 5,000 rpm f o r 10 minutes.  antiserum in P e t r i  The supernatant  dishes  by the m i c r o p r e c i p i t i n method  (van S l o g t e r e n , 1955) with ant i body^-ant i gen mixtures co-  vered with mineral o i l . The h i g h e s t antiserum each homologous v i r u s  a g a i n s t CP-TMV was 1/1024.  IV.  Mixed A.  dilution  that reacted with  (0.1 mg/ml) a n t i g e n was taken as the t i t r e o f the  The t i t r e o f the antiserum  of the v i r u s  was mixed  volume o f g l y c e r i n e and s t o r e d a t -20°C. The t i t r e o f the  f o r each v i r u s was estimated  antiserum.  The c o l l e c -  t o stand at 37°C f o r one hour  and o v e r n i g h t at 4°C to a l l o w c l o t t i n g .  with an equal  from  a g a i n s t CP-SBMV was 1/512 and that  N e i t h e r normal serum nor PBS reacted with any  antigens.  i n f e c t i o n s o f CP-SBMV and CP-TMV i n cowpea.  I n t e r a c t i o n s in C a l i f o r n i a blackeye and Botswana l o c a l  variety,  V45~Bots. cowpeas. 1.  Inocu1 at ions.  California  blackeye cowpea or V45~Bots. cowpea  s e e d l i n g s were s e l e c t e d f o r u n i f o r m i t y 8 to 10 days a f t e r sowing. were e i t h e r  i n o c u l a t e d at that time or covered w i t h black p l a s t i c f o r 24  hours at 28 to 30°C t o enhance s u s c e p t i b i l i t y experiments 0.05 mg/ml used  (final  in s i n g l e and double  to v i r u s  infection.  c o n c e n t r a t i o n ) o f each p u r i f i e d  infections.  A l l i n o c u l a contained  and were a p p l i e d with c o t t o n swabs ( Q - t i p s ) . kept  Plants  In a l l  v i r u s was \% c e l i t e  The i n o c u l a t e d p l a n t s were  in the greenhouse at 24 to 32°C or in the growth room a t 24 to 27°C.  52  (a)  Simultaneous  inoculation.  When simultaneous  i n o c u l a t i o n s were  made one group o f s e e d l i n g s was mock-inocu1ated with phosphate b u f f e r ( c o n t r o l ) , another group was i n o c u l a t e d CP-TMV s e p a r a t e l y Only the primary after  s i n g l y with e i t h e r CP-SBMV or  and the l a s t group with a mixture o f CP-SBMV and CP-TMV. leaves were i n o c u l a t e d and they were r i n s e d  immediately  i n o c u l a t i o n with running tap water.  (b)  Sequential  inoculations.  When s e q u e n t i a l  made p l a n t s were not p r e v i o u s l y p r e - c o n d i t i o n e d were s e l e c t e d  f o r uniformity  and Inoculated  i n o c u l a t i o n s were  in the dark.  as f o l l o w s :  was mock-inocu1ated w i t h phosphate b u f f e r o n l y ;  Plants  the f i r s t l o t  the second one was  singly  i n o c u l a t e d w i t h e i t h e r CP-SBMV or CP-TMV; the t h i r d  doubly  i n o c u l a t e d with a mixture o f the two v i r u s e s , and the f o u r t h l o t ,  which was to be sequentia 11y inocu1ated, was i n o c u l a t e d e i t h e r CP-SBMV o r CP-TMV.  A l l the i n o c u l a t e d  l o t was  s i n g l y with  leaves were r i n s e d with  tap water and p l a n t s moved to the greenhouse o r t o the growth room. At v a r i o u s lot  intervals after i n i t i a l  were s e q u e n t i a l l y  quentially  inoculated  the p r e v i o u s l y  i n o c u l a t e d w i t h e i t h e r CP-SBMV or CP-TMV. plants  inoculated  after, the p r e v i o u s  running  was s t o r e d  the second v i r u s was a p p l i e d d i r e c t l y on  i n o c u l a t i o n c e l i t e was excluded a l l primary  tap water t o remove r e s i d u a l at-20°C u n t i l  In s e -  l e a f and i f t h i s was done l e s s than 12 hours  When t i s s u e was harvested in  i n o c u l a t i o n p l a n t s o f the f o u r t h  needed.  from the second  leaves were washed  inoculum.  overnight  inoculum. A l l harvested  tissue  53  2.  Assay.  Assay of n u c l e o p r o t e i n was determined  by a n a l y t i c a l  sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n and by e l e c t r o n (a)  Sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n assay.  p r o t e i n content o f CP-SBMV or CP-TMV i n s i n g l y to that  in doubly  infected  infected  microscopy. When nucleot i s s u e was compared  t i s s u e the t i s s u e was p a r t i a l l y c l a r i f i e d as  fol1ows: (i)  Frozen t i s s u e was ground w i t h a mortar and p e s t l e Tris-HCl  b u f f e r pH 6.5  the stage o f l e a f (ii)  (iii)  through k l a y e r s o f c h e e s e c l o t h and to stand o v e r n i g h t a t 4°C.  The f i l t r a t e was mixed w i t h an equal volume o f c h l o r o f o r m and by s t i r r i n g  f o r 3 to 5 minutes  The emulsion was c e n t r i f u g e d 5°C)  (v)  analysis),  immediately or l e f t  emulsified (iv)  (lg/0,5 - 2.0 ml o f b u f f e r depending on  The homogenate was s t r a i n e d clarified  i n 0.2 M  a t 4°C.  (SS-34 r o t o r , 10,000 rpm, 20 minutes,  and the supernatant saved,  The c l e a r supernatant was used  in e s t i m a t i o n o f v i r u s c o n c e n t r a -  tion. T h i s method gave reasonably c l e a n e x t r a c t s w i t h l i t t l e or no aggreg a t i o n of CP-TMV, compared t o o t h e r methods (Hamilton and Dodds, 1970). To determine  the c o n c e n t r a t i o n o f each v i r u s  infections, c l a r i f i e d  sap (0.2 or 0.4 ml) was layered on sucrose d e n s i t y  g r a d i e n t columns and c e n t r i f u g e d The c e n t r i f u g e d  i n s i n g l e and double  as p r e v i o u s l y d e s c r i b e d ( S e c t i o n  sucrose d e n s i t y g r a d i e n t columns were scanned w i t h  III.C.2). ultra-  5k  v i o l e t absorbance monitor a t 25k nm ( S e c t i o n c o n c e n t r a t i o n o f each sample was determined and p l o t t i n g  the number o f squares under  prepared standard curve  (Section  each absorbance  I II.C.2).  E l e c t r o n microscopy assay.  determine  inoculated lation  10 days a f t e r  sowing.  in 5% g1utara1dehyde  base  in c e l l s o f s i n g l y  P l a n t s used  Sampling  was done 12 days a f t e r inocu-  and in another 20 days a f t e r infected  in 0.1 M potassium phosphate  rinsed  in  i n Palade's b u f f e r f o r 60 minutes.  were dehydrated and  then  in graded  in phosphate  b u f f e r and then  s e r i e s o f ethanol  in p r o p y l e n e o x i d e .  infiltrated  they were embedded  T h i n s e c t i o n s were c u t on an LKB Ultratome  in a P h i l i p s  lead c i t r a t e  leaves buffer  post-fixed The p i e c e s  in propylene in Epon 812.  I I I w i t h a diamond  T h i n s e c t i o n s were mounted on copper g r i d s w i t h a c o l l o d i o n super f i l m and s t a i n e d w i t h 5% uranyl  inocu-  (50, 70, 35% and a b s o l u t e )  They were then  oxide; Epon 812 (1:1, v/v) a f t e r which  with Reynolds'  infected  in t h i s study were  pH 7-2 f o r 90 minutes, ] % osmium t e t r o x i d e  lines.  in the same c e l l s o f s i m u l -  Small p i e c e s o f i n o c u l a t e d and s y s t e m i c a l l y  were f i x e d  peak on the  E l e c t r o n microscopy was used to  blackeye cowpea.  in one s e r i e s o f experiments  lation.  to p r o j e c t  i n f e c t e d and o f each v i r u s  leaves o f C a l i f o r n i a  the peak  P r o f i l e s o f g r a d i e n t s layered  the presence o f CP-SBMV and CP-TMV  t a n e o u s l y doubly  C. 2) and the v i r u s  by c o u n t i n g squares under  with e x t r a c t s from h e a l t h y t i s s u e were used (b)  III.  knife.  carbon  a c e t a t e ( i n 50% methanol) and then  (Reynolds, 1963)•  The s e c t i o n s were examined  EM 300 t r a n s m i s s i o n microscope a t a v o l t a g e output o f 60 KV.  55  B.  Movement o f CP-SBMV and CP-TMV from i n t o preformed 3rd t r i f o l i a t e  The  experiments  leaves o f C a l i f o r n i a  in t h i s s e c t i o n were aimed a t s t u d y i n g  of  CP-SBMV and CP-TMV in the 3rd t r i f o l i a t e  of  arrival  trifoliate 1. to  o f each v i r u s  in these  leaves.  leaves were trimmed b e f o r e  Trimming.  i n o c u l a t e d primary  When the f i r s t  to a l e n g t h o f 0.5 to 1 cm b e f o r e the  1st and 2nd t r i f o l i a t e  had  reached 2.  inoculation.  right  leaves were then allowed  hand h a l f  leaves were trimmed  the 3rd t r i f o l i a t e  ( c o n t r o l ) ; group  leaves  leaves  ( i i ) p l a n t s were i n o c u l a t e d on the  l e a f with 0.05 mg/ml CP-SBMV and on the l e f t  hand  half  ( i i i ) p l a n t s were i n o c u l a t e d with 0.05 mg/ml o f  hand h a l f  with b u f f e r ; group  ( i v ) p l a n t s were s i m u l t a n e o u s l y  parate half  to grow  i n o c u l a t i o n ; however i f by the time  CP-TMV on the l e f t  l e a f and on the r i g h t  hand h a l f  leaf  i n o c u l a t e d on se-  leaves with CP-SBMV and CP-TMV as f o l l o w s :  the r i g h t  hand  l e a f was i n o c u l a t e d w i t h 0.05 mg/ml o f CP-SBMV and the l e f t hand  h a l f was immediately ted  razor blade o r a  I n o c u l a t i o n s were u s u a l l y done at 14 days a f t e r  l e a f with b u f f e r ; group  half  leaves were 1.5  Group ( i ) p l a n t s were mock-inocu1ated on the primary  b u f f e r alone  and 2nd  i n o c u l a t i o n was done the f o l l o w i n g day.  Inocu1 at ions.  sowing. with  this size  cowpea.  interactions  T h e r e f o r e the f i r s t  and 2nd t r i f o l i a t e  The 3rd t r i f o l i a t e  blackeye  leaves based on the sequence  3-0 cm long they were trimmed o f f w i t h a flamed  pair of scissors.  leaves  i n o c u l a t e d with 0.05 mg/ml o f CP-TMV.  All  inocula-  leaves were r i n s e d w i t h water and the p l a n t s were put i n the  24 ;to 27°C growth room f o r the d u r a t i o n of the experiment.  At v a r i o u s  56  intervals after a flamed The  i n o c u l a t i o n the 3rd t r i f o l i a t e  leaves were removed  razor blade from t h r e e t o f o u r p l a n t s per treatment  removed  in P e t r i  leaves were f l o a t e d on h a l f - s t r e n g t h Hoagland s 1  d i s h e s and incubated  were s t o r e d at -20°C u n t i l  with  per i n t e r v a l . solution  a t 2k to 27°C f o r 48 hours a f t e r which  a l l samples could be assayed  together.  they  In-  f e c t i v i t y assay o f the leaves was done by g r i n d i n g leaves o f each harvest in a small lesion C.  amount o f phosphate b u f f e r and i n o c u l a t i n g i t to the l o c a l  indicator  E f f e c t o f sequence o f a r r i v a l 3rd  1.  hosts.  trifoliate  InoculatIons.  o f each v i r u s i n t h e preformed  l e a f on the c o n c e n t r a t i o n o f the o t h e r . P l a n t s o f the same age trimmed as above ( S e c t i o n  IV.B.1) were i n o c u l a t e d e s s e n t i a l l y as above ( S e c t i o n IV.B.2) except that a f i f t h group  (v) was added and i n o c u l a t e d as f o l l o w s :  of  t h e p l a n t s were i n o c u l a t e d on the r i g h t hand h a l f  of  CP-SBMV and the other h a l f  the other with  l e a f was l e f t  At v a r i o u s  l e a f w i t h 0.05 mg/ml  uninocu1ated;  h a l f o f the p l a n t s were i n o c u l a t e d on the l e f t  0.05 mg/ml o f CP-TMV and the o t h e r intervals after  group were  left  right  hand h a l f  hand h a l f  similarly, hand h a l f  l e a f was l e f t  leaf  uninocu1ated.  inoculation,plants of this  cha11enge-inocu1ated with e i t h e r CP-TMV  the u n i n o c u l a t e d uninoculated  the f i r s t  half  one h a l f  fifth  (0.05 mg/ml) on  l e a f or CP-SBMV (0.05 mg/ml) on the  leaf.  The p l a n t s were l e f t  i n the 2k to 27°C  growth room f o r the d u r a t i o n o f the experiment. 2.  Assay.  Nucleoprotein  assay  o f s i n g l y and doubly  infected  tissue  57  was by a n a l y t i c a l D.  sucrose  density~~gradient  c e n t r i f u g a t i o n (Section  I n t e r a c t ions o f CP~SBMV and CP-TMV  in C a l i f o r n i a  IV.A.2  blackeye cowpea  under synchronous c o n d i t i o n s . 1.  Trimming.  California  blackeye cowpea p l a n t s  s o l u t i o n as p r e v i o u s l y d e s c r i b e d and  3rd t r i f o l i a t e  leaves.  raised  were trimmed t o leave o n l y  The 3rd t r i f o l i a t e  in Hoagland's the primary  leaves a t the time d f  trimming were 0.5 to 1.5 cm long. 2.  Inocu1 at ions.  culation. treatment.  P l a n t s s e l e c t e d f o r u n i f o r m i t y were p r e - c o n d i t i o n e d One group was i n o c u l a t e d w i t h  l e a f o f each primary other with  U s u a l l y p l a n t s were trimmed the day before  l e a f and with  group was i n o c u l a t e d w i t h b u f f e r on the r i g h t  neously  i n o c u l a t e d with  CP-SBMV on the r i g h t  with  A l l inoculated  viruses  hand h a l f  hand h a l f l e a f ; the  hand h a l f l e a f and  Control  hand h a l f l e a f and w i t h p l a n t s were  f o r 42 hours  i n t o 3rd t r i f o l i a t e  (Section  leaves)  a photoperiod  inoculated  leaves were r i n s e d with  i n o c u l a t e d p l a n t s were held  growth chamber w i t h  by dark  hand h a l f l e a f ; and the l a s t group was s i m u l t a -  hand h a l f l e a f .  The  b u f f e r on the l e f t  CP-TMV on the l e f t  CP-TMV on the l e f t buffer only.  CP-SBMV on the r i g h t  ino-  water.  (based on movement o f  IV.B.) at 25 t o 27°C  o f 18 hours and 13,000 l u x .  in a  After  i n c u b a t i o n a t 2 5 to 27°C f o r 42 hours some p l a n t s from each i n o c u l a t i o n treatment were kept a s i d e and the r e s t were moved t o a p o l y s t y r o f o a m  cham  ber.  From p l a n t s kept a s i d e a small  liate  leaves was c u t w i t h a flamed r a z o r blade and the l e a f p i e c e s were  incubated  in P e t r i  dishes  p i e c e o f t i s s u e from the 3rd t r i f o -  f o r 48 hours.  A f t e r this period  the t i s s u e  58  was  homogenized  in a spot p l a t e and assayed on the r e s p e c t i v e  i n d i c a t o r hosts f o r CP-SBMV and CP-TMV. in the styrofoam chamber lated 3rd  trifoliate  chamber was The  were incubated at 27  temperature  S c h l e g e l 1973)  to 28°C w i t h heat f u r n i s h e d  by a r h e o s t a t . treatment  was  T h i s was treated  maintained  (R.H.) of 75  by h e a t i n g a l a r g e water bath  15 W l i g h t  to 8 0 % .  of v i r u s  Only the 3 r d  in the 3 r d  by a n a l y t i c a l  trifoliate  to space  p l a n t s were r a i s e d  Third  trifolate  trifoliate  at v a r i o u s i n t e r v a l s u n t i l  leaves was  deter-  sucrose d e n s i t y g r a d i e n t  leaves were used  beginning at the time p l a n t s were s h i f t e d  from the styrofoam chamber.  Due  trifoliate  throughout.  i n f e c t i v i t y o f d i s c s and  I n f e c t i v i t y assay.  The  p r e l i m i n a r y o b s e r v a t i o n s showed that i f  leaves became n e c r o t i c and a b s c i s e d .  Assay. Accumulation  (zero time) and  bulbs  in the growth room.  in the styrofoam chamber h e a l t h y c o n t r o l  centrifugation. (a)  by two  f o r 5 days a f t e r which they were moved to the 2k  in the 2k to 27°C growth room  mined by  styrofoam  in the dark.  p l a n t s were moved to a dry atmosphere some of the 3 r d  limitation  3.  The  The p l a n t s were s u b j e c t e d to a d i f f e r e n t i a l  necessary because  leaves and primary  noninocu-  leaves which were i n s i d e the styrofoam chamber  to 27°C growth room w i t h r e l a t i v e humidity R.H.  with t h e i r  leaves extending out of the chamber.  primary  lesion  r e s t of the p l a n t s were put  then p l a c e d in a p l a n t growth chamber at 10°C  inoculated  controlled  (Dawson and  The  local  f o r assay.  leaves were harvested  to the 2k to 27°C growth room \kk hours s i n c e the  Samples f o r i n f e c t i v i t y  shift  assay were removed  59  from leaves w i t h a no. 3 cork borer each sampling. be assayed  The d i s c s were s t o r e d at -20°C u n t i l  together.  Frozen  and were f u r t h e r d i l u t e d I n f e c t i v i t y was assayed block d e s i g n  (7 mm diameter) t o g i v e 8 d i s c s f o r  samples were ground  treatments  were assayed  was assayed  two leaves  (KIeczkowski,  1950) f o r 8  10 v i r u s c o n c e n t r a t i o n s ,  T i s s u e s from h e a l t h y c o n t r o l and z e r o  on primary  (w/v).  by the h a l f - l e a f method based on a randomized  d i f f e r e n t v i r u s c o n c e n t r a t i o n s extended t o s u i t 18 times.  in phosphate b u f f e r  t o g i v e 1/10 t o 1/10,000 d i l u t i o n s  f o r plants with  replicated  a l l samples c o u l d  separately.  time  I n f e c t i v i t y o f CP-SBMV and CP-TMV  leaves o f GA 21 and  N^. g 1 ut i nosa, trimmed t o  leave two middle l e a v e s , r e s p e c t i v e l y . (b)  Nucleoprotein  leaves a f t e r  E.  (Section  by a n a l y t i c a l  sucrose d e n s i t y g r a d i e n t  assayed centrifu-  IV.A.2.a).  Analysis f o r structural doubly  1.  T i s s u e which remained from 3rd t r i f o l i a t e  removal o f d i s c s was s t o r e d a t -20°C and was l a t e r  for nucleoprotein y i e l d gation  assay.  infected  Inoculations.  (simultaneously)  i n t e r a c t i o n s in C a l i f o r n i a  blackeye  cowpea  by CP-SBMV and CP-TMV. Cowpea p l a n t s were i n o c u l a t e d s i n g l y and doubly  with CP-SBMV and CP-TMV as p r e v i o u s l y d e s c r i b e d  (Section  IV.A.I.a). 2.  Assay.  were harvested  Twenty days a f t e r  i n o c u l a t i o n the 3rd t r i f o l i a t e  and s t o r e d at -20°C u n t i l  leaves  needed.  (a) Pur i f i cat ion. (i)  Frozen  t i s s u e was p u l v e r i z e d and homogenized  b u f f e r pH 6.5 with a Waring  blender.  in 0.2 M T r i s - H C l .  60  (ii)  The  homogenate was  (iii)  The  f i l t r a t e was  stirring (iv)  The  s t r a i n e d through k  emulsified  with c h l o r o f o r m  cheesecloth,  (1:1  v/v)  by  k°C.  f o r 2 to 5 minutes at  emulsion was  l a y e r s of  broken by c e n t r i f u g i n g at  10,000 rpm  for  20 minutes in a S o r v a l l SS-34 r o t o r , (v)  The  supernatant was  centrifuged  at 28,000 rpm  in a Spinco no.  30  r o t o r f o r 3 hours, (vi)  The pH  (vii)  6.5  The  The  p e l l e t was  containing  and  of  the v i r u s e s  CP-TMV, r e s p e c t i v e l y .  extracts  the n a t u r a l  one  from s i n g l y  clarified  gradient  ( n a t u r a l mixture) was  t i o n as  Igepon T -  of each v i r u s  sucrose d e n s i t y  the c o n c e n t r a t i o n plants  0.1%  overnight  v i r u s suspension was  concentration  analytical  extracted  was  t i o n was diluted  infectivity  Serum n e u t r a l i z a t i o n of performed f o l l o w i n g 1/8  in PBS  to 0.25  and  (Section  from doubly 0.5  IV.  by  IV.A.2.a) and infected  mg/ml f o r CP-SBMV  mixture of the  The  f o r . 1 0-minutes.  estimated  same c o n c e n t r a volumes of  the  mixtures were then analysed  n e u t r a l i z a t i o n assay and  also  by  gradients.  infectivity.  Infectivity  the method of Dodds (1972).  b e f o r e use.  prepared as above ( S e c t i o n  10,000: rpm  made by mixing a p p r o p r i a t e  i n f e c t i v i t y d i s t r i b u t i o n In sucrose (b)  at  in the e x t r a c t s  artificial  buffer  73.  centrifugation  infected plants.  f o r genomic masking by  M Tris-HCl  in the e x t r a c t s was  adjusted  An  in 0.02  Virus antigens p a r t i a l l y D.2.a) were n e u t r a l i z e d as  neutralizaA l l sera purified follows:  were and  61  one mi 1 1 i 1 i t r e - a l i q u o t s o f n a t u r a l ( i n v i v o mixture) (in  v i t r o mixture)  each  incubated  mixtures,  as w e l l as o f each v i r u s s e p a r a t e l y , were  in 0.2 ml o f CP-SBMV antibody, CP-TMV antibody,  serum and PBS at room temperature cal  and a r t i f i c i a l  (25°C) f o r one hour on a F i s h e r c l i n i -  r o t a t o r and then at 4°C f o r three hours.  The a n t i g e n - a n t i b o d y mix-  t u r e s were then c e n t r i f u g e d a t 10,000 rpm f o r 10 minutes. natants were c o l l e c t e d of  from each mixture,  at  combined w i t h  serum, or b u f f e r and incubated o v e r n i g h t .  lexes were then p r e c i p i t a t e d  by low speed  The super-  1/10 volumes  The a n t i g e n - a n t i b o d y comp-  c e n t r i f u g a t i o n as b e f o r e , and  t h i s time no f u r t h e r p r e c i p i t a t i o n was observed.  a n t i g e n s were then used  The n e u t r a l i z e d  f o r i n f e c t i v i t y by the o p p o s i t e h a l f  l e a f method  on GA 21 and N_. g l u t i n o s a f o r CP-SBMV and CP-TMV, r e s p e c t i v e l y . nia  blackeye cowpea was a l s o  rations its  (c)  local  lesion  RNA could  CP-TMV, p a r t i a l l y  not r e c o g n i z e  host.  Sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n .  n a t u r a l mixtures  Califor-  i n o c u l a t e d s e p a r a t e l y by each o f the prepa-  in case the heterologous 1y encapsidated  specific  normal  Artificial  and  of the two v i r u s e s and p r e p a r a t i o n s of CP-SBMV and o f p u r i f i e d as above ( S e c t i o n IV.D.2.a) were l a y e r e d on  sucrose g r a d i e n t columns and c e n t r i f u g e d a t 39,000 rpm f o r 105 minutes at 5°C  in a Beckman SW 41 r o t o r .  F r a c t i o n s were c o l l e c t e d with a s y r i n g e by  p u n c t u r i n g on the s i d e o f the tube w i t h a needle 1973) a t p o s i t i o n s c o r r e s p o n d i n g and  t o the l i g h t  (Peterson and Brakke,  s c a t t e r i n g zones o f CP-SBMV  CP-TMV and to the zones between CP-SBMV and CP-TMV.  The c o l l e c t e d  62  f r a c t i o n s were d i a l y s e d o v e r n i g h t  in phosphate b u f f e r and i n o c u l a t e d  to GA 21 and N_. g l u t i n o s a by the o p p o s i t e nia tely  h a l f l e a f method.  blackeye cowpea and P i n t o bean were a l s o  inoculated  by each o f the f r a c t i o n s because both v i r u s e s  V.  I n t e r a c t i o n s o f CP-SBMV and CP-TMV  but separa-  infect  blackeye cowpea s y s t e m i c a l l y and CP-TMV i n f e c t s P i n t o  Califor-  California  systemically.  in P i n t o  bean.  When f r a c t i o n s c o l l e c t e d a f t e r sucrose d e n s i t y g r a d i e n t f u g a t i o n were assayed on P i n t o as d e s c r i b e d was  above ( S e c t i o n  i n a d v e r t e n t l y observed that f r a c t i o n s c o l l e c t e d between  scattering mixtures of P i n t o ,  zones o f CP-SBMV and CP-TMV  induced n e c r o t i c r e d d i s h s i m i l a r to those induced  in both n a t u r a l  local  IV.D.2.c), i t light  and a r t i f i c i a l  l e s i o n s on primary  leaves  by the bean s t r a i n o f SBMV on P i n t o .  These symptoms were always r e p r o d u c i b l e doubly  centri-  when P i n t o primary  leaves  were  i n o c u l a t e d with e i t h e r p u r i f i e d or crude sap e x t r a c t s o f a mix-  t u r e o f CP-SBMV and CP-TMV but not by CP-SBMV A.  Inoculations.  1.  Inoculations  with  c u l a t e d w i t h known c o n c e n t r a t i o n s  o f P i n t o were s i n g l y  of e i t h e r p u r i f i e d  or phosphate b u f f e r alone.  The i n o c u l a t e d  with running tap water t o  remove r e s i d u a l  concentrations  or b u f f e r 72 or 96 hours a f t e r i n i t i a l  leaves  doubly  inoculated  In one ino-  CP-SBMV or CP-TMV  leaves were thoroughly  rinsed  inoculum, and c h a l l e n g e -  o f e i t h e r CP-SBMV, inoculation.  s e r i e s of experiments, time-course s y n t h e s i s P i n t o primary  alone.  i n t a c t CP-SBMV and i n t a c t CP-TMV.  s e r i e s o f experiments the primary leaves  i n o c u l a t e d with v a r y i n g  inoculated  CP-TMV  In another  o f CP-SBMV was s t u d i e d in  simultaneously  w i t h CP-SBMV  63 and  CP-TMV and s i n g l y  i n o c u l a t e d w i t h the former  leaves o f one group o f p l a n t s were s i n g l y those o f another  group were doubly  with  tap  i n o c u l a t i o n , the primary  leaves  s o l u t i o n f o r 10 minutes and r i n s e d  water f o r 24 hours a f t e r which d i s c s were c u t with a no. 10 a l l could be assayed  together.  I n o c u l a t i o n s w i t h CP-SBMV-RNA and i n t a c t CP-TMV.  P i n t o primary prepared  C o n t r o l p l a n t s were i n o c u l a t e d w i t h  in a detergent  cork borer and s t o r e d a t -20°C u n t i l 2.  i n o c u l a t e d w i t h CP-SBMV and  At i n t e r v a l s a f t e r  were harvested and soaked  The primary  i n o c u l a t e d s i m u l t a n e o u s l y w i t h 0.05 mg/ml  o f CP-SBMV and 0.05 mg/ml o f CP-TMV. phosphate b u f f e r o n l y .  virus.  leaves were s i n g l y  i n o c u l a t e d with  CP-SBMV-RNA  from e i t h e r p u r i f i e d v i r u s or from sap o f i n f e c t e d  ( S e c t i o n V.B.a and b ) , or they were doubly i n t a c t CP-TMV.  When simultaneous  leaves.  i n o c u l a t e d w i t h CP-SBMV-RNA and  i n o c u l a t i o n s were made,  a one ml  a l i q u o t o f CP-SBMV-RNA was mixed w i t h one ml (0.1 mg/ml) o f i n t a c t CP-TMV and  a p p l i e d t o P i n t o primary  l e a v e s . For s i n g l e  i n o c u l a t i o n s the a l i q u o t  of CP-SBMV-RNA was d i l u t e d with an equal volume o f phosphate b u f f e r and applied  t o the primary  leaves.  In s e q u e n t i a l i n o c u l a t i o n s P i n t o  leaves were i n o c u l a t e d with the d i l u t e d  primary  CP-SBMV-RNA or 0.05 mg/ml of i n t a c t  CP-TMV o r b u f f e r o n l y and l a t e r cha11enge-inocu1ated at v a r i o u s i n t e r v a l s : with  e i t h e r 0.05 mg/ml o f i n t a c t CP-TMV o r the d i l u t e d  the time-course e i t h e r doubly  CP-SBMV-RNA.  s y n t h e s i s o f CP-SBMV was s t u d i e d , the primary  When  leaves were  i n o c u l a t e d s i m u l t a n e o u s l y with CP-SBMV-RNA and i n t a c t CP-TMV or  s i n g l y with CR-SBMV-RNA. A l l i n o c u l a t e d leaves were r i n s e d w i t h water.  At  6k  intervals after  i n o c u l a t i o n the primary  leaves were harvested and r i n s e d  in running water f o r 8 hours or o v e r n i g h t , b l o t t e d , a n d d f s e s were c u t w i t h a no.10 cork borer and s t o r e d at -20°C u n t i l  a l l samples could be assayed  together. 3.  Assay.  Assays  f o r CP-SBMV on P i n t o primary  l a t e d as mentioned above, were made by i n f e c t i v i t y ,  leaf tissue,  inocu-  s e r o l o g y and e l e c t r o n  microscopy. (a)  I n f e c t i v i t y assay.  on GA 21 whole leaves  The i n f e c t i v i t y assay f o r CP-SBMV was made  (replicated  in order to a v o i d contamination inocula.  o f the o p p o s i t e h a l f - l e a f  leaves  by one o f the  in a mortar with a p e s t l e with a small amount o f sand and  1:2 (w/v) i n phosphate b u f f e r and a p p l i e d to GA 21 primary  (b) sion  r a t h e r than on h a l f  T i s s u e s from d i s c s s t o r e d as mentioned above ( S e c t i o n V.A.I and 2)  were ground diluted  20 times)  Serological  (Ouchterlony,  assay.  leaves.  The double d i f f u s i o n method o f immunodiffu-  1958) was used  to d e t e c t a n t i g e n s  in t i s s u e  extracts.  The d i f f u s i o n medium c o n s i s t e d o f 10 ml o f 0.75% Noble agar, 0.02% NaN^  in PBS i n p l a s t i c P e t r i  (total  (8.5 cm d i a m e t e r ) .  Several  discs  wt, 2.0 - 2.5 g) c o n t a i n i n g c o a l e s c i n g l e s i o n s were cut w i t h a no. 5  cork borer from detergent  presence  P i n t o primary  leaves that had been washed with  f o r 10 minutes and r i n s e d  They were ground  The  dishes  f o r 2k hours w i t h running  10% Lux tap water.  in PBS (1 g/0.2 ml) in a mortar with a p e s t l e  of p u r i f i e d  sand  (The B r i t i s h Drug Houses L t d . , Poole,  homogenate was then squeezed through  a double  i n the England).  layer of M i r a c l o t h ,  65  heated a t kO C f o r 60 minutes,  10,000  deposited or  10 minutes.  rpm f o r  in w e l l s , 5 mm from a c e n t r a l  no more p r e c i p i t i n  leaves showing l o c a l  well, containing either  Virus p u r i f i e d  examined by EM but because an attempt  assay.  Seven to 10 days a f t e r  l e s i o n s were prepared  as f o r CP-SBMV  i t was necessary t o separate  was r e a c t e d w i t h  i t s homologous antiserum  in a beaker o f 0.05 M NaCl (Dr. J.H. Tremaine,  I M.B.I) was  o f CP-TMV even  after  t o be CP-SBMV  in agar g e l double  the whole P e t r i  personal communication).  diffusion.  d i s h was subin d i s t i l l e d  The bands were  in a drop o f 2% u r a c y l  acetate  The homogenate was mounted on g r i d s and r i n s e d 5  times w i t h 2% u r a c y l with a P h i l i p s  believed  o v e r n i g h t and r i n s e d  out with a r a z o r blade and ground  negative s t a i n .  B.  IV.A.2.b) and  CP-SBMV by p r e c i p i t a -  The f r a c t i o n  A f t e r CP-SBMV-antibody bands had formed  sliced  Inoculation  f o r EM or p u r i f i c a -  (Section  o f the preponderance  i t with homologous antiserum.  water  later  to separate the two v i r u s e s by sucrose d e n s i t y g r a d i e n t  centrifugation,  merged  CP-SBMV  The g e l s were incubated at room  S e c t i o n s f o r EM were prepared as before ( S e c t i o n  so examined.  ting  1:128.  ml) o f supernatant were  formed.  E l e c t r o n microscopy  primary tion.  to  by c e n t r i f u g i n g at  (25°C) and examined f o r :timmunoprecipi t a t e s 2k hours  temperature  (c)  (0.02  Aliquots  CP-TMV antiserum d i l u t e d  until  and then c l a r i f i e d  acetate negative s t a i n .  The g r i d s were viewed  EM 200 or 3 0 0 .  Extraction of r i b o n u c l e i c  acid.  I n f e c t i o u s RNA was e x t r a c t e d from e i t h e r p u r i f i e d  virus preparation  66  or crude sap o f i n f e c t e d C a l i f o r n i a blackeye cowpea. 1.  E x t r a c t i o n o f RNA from p u r i f i e d  RNA was e x t r a c t e d as f o l l o w s  (Santalli  virus preparation. et a l , 1961):  was added t o 6 ml o f 0.05 M Kh^PO^, 0.13 M NaCl NaOH) which had been heated at t h i s temperature in  f o r one minute.  fuged at 6,000 rpm f o r 10 minutes.  ( a d j u s t e d t o pH 7-0 with  The s o l u t i o n was immediately p l a c e d  After c h i l l i n g The RNA  the mixture was c e n t r i -  in the supernatant was p r e c i -  by adding two volumes o f a b s o l u t e ethanol c o n t a i n i n g a few drops  of 3 M NaAc and allowed t o stand on i c e f o r 30 minutes. ted by c e n t r i f u g i n g  f o r 30 minutes  a t 9.000 rpm.  in 2.5 ml o f 0.01 M potassium phosphate nite  (2 ml, 3 mg/ml)  t o 96.5 £ 0.5°C and the mixture was maintained  i c e and c o o l e d f o r 5 minutes.  pitated  virus  The i n f e c t i o u s  The RNA was c o l l e c -  The p e l l e t was e x t r a c t e d  b u f f e r pH 7.1 c o n t a i n i n g bento-  (100 ^jg/ml) and the suspension was then used  immediately  f o r ino-  c u l a t ion. 2.  E x t r a c t i o n o f RNA from crude sap (Dawson and Kuhn, 1972).  grams o f f r e s h l y harvested l e a f t i s s u e o f C a l i f o r n i a previously  i n o c u l a t e d with CP-SBMV was blended  Twenty  blackeye cowpea  in a Waring blender in an  e x t r a c t i o n medium c o n t a i n i n g 20 ml o f water - s a t u r a t e d phenol, \% SDS (sodium dodecyl s u l p h a t e ) , \% b e n t o n i t e 0.01 M EDTA, as m o d i f i e d by Kuhn and Adams (1976). The  The emulsion was c e n t r i f u g e d at 6,500 rpm f o r 10 minutes.  supernatant aqueous phase was mixed t h r e e times with petroleum e t h e r  to remove phenol. of 35% i c e - c o l d  The f i n a l  aqueous phase was p r e c i p i t a t e d with 2 volumes  ethanol and c e n t r i f u g e d  f o r 10 minutes  at 6,500 rpm.  The  67  RNA p e l l e t was e x t r a c t e d containing bentonite  in 0.01 "M'potassium phosphate b u f f e r pH 7-1  (100^ig/ml).  The resuspended RNA was d i v i d e d  1 m l - a l i q u o t s and s t o r e d at -20°C u n t i l way  was used VI.  used.  Seed t r a n s m i s s i o n o f CP-SBMV and CP-TMV Propagation  The  California  Botswana blackeye,  hosts f o r CP-SBMV and CP-TMV blackeye  cowpea and three Botswana l o c a l -  1.  i n o c u l a t e d s i n g l y or doubly and  in the greenhouse as d e s c r i b e d before  (Section I I I ) ,  D i s t r i b u t i o n o f CP-SBMV and CP-TMV i n seed blackeye  The d i s t r i b u t i o n  r i v e d from p l a n t s s i n g l y o r doubly  o f CP-SBMV o r CP-TMV  the peduncle o f the flower with a date The pods were then  days o l d ; these stages v i n e - d r y mature, 2.  harvested  represented  i n t o seed  tag as soon as a small  by..tagging pod was  when 15 to 20, 30 to 35 and 45 to 55  green  immature, dough stage  immature  Seeds were removed from the pods and  coats and embryos.  were e a s i l y peeled o f f from the embryos shoot) without  T h i s was done  respectively.  Decontamination procedures.  separated  i n seeds de-  i n f e c t e d by CP-SBMV and CP-TMV was  determined a t v a r i o u s stages o f seed m a t u r i t y .  and  parts of C a l i f o r n i a  cowpea and 3 Botswana cowpeas.  Harvesting.  formed.  varieties,  V26-Bots (khaki c o l o u r ) and V45 Bots (dark brown  c o l o u r ) cowpeas were grown in 21-cm pots,  B.  in t h i s  in most experiments.  A.  raised  The RNA e x t r a c t e d  into  soaking.  In immature seed  the seed  coats  (cotyledons and r a d i c l e - p l u m u l e  However, mature seed were soaked  overnight  68  in d i s t i l l e d water b e f o r e seed c o a t s c o u l d be removed. seed p a r t s were s e p a r a t e l y decontaminated for  2k hours o r they were soaked  10 minutes tap  and then  water.  suspension artificially  in 5% t r i s o d i u m phosphate  contaminated  ( 1 0 ug/ml) f o r 10 to 15 minutes. contaminated  plumule 3-  The h e a l t h y embryos  H a l f o f the p o p u l a t i o n o f  embryos was then decontaminated  by tap water  The remainder was l e f t  sharp f o r c e p s was used  to d i s s e c t  shoots which were then r i n s e d  i n water.  Assay o f embryos and s e e d l i n g s d e r i v e d from seeds produced on i n f e c t e d w i t h CP-SBMV and CP-TMV.  D i r e c t assay o f seed p a r t s on i n d i c a t o r h o s t s .  samples c o n t a i n i n g f i v e pooled  or  in phosphate  buffer.  singly Occasional  r a d i c l e - p l u m u l e s o r seed coats were homo-  in 0.1 and 0.25 ml, r e s p e c t i v e l y .  t o primary  The homogenates were  leaves o f GA 21 and to middle  Xanthi and the leaves were r i n s e d (b)  A  the c o t y l e d o n s and r a d i c l e -  f o r 30 minutes  in 0.2, 0.05 and 0.25 ml, r e s p e c t i v e l y ,  ted  undeco-  before d i s s e c t i o n .  Embryos, r a d i c l e - p l u m u l e s and seed c o a t s were homogenized  genized  in running  When c o t y l e d o n s and r a d i c l e - p l u m u l e shoots were assayed  plants (a)  for  w i t h v i r u s by soaking them i n v i r u s  s e p a r a t e l y , the whole embryo was decontaminated flamed  (Na^PO^)  r i n s e d o v e r n i g h t or a t l e a s t f o r 12 hours  wash or by soaking them in 5% Na^PO^. taminated.  by e i t h e r a tap water wash  C o n t r o l s included h e a l t h y seed p a r t s .  were a r t i f i c i a l l y  The separated  inocula-  leaves o f N_. g 1 ut i nosa  immediately w i t h  water.  S e e d l i n g assay.  Undecontaminated embryos,, decontaminated  embryos o f mature seeds and  69 i n t a c t mature seed were sown germinate.  in s t e a m - s t e r i l i z e d  In e a r l y experiments,  However, i t was l a t e r  i n d i v i d u a l l y on the r e s p e c t i v e realized  CP-SBMV were as r e l i a b l e as the i n f e c t i v i t y riments the c r i t e r i o n singly  doubly  for infection  from seed produced  infected  those o f the o t h e r .  indi-  symptoms f o r  assay so in subsequent  expe-  i n s e e d l i n g s from seed o f  symptoms.  by p l a n t s s i n g l y  by CP-SBMV were always  c a t o r hosts f o r each v i r u s  that v i s u a l  by CP-SBMV  i n f e c t e d p l a n t s was based on v i s u a l  lings derived  and allowed t o  t i s s u e samples o f 2nd and 3 r d t r i f o l i a t e  leaves o f a l l s e e d l i n g s were assayed cator hosts.  soil  However, a l l seed-  infected  by CP-TMV or  assayed on the r e s p e c t i v e  indi-  in case symptoms o f one v i r u s were masked by  Primary  leaves were never assayed.  S e e d l i n g s were  never handled except when assayed. C.  E f f e c t o f g e r m i n a t i o n ori CP-SBMV and CP-TMV  1.  Germination o f seed.  Seeds from p l a n t s p r e v i o u s l y  s i n g l y by CP-SBMV or CP-TMV were sown Two to f o u r days a f t e r coats s e p a r a t e d . contaminated was  left 2.  i n seed c o a t s .  sowing  in s t e a m - s t e r i l i z e d  infected river  seeds were removed and embryos and seed  One h a l f o f the p o p u l a t i o n o f the seed c o a t s was de-  o v e r n i g h t or f o r 2k hours  in running tap water;  the remainder  undecontaminated. I n f e c t i v i t y assay.  germinated  I n f e c t i v i t y o f the seed c o a t s o b t a i n e d from  seed was compared w i t h that o f ungerminated  seed c o a t s were each ground  seed c o a t s .  The  i n 0.25 ml o f b u f f e r and assayed by the h a l f -  l e a f method on GA 21 f o r CP-SBMV and N. g l u t i n o s a f o r CP-TMV. of  sand.  i n o c u l a t i o n was as f o l l o w s :  The scheme  ( i ) e x t r a c t s of washed seed c o a t s (from  70  germinated  o r ungerminated seeds) were i n o c u l a t e d to o p p o s i t e halves of  P i n t o primary  leaves;  and ( i i ) those from unwashed seed  coats were  s i m i l a r l y inoculated. D.  E f f e c t o f healthy mature seed e x t r a c t s on i n f e c t i v i t y o f CP-SBMV and CP-TMV.  1.  E x t r a c t ion.  distilled  Healthy mature seeds were soaked o v e r n i g h t in  water and seed  coats were removed.  a Waring blender with 0.01 M potassium 1 ml )• The  The homogenate was s t r a i n e d  f i l t r a t e was d i l u t e d  The embryos were ground in  phosphate b u f f e r pH 7-1 (one embryo/  through  a double  1/10 and 1/100 ( v / v ) .  f i l t r a t e and i t s d i l u t i o n made to a f i n a l  layer of M i r a c l o t h .  V i r u s was added to the  c o n c e n t r a t i o n o f 10jjg/rn];  b u f f e r c o n t a i n i n g the same v i r u s c o n c e n t r a t i o n was used 2. inoculated tests,  I n f e c t i v i t y assay. The v i r u s p r e p a r a t i o n s made as above were t o GA 21 and N_. g l u t i n o s a  respectively  method, r e p l i c a t e d VII. The duction.  , in a.completely  f o r CP-SBMV and CP-TMV  infectivity  randomized design by the h a l f  P r o d u c t i o n o f seed California  for yield  analysis.  blackeye cowpea was used  Seeds were sown  e x c l u s i v e l y f o r seed  peduncles  pro-  in 21-cm pots and a f t e r germination s e e d l i n g s The treatments  were arranged  in a  randomized design on a greenhouse bench. Because cowpea  indefinitely,  leaf  8 times.  were thinned to 2 p l a n t s per pot. completely  as a c o n t r o l .  o f f l o w e r s were tagged  f l o w e r s were the o n l y ones harvested  foryield  and pods produced analysis.  harvested when v i n e - d r y at 55 t o 65 days a f t e r f l o w e r i n g .  bears  on these'  Pods were  ;  71  RESULTS  I .  Estimation of concentration of p u r i f i e d  v i r u s by a n a l y t i c a l  sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . Absorbance p r o f i l e s o f p u r i f i e d  CP-SBMV and o f p u r i f i e d  CP-TMV a r e  shown in F i g u r e 1 and F i g u r e 2, r e s p e c t i v e l y and when the area under the peaks was c a l c u l a t e d linear left  and p l o t t e d a g a i n s t v i r u s c o n c e n t r a t i o n (mg/ml), a ( F i g u r e s 3 and h).  r e l a t i o n s h i p was obtained  The shoulder on the  hand s i d e o f the CP-TMV absorbance p r o f i 1 e , p r o b a b l y , r e s u l t e d  a g g r e g a t i o n of the v i r u s .  This  i s the best p r o f i l e p a t t e r n one c o u l d get  with t h i s s t r a i n o f TMV, which seems prone to a g g r e g a t i o n 1965) and i t d i d not seem to a f f e c t relationship. in T a b l e and  (Rees and Short,  the r e p r o d u c i b i l i t y o f the l i n e a r  The absorbance area v a l u e s ,  II and these were used  to plot  in a r b i t r a r y u n i t s , a r e given  the standard  curves  4 ) . The standard curves were d e r i v e d from data averaged  periments I I.  from  (Figures 3 from  two ex-  f o r each v i r u s . E f f e c t o f s i n g l e and double  i n f e c t i o n s on C a l i f o r n i a  blackeye cowpea and Botswana cowpea v a r i e t i e s .  gly  A.  Comparison o f symptoms.  1.  Symptoms in C a l i f o r n i a  blackeye cowpea.  When s e e d l i n g s were s i n -  i n o c u l a t e d with CP-SBMV, d i f f u s e c h l o r o t i c spots developed  culated  primary  developed  leaves 4 to 5 days a f t e r  a l s o on primary  on the ino-  i n o c u l a t i o n . The c h l o r o t i c  leaves that were doubly  spots  i n o c u l a t e d with CP-SBMV  72  Figure l. :  Absorbance p r o f i l e s f o l l o w i n g c e n t r i f u g a t i o n o f 0.2 ml o f p u r i f i e d CP-SBMV through sucrose d e n s i t y g r a d i e n t columns in a Beckman SW 41 r o t o r , at 39,000 rpm at 5 C f o r 105 minutes and scanned a t 254 nm (0.5 absorbance range). Direction of sed'imenitation i s t o the l e f t .  A.  CP-SBMV at 0.50 mg/ml b e f o r e  centrifugation.  B.  CP-SBMV a t 0.25 mg/ml b e f o r e  centrifugation.  C.  CP-SBMV at 0.10 mg/ml b e f o r e  centrifugation.  D.  CP-SBMV a t 0.05 mg/ml b e f o r e  centrifugation.  0.16  0.14  0.12  0.10 E LTV CN  0.08  0.06  o c ro  XI  i_  o  0.04  in  < 0.02  0.0  0.04  0.02  0.0  7k  F i g u r e 2.  Absorbance p r o f i l e s f o l l o w i n g c e n t r i f u g a t i o n o f 0.2 ml o f p u r i f i e d CP-TMV through sucrose d e n s i t y g r a d i e n t columns in a Beckman SW k] r o t o r , at 39,000 rpm a t 5°C f o r 105 minutes and scanned a t 254 nm (0.5 absorbance range). Direction of sedimentation i s to the l e f t .  A.  CP--TMV at  1.00 mg/ml b e f o r e  centrifugation.  B.  CP-•TMV at 0.50 mg/ml before  centrifugation.  C.  CP--TMV at 0.25 mg/ml b e f o r e  centrifugation.  D.  CP--TMV at 0.125 mg/ml before  centrifugation  75  76  F i g u r e 3-  R e l a t i o n s h i p between CP-SBMV c o n c e n t r a t i o n (mg/ml) and absorbance area o f CP-SBMV in sucrose d e n s i t y g r a d i e n t columns a f t e r c e n t r i f u g a t i o n i n Beckman SW 41 r o t o r , a t 39,000 rpm at 5 C f o r 105 minutes. The amount o f v i r u s in a suspension l a y e r e d on sucrose d e n s i t y g r a d i e n t columns was 0.2 ml f o r each c o n c e n t r a t i o n and the columns were scanned a t 254 nm (0.5 absorbance range). The c o n c e n t r a t i o n o f v i r u s in the g r a d i e n t columns i s expressed in a r b i t r a r y absorbance area un i t s .  78  F i g u r e k.  R e l a t i o n s h i p b e t w e e n CP-TMV c o n c e n t r a t i o n (mg/ml) a n d a b s o r b a n c e a r e a o f CP-TMV i n s u c r o s e d e n s i t y g r a d i e n t c o l u m n s a f t e r c e n t r i f u g a t i o n i n Beckman SW 41 r o t o r , a t 3 9 , 0 0 0 rpm a t 5 C f o r 105 m i n u t e s . T h e amount o f v i r u s i n a s u s p e n s i o n l a y e r e d on s u c r o s e d e n s i t y g r a d i e n t c o l u m n s was 0.2 ml f o r e a c h c o n c e n t r a t i o n a n d t h e c o l u m n s w e r e s c a n n e d a t 25b nm (0.5 a b s o r b a n c e r a n g e ) . The c o n c e n t r a t i o n o f v i r u s i n t h e gradient columns i s expressed i n a r b i t r a r y absorbance area un i t s .  80  Table  II.  R e l a t i o n s h i p between CP-SBMV and CP-TMV concent r a t i o n s arid t h e i r absorbance areas ( a r b i t r a r y u n i t s ) a f t e r c e n t r i f u g a t i o n o f 0.2 ml o f each v i r u s through sucrose d e n s i t y g r a d i e n t columns at v a r i o u s concentrations  Virus concentration  (mg/ml)  Absorbance area  units  CP-SBMV  CP-TMV  CP-SBMV  CP-TMV  0.50  1.000  139  87  0.25  0.500  68  kl  0.10  0.250  27  22  0.05  0.125  13  11  Before c e n t r i f u g a t i o n .  81  and  CP-TMV.  No symptoms developed  c u l a t e d , 8 t o 10 days a f t e r  on the primary  sowing, with CP-TMV.  i r r e g u l a r p u r p l e l e s i o n s developed In  systemically infected  on the i n o c u l a t e d primary  trifoliate  CP-SBMV a l o n e , v e i n c l e a r i n g was induced was  ino-  However, when o l d e r p l a n t s  \k days) were i n o c u l a t e d with CP-TMV a l o n e , or doubly with  (ca  CP-SBMV,  leaves.  leaves o f p l a n t s i n o c u l a t e d with in the newly-formed  leaves which  replaced l a t e r by mosaic with m o t t l i n g and a t times d i s t o r t i o n and  c h l o r o t i c spots appeared to  leaves o f s e e d l i n g s  recover from  ( F i g u r e 5B).  severe symptoms.  Systemically infected  i n o c u l a t e d w i t h CP-TMV o n l y developed  symptoms o f CP-TMV  leaves,  in the newly-developed  l e a f c l e a r i n g and b l i s t e r s developed  symptoms were sometimes expressed  infection  When primary  leaves o f p l a n t s  in double  Double  Although  Leaf s i z e was a l s o  leaves  i n o c u l a t e d with CP-TMV were cha11enge-inocu1ated  with  increase,in  the i n t e r v a l  When CP-SBMV-inocu1ated primary  c u l a t e d with CP-TMV 2k and 72 hours a f t e r systemic symptoms, accompanied trifoliate  reduced  ( F i g u r e 5D).  leaves decreased  inoculations.  CP-SBMV  i n f e c t i o n , CP-TMV symptoms  with CP-SBMV, the number of c h l o r o t i c spots which developed lated  infection  leaves and in l a t e r  ( F i g u r e SO).  u s u a l l y dominated and masked those o f CP-SBMV. by double  l a t e r seemed  mosaic and m o t t l i n g accompanied by  ( F i g u r e 5 C ) and d i s t o r t i o n o f the l e a v e s .  blisters caused  Leaves that appeared  between the two  leaves were  initial  on the inocu-  chal1enge-ino-  i n o c u l a t i o n , more severe  by n e c r o s i s , developed  on  newly-developed  leaves than on those of p l a n t s s i m u l t a n e o u s l y  i n o c u l a t e d with  82  F i g u r e 5-  T h i r d t r i f o l i a t e leaves o f C a l i f o r n i a b l a c k e y e cowpea s i n g l y and doubly i n f e c t e d by CP-SBMV and CP-TMV. Photographed 20 days a f t e r i n o c u l a t i o n (28 days a f t e r sowing).  A.  Healthy t r i f o l i a t e l e a f - w i t h phosphate b u f f e r .  from p l a n t s  B.  CP-SBMV-infected  C.  CP-TMV-infected t r i f o l i a t e  D.  CP-SBMV/CP-TMV-(doubly)-infected  trifoliate  mock-inocu1ated  leaf. leaf. trifoliate  leaf.  83  the two v i r u s e s .  When CP-TMV i n f e c t e d  with CP-SBMV a t s i m i l a r foliate  i n t e r v a l s , severe symptoms developed on the t r i -  leaves that were not formed  the sequences,  leaves were cha11enge-inocu1ated  a t the time o f i n o c u l a t i o n .  CP-TMV symptoms dominated  those  In a l l  induced by CP-SBMV in t r i -  1 eaves.  foliate 2.  Symptoms  lations  in Botswna cowpea v a r i e t i e s .  CP-SBMV in s i n g l e inocu-  induced the c h l o r o t i c spots on the i n o c u l a t e d primary  leaves o f  Botswana blackeye and V26-Bots cowpea v a r i e t i e s , but CP-TMV d i d not cause any obvious symptoms caused  on the i n o c u l a t e d  leaves.  symptoms s i m i l a r t o those i t induced  except that they were l e s s severe.  CP-SBMV systemic  in C a l i f o r n i a  CP-SBMV s i n g l e  cause symptoms on the i n o c u l a t e d primary  leaves or t r i f o l i a t e leaves o f  -  CP-TMV r e a d i l y  t i e s , causing symptoms s i m i l a r Double i n o c u l a t i o n varieties  t i e s developed was doubly  leaves by i n f e c -  i n f e c t e d a l l the three Botswana cowpea v a r i e to those i n the C a l i f o r n i a  resulted  by both v i r u s e s .  blackeye cowpea,  i n o c u l a t i o n d i d not  V 4 5 B o t s , although the v i r u s c o u l d be d e t e c t e d in primary t i v i t y assay.  infection  blackeye cowpea.  in systemic i n f e c t i o n o f the three  Doubly  i n o c u l a t e d primary  leaves,, o f a l l v a r i e -  c h l o r o t i c spots c h a r a c t e r i s t i c o f CP-SBMV.  i n o c u l a t e d with both v i r u s e s , s y s t e m i c a l l y  When V45-Bots  infected  leaves  showed c h l o r o t i c s p o t s , mosaic and m o t t l i n g symptoms c h a r a c t e r i s t i c o f CP-SBMV, but these were not masked or dominated contrast  to symptoms  in C a l i f o r n i a  B.  E f f e c t on p l a n t  1.  Plant growth i n C a l i f o r n i a  p l a n t s , s i n g l y or doubly  by CP-TMV symptoms, in  blackeye cowpea.  growth. blackeye cowpea.  Primary  i n o c u l a t e d , weighed the same as those  leaves o f inoculated  84  with  buffer only.  CP-SBMV  in s i n g l y i n f e c t e d p l a n t s caused mild growth  r e d u c t i o n , compared t o b u f f e r - i n o c u 1 a t e d in  For i n s t a n c e , CP-SBMV  s i n g l e i n f e c t i o n s reduced f r e s h weight and height o f p l a n t s by 6.8 and  3.7%,  r e s p e c t i v e l y (not s t a t i s t i c a l l y  significant III). in  plants.  reductions of  28.4  and  s i g n i f i c a n t ) , w h i l e CP-TMV caused  45-6%,  6 and  r e s p e c t i v e l y (Figure  Although double i n f e c t i o n d i d not cause any s i g n i f i c a n t  height over that a l r e a d y caused by CP-TMV alone,  Table  reduction  i t , however, reduced  f r e s h weight s i g n i f i c a n t l y over that o f p l a n t s s i n g l y i n f e c t e d by CP-TMV ( F i g u r e 6 and T a b l e  III).  The number o f t r i f o l i a t e  leaves produced on  doubly or s i n g l y i n f e c t e d or b u f f e r - i n o c u l a t e d p l a n t s was about the same (Table I I I ) . 2.  P l a n t growth in V45~Bots cowpea v a r i e t y > .  Double  infection  reduced weight and height o f V45~Bots cowpea, as d i d CP-TMV  infection  v  alone,  whereas no apparent d i f f e r e n c e was observed between b u f f e r - i n o c u 1 a t e d and CP-SBMV-singly C.  inoculated plants  (Table IV).  Comparison o f e f f e c t s o f CP-SBMV and CP-TMV on y i e l d and seed character i s t i cs.  1.  Y i e l d of C a l i f o r n i a  blackeye  cowpea.  Double i n f e c t i o n  reduced number o f pods and seeds per p l a n t by bO.b and bb.3%, compared t o y i e l d o f h e a l t h y r e d u c t i o n s o f 6.6 and 8.3% single  infection  respectively  plants.  respectively,  caused o n l y  slight  in pods and seeds, r e s p e c t i v e l y , whereas CP-TMV  significantly  (Table V ) .  CP-SBMV alone;  significantly  reduced pod and seed y i e l d  by 36.8 and  30.3%,  F i g u r e 6.  C a l i f o r n i a b l a c k e y e cowpea p l a n t s s i n g l y and doubly i n f e c t e d by CP-SBMV and CP-TMV. Photographed 20 days a f t e r i n o c u l a t i o n (28 days a f t e r sowing).  A.  Healthy p l a n t —  mock-inocu1ated  B.  CP-SBMV-infected  C.  CP-TMV-infected p l a n t .  D.  CP-SBMV/CP-TMV-(doubly)-infected  w i t h phosphate  plant.  plant.  buffer.  86  Table I I I . E f f e c t o f s i n g l e ( S l ) and simultaneous double i n f e c t i o n s (Dl) o f CP-SBMV and CP-TMV on growth o f C a l i f o r n i a b l a c k e y e cowpea  Treatment  Height per p l a n t (cm)  Buffer  92.75  CP-SBMV-SI  83-75  CP-TMV-SI Dl  Percent reduction  Fresh wt. per p l a n t (g)  0.0  16.2  C  9-7  15.1  50.50  d  45.6  11.6  42.75  d  53.9  9.8 ?  Readings made 24 days a f t e r  inoculation  Only t r i f o l i a t e  C  ?  .  C  d  Percent reduction  No. o f leaves per p l a n t  0..0  8..0  6..8  9. 0  28. ,4  8. 0  39. 5  8. 0  (32 days a f t e r p l a n t i n g )  leaves were counted.  Values i n each column not f o l l o w e d by t h e same l e t t e r v a r y c a n t l y a t P = 0.05 by Duncan's M u l t i p l e Range (DMR) t e s t .  signifi-  87  Table  IV.  E f f e c t o f s i n g l e and simultaneous double i n f e c t i o n s by CP-SBMV and CP-TMV on growth o f Botswana v a r i e t y of V45-Bots cowpea  Treatment  Height per p l a n t (cm)  Percent height reduct ion  Fresh wt. per p l a n t (g)  Percent wt. reduction  Buffer  29.0  0.0  11.0  0.0  CP-SBMV  29.3  -1.0  10.7  2.7  CP-TMV  21.3  30.0  8.0  27.3  CP-SBMV + CP-TMV  23.3  20.0  8.9  20.0  Readings made 20 days a f t e r  inoculation  (28 days a f t e r sowing)  88  T a b l e V. E f f e c t o f s i n g l e a n d s i m u l t a n e o u s d o u b l e i n f e c t i o n s o f CP-SBMV and CP-TMV on pod and s e e d y i e l d o f C a l i f o r n i a b l a c k e y e cowpea  Treatment  Pods  Buffer  13.6  CP-SBMV  12.7  CP-TMV  8.6  CP-SBMV + CP-TMV  8. 1  a  P o d s and s e e d s  Number p e r p l a n t % Reduction Seed  b b c c  % Reduction  b  0.0  87.• 5  6.6  80..3  36.8  61..0  4o.4  48..8 c  h a r v e s t e d 60 d a y s a f t e r  b c  0.0 8.3 30.3 44.3  flowering.  it V a l u e s i n e a c h c o l u m n n o t f o l l o w e d by t h e same l e t t e r s i g n i f i c a n t l y a t P = 0.05 by DMR t e s t .  vary  89  2.  Seed c h a r a c t e r i s t i c s  i n C a l i f o r n i a blackeye cowpea.  Seeds produced  by p l a n t s t h a t had been b u f f e r - i n o c u l a t e d and by p l a n t s s i n g l y i n f e c t e d by CP-SBMV had a normal seed coat c o l o u r . singly  However, seeds produced by p l a n t s  i n f e c t e d by CP-TMV and by p l a n t s doubly  CP-TMV had mottled  and d i s c o l o u r e d seed coat, w i t h some i n t e r n a l n e c r o s i s .  Double i n f e c t i o n , however, caused more seed 3.  Seed c h a r a c t e r i s t i c s  coat m o t t l i n g than CP-TMV alone.  i n Botswana cowpea v a r i e t i e s .  i n f e c t i o n d i d not cause seed coat m o t t l i n g o f seeds o f V26-Bots from p l a n t s s i n g l y seed c o a t s .  i n f e c t e d by CP-SBMV and  CP-SBMV s i n g l e  i n Botswana blackeye,  but 50%  i n f e c t e d by CP-SBMV had mottled  CP-TMV and double i n f e c t i o n caused more seed  coat d i s c o l o u r a -  tion. D.  D i s t r i b u t i o n and c o n c e n t r a t i o n o f CP-SBMV and CP-TMV  nucleoproteins  in cowpea. Analytical  sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n proved  f o r s e p a r a t i o n o f the v i r u s e s i n a n a t u r a l mixture sap  ( F i g u r e s 7 and 8 ) .  of p a r t i a l l y  to be s u i t a b l e clarified  F u r t h e r , the T r i s - H C l b u f f e r pH 6 . 5 a l s o proved to  be s u i t a b l e as i t caused minimal or no aggregation  o f CP-TMV  ( F i g u r e s 7 and  8). 1.  D i s t r i b u t i o n and c o n c e n t r a t i o n o f CP-SBMV and CP-TMV i n o c u l a t e d primary  (a) be d e t e c t e d tially after  leaves o f C a l i f o r n i a blackeye  S y n t h e s i s a f t e r simultaneous by a n a l y t i c a l  clarified  sap  inoculation.  inoculations.  in the  cowpea.  CP-SBMV c o u l d not  sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n o f par-  prepared  from primary  leaves when analysed  48 hours  However, CP-SBMV was d e t e c t e d a t 9 6 to 1 9 2 hours a f t e r  90  Figure  7-  R e l a t i v e absorbance p r o f i l e s f o l l o w i n g c e n t r i f u g a t i o n , through sucrose d e n s i t y g r a d i e n t columns, o f 0.2 ml o f c l a r i f i e d e x t r a c t s o f primary leaves o f C a l i f o r n i a blackeye cowpea i n o c u l a t e d s i n g l y w i t h e i t h e r CP-SBMV or CP-TMV or w i t h CP-SBMV and CP-TMV and harvested 8 days a f t e r i n o c u l a tion. An equal amount (20 g f r . wt.) o f t i s s u e from each inoculum treatment was used and d i l u t e d 1/2 (w/v), in t h i s p a r t i c u l a r experiment. The g r a d i e n t s were c e n t r i f u g e d in a Beckman SW :4l r o t o r , a t 39,000 rpm a t 5°C f o r 105 minutes and scanned a t 254 nm (0.5 absorbance range) s c a l e . Direct i o n o f s e d i m e n t a t i o n i s to the l e f t .  A.  P r o f i l e s o f e x t r a c t s of t i s s u e and CP-TMV.  i n o c u l a t e d w i t h CP-SBMV  B.  P r o f i l e o f e x t r a c t s o f t issue  i n o c u l a t e d w i t h CP-SBMV.  C.  P r o f i l e o f e x t r a c t s o f t issue  i n o c u l a t e d w i t h CP-TMV.  D.  P r o f i l e o f e x t r a c t s o f t i s s u e mock-inocuIated w i t h buffer only.  Relative  absorbance at 254 nm  92  F i g u r e 8.  R e l a t i v e absorbance p r o f i l e s f o l l o w i n g c e n t r i f u g a t i o n , through sucrose d e n s i t y g r a d i e n t columns, o f 0.2 ml o f c l a r i f i e d e x t r a c t s o f 3rd t r i f o l i a t e leaves which developed a f t e r inoc u l a t i o n o f C a l i f o r n i a blackeye cowpea with e i t h e r CP-SBMV or CP-TMV o r w i t h CP-SBMV and CP-TMV and harvested 20 days a f t e r i n o c u l a t i o n o f the primary leaves. An equal amount (4 g f r . wt.) o f t i s s u e from each treatment inoculum was used and d i l u t e d 1/3 (w/v), in t h i s p a r t i c u l a r experiment; The g r a d i e n t s were c e n t r i f u g e d in a Beckman SW 41 r o t o r , a t 39,000 rpm a t 5 C f o r 105 minutes and scanned a t 254 nm (0.5 absorbance range). D i r e c t i o n o f s e d i m e n t a t i o n i s to the l e f t .  A.  P r o f i l e s o f e x t r a c t s of t r i f o l i a t e t e m i c a l l y by CP-SBMV and CP-TMV.  tissue  infected sys-  B.  P r o f i l e of extracts of t r i f o l i a t e t e m i c a l l y by CP-SBMV.  tissue  infected sys-  C.  P r o f i l e of extracts m i c a l l y by CP-TMV.  D.  P r o f i l e of extracts of t r i f o l i a t e tissue a f t e r leaves were mock-inocu1ated w i t h b u f f e r o n l y .  of t r i f o l i a t e  tissue  infected  syste-  primary  R e l a t i v e absorbance at 254 nm  3h  i n o c u l a t i o n , and i t was always reduced inoculation to that  caused  reductions  in s i n g l y i n f e c t e d t i s s u e ( F i g u r e 7 and T a b l e V I ) .  t o that  (b)  Synthesis  inoculated intervals  (0.0,  a f t e r sequential  i n o c u l a t i o n by 24 to 27%, be d e t e c t e d  inoculations.  0.5, 6.0 and 24.0 hr) a f t e r to o n l y  primary l e a v e s .  (Figure 9)-  the  Although the com-  as e a r l y as 48  ( F i g u r e 7 and T a b l e V I ) . When primary  In c o n t r a s t  When the r e v e r s e  CP-TMV was i n o c u l a t e d synthesis  initial  about 59 to 65% o f that  by about 50% o f i t s c o n c e n t r a t i o n  that  Double  leaves  w i t h CP-SBMV, were cha11enge-inocu1ated w i t h CP-TMV a t v a r i o u s  synthesized  culated  in double  in s i n g l e i n o c u l a t i o n , i t could  hours a f t e r i n o c u l a t i o n  was  infected tissue.  o f 56 to 68% in the amount o f CP-SBMV compared  amount o f CP-TMV was a l s o reduced pared  i n doubly  i n o c u l a t i o n , CP-SBMV  synthesized  the c h a l l e n g i n g  in s i n g l y  CP-TMV was reduced  i n s i n g l y i n f e c t e d primary  leaf  sequence was made on t h e primary  first  ino-  tissue  l e a v e s , so  and then cha11enge-inocu1ated w i t h CP-SBMV,  o f CP-SBMV was o n l y  29 to 59% o f that  in s i n g l y  inoculated  leaves. 2.  D i s t r i b u t i o n and c o n c e n t r a t i o n cowpea v a r i e t y  leaves  i t was n o t i c e d  that  s t u d i e s w i t h seeds o f cowpea v a r i e t i e s the v a r i e t y V 4 5 B o t s d i d not show symp_  toms when i n o c u l a t e d w i t h CP-SBMV a l o n e and no v i r u s could the  trifoliate  leaves  It was t h e r e f o r e  o f Botswana  V45-Bots.  During the seed t r a n s m i s s i o n from Botswana,  in primary  o f such p l a n t s  by i n f e c t i v i t y  be d e t e c t e d in  and s e r o l o g i c a l t e s t s .  reasoned that CP-TMV might c o n d i t i o n t h i s cowpea v a r i e t y  T a b l e VI.  R e l a t i v e amounts o f CP-SBMV and CP-TMV nuc1eoprotein in s i n g l e (SI) and double (Dl) i n f e c t i o n s o f primary leaves o f C a l i f o r n i a blackeye cowpea harvested a t various intervals a f t e r inoculation  Nucleoprotein content (mg/g f r . wt.) a t v a r i o u s i n t e r v a l s (hr) a f t e r i n o c u l a t i o n  Inoculum  48  96  144  196  Buffer  0.0  0.0  0.0  0.0  CP-SBMV-SI  0.0  0.03  0.13  0.16  CP-SBMV-DI  0.0  0.01  0.05  0.07  Percent o f CP-SBMV-DI as o f CP-SBMV-SI  0.0  CP-TMV-Sl"  0.08  CP-TMV-DI  0.06  Mean o f 4  experiments  75-0  38.5  43.8  0.34  0.85  1.49  0.25  0.65  1.14  73-5  Percent o f CP-TMV-DI as o f CP-TMV-SI  33.3  76.5  76.5  96  F i g u r e 9.  R e l a t i v e amounts o f CP-SBMV and CP-TMV n u c l e o p r o t e i n s (mg/g f r . wt.) i n primary leaves o f C a l i f o r n i a b l a c k eye cowpea i n o c u l a t e d s i n g l y and s e q u e n t i a l l y with either v i r u s at various i n t e r v a l s . T i s s u e was analysed 192 hours a f t e r i n i t i a l i n o c u l a t i o n .  A.  Y i e l d o f CP-SBMV in leaves cha11enge-inocu1ated with CP-TMV (T/S) or a f t e r super i n o c u l a t i o n t o leaves p r e - i n o c u l a t e d with CP-TMV (S/T) at v a r i o u s i n t e r v a l s a f t e r i n t i a l i n o c u l a t i o n and i n s i n g l e inoculation ( S l ) .  B.  Y i e l d o f CP-TMV in leaves c h a l l e n g e - i n o c u l a t e d .;; w i t h CP-SBMV (S/T) or a f t e r s u p e r i n o c u l a t ion t o leaves p r e - i n o c u l a t e d with CP-SBMV (T/S) a t various intervals a f t e r i n i t i a l inoculation and in s i n g l e i n o c u l a t i o n ( S l ) .  A  98  to become s y s t e m i c a l l y s u s c e p t i b l e t o CP-SBMV. In e x t r a c t s obtained trifoliate  leaves  o f doubly  could  be d e t e c t e d  vided  an o p p o r t u n i t y (a)  inoculated  V45 Bots p l a n t s , both v i r u s e s _  by i n f e c t i v i t y and s e r o l o g i c a l t e s t s .  Synthesis  for further  This  inoculations.  When V45 Bots cowpea _  p l a n t s were s i n g l y i n o c u l a t e d with CP-SBMV, the v i r u s could  sucrose d e n s i t y ted 2 - f o l d by  leaves  gradient  c e n t r i f u g a t i o n , even when e x t r a c t s were However, CP-SBMV could  i n f e c t i v i t y and sucrose d e n s i t y g r a d i e n t  (Tables  leaves  that CP-SBMV m u l t i p l i e d CP-SBMV recovery therefore  of CP-SBMV  i t was d i f f i c u l t  in these l e a v e s .  t i s s u e o f primary  leaves  than  D i s t r i b u t i o n and c o n c e n t r a t i o n  Synthesis leaves  primary  in s y s t e m i c a l l y  in doubly  leaves  i n o c u l a t e d with CP-TMV.  leaves was e r r a t i c and (Table V I I I ) about the  CP-TMV y i e l d was g r e a t e r in doubly  indicated  inoculated  in s i n g l y  leaves.  o f CP-SBMV and CP-TMV  in t r i f o l i a t e  blackeye cowpea.  a f t e r simultaneous  s y n t h e s i s o f CP-SBMV  in the i n o c u l a t e d  t o draw any c o n c l u s i o n  leaves o f C a l i f o r n i a (a)  in unconcentra-  i n o c u l a t e d with both v i r u s e s  in these leaves when doubly  from s i n g l y i n o c u l a t e d  rate of i t s synthesis  3.  r e a d i l y be detected  centrifugation  that were doubly  concentra-  VII and V I M ) .  A time-course s y n t h e s i s  infected  be detected  by i n f e c t i v i t y assay but not by a n a l y t i c a l  (w/v) by 10% PEG.  ted e x t r a c t s o f primary  r e s u l t pro-  i n v e s t i g a t i o n o f t h i s type o f i n t e r a c t i o n .  a f t e r simultaneous  in e x t r a c t s o f primary  from  i n f e c t e d 1 s t , 2nd, 3rd and 4th t r i f o l i a t e i n o c u l a t i o n s o f primary  infected t r i f o l i a t e  leaves.  The  leaves was g r e a t l y enhanced.  99  Table VII.  Leaf  N u c l e o p r o t e i n y i e l d o f CP-SBMV and CP-TMV in s i n g l y (SI) and doubly (Dl) i n o c u l a t e d ( s i m u l t a n e o u s l y ) primary leaves and s y s t e m i c a l l y i n f e c t e d 3rd and 4th t r i f o l i a t e leaves o f Botswana cowpea v a r i e t y V45~Bots, analysed a t v a r i o u s i n t e r v a l s by a n a l y t i c a l sucrose d e n s i t y g r a d i e n t c e n t r i fugation  type  Leaf agg (days)  Nucleoprotein CP- SBMV SI Dl  Pr imary  20  0  3rd  20  o  Pr imary  30  4th  30  a  trifoliate  trifoliate  (mg/g. f r . wt) CP-TMV SI Dl -  0.24  1.39  1.04  c  0.20  0.32  0.30  o  c  0.20  o  e  0. 12  _d  -  1.34 0. 10  Mean o f two e x p e r i m e n t s  ^Days s i n c e Concentrated range). d  yield  inoculation. 2- t o 1 0 - f o l d  a n d s c a n n e d a t 254 nm (0.05 a b s o r b a n c e  Not tested  Concentrated  20-fold  a n d s c a n n e d a t 254 nm (0.05 a b s o r b a n c e r a n g e ) .  100  Table VIII.  I n f e c t i v i t y o f CP-SBMV in e x t r a c t s from primary leaves of cowpea v a r . V 4 5 B o t s , a t d i f f e r e n t i n t e r v a l s a f t e r inoc u l a t i o n w i t h CP-SBMV alone or with CP-TMV -  Mean number o f l o c a l l e s i o n s per h a l f l e a f o f GA 21 a t v a r i o u s i n t e r v a l s (days) a f t e r i n o c u l a t i o n Inoculum  1  3  6  10  20  Expt. No. 1 Buffer-inoculated CP-SBMV-inoculated  extracts  o  62  extracts  273  CP-SBMV + CP-TMV  Expt. No. 2 Buffer-inoculated CP-SBMV-inoculated  extracts extracts  CP-SBMV + CP-TMV  a  Not  tested  ^No v i r u s  detected-  0  0  0  0  0  0  0  71  0  k  22  130  b  101  by  the presence o f CP-TMV.  from doubly infected  infected  leaves  About f i v e times as much CP-SBMV was o b t a i n e d  3rd t r i f o l i a t e  (Figure  8).  1 eaves, compared to that  o f CP-TMV, as i t was s y n t h e s i z e d  and  doubly  doubly  infected  trifoliate than  on  trifoliate  declined  inoculation,  leaves which appeared about 7 and 10  respectively.  infections  Synthesis  in systemically inoculation  in the 3rd t r i f o l i a t e  amount of CP-SBMV  infected  However, the i n f l u e n c e  infected  o f primary  leaves,  in doubly  When CP-SBMV was i n o c u l a t e d  3rd t r i f o l i a t e leaves.  inoculation  l a t e d on the primary  infected  interval  between  to primary  i n o c u l a t i o n was reduced. primary  that  in s i n g l y  8 and 10). leaves,72 hours a f t e r o f CP-SBMV  initial  in the 3rd  simultaneously  The r a t e o f CP-SBMV increase  preceded CP-TMV in the i n o c u l a t e d  leaves were  leaves r e l a t i v e t o that  in comparable leaves o f p l a n t s  leaves.  The syntheses o f  o f primary  i n o c u l a t i o n with CP-TMV, there was g r e a t e r s y n t h e s i s leaves than  leaves a f t e r  because i t was in these leaves  leaves was the h i g h e s t , ( F i g u r e s  trifoliate  o f CP-TMV  8 and 10).  (Figures  CP-SBMV and CP-TMV a f t e r s e q u e n t i a l  the  rapidly,  o f CP-SBMV seemed t o be even more pronounced as each v i r u s  sequential  studied  in both s i n g l y and  which a t the time o f i n o c u l a t i o n were e l o n g a t i n g  in s i n g l e  (b)  Both v i r u s e s ,  in both s i n g l y  t i s s u e s , seemed to be s y n t h e s i z e d more in the 1st and 2nd  leaves,  the s y n t h e s i s  to about the same extent  leaves.  in the 3rd and 4th t r i f o l i a t e  days a f t e r  singly  Double i n f e c t i o n d i d not seem t o a f f e c t synthe-  sis  infected  from  declined  inocuas the  On the o t h e r hand, when CP-SBMV leaves by 24 or 72 hours, only  102  F i g u r e 10.  R e l a t i v e amounts o f CP-SBMV and CP-TMV n u c l e o p r o t e i n s (mg/g f r . wt.) in t h e 1st, 2nd, 3rd and 4th t r i f o l i a t e , leaves o f C a l i f o r n i a blackeye cowpea analysed 10, 15, 20 and 25 days, r e s p e c t i v e l y , a f t e r s i n g l e and simultaneous double i n o c u l a t i o n s o f primary l e a v e s . The numbers on top o f the f i g u r e correspond to the f i r s t to f o u r t h t r i f o 1iate leaves.  A  Dl.  CP-•SBMV from  doubly i n f e c t e d  tissue.  A  SI.  CP-•SBMV from  singly infected  tissue.  B  Dl.  CP-•TMV from  doubly i n f e c t e d  tissue.  B  SI.  CP-•TMV from  singly infected  tissue.  1st  2nd  I  I  3rd  4th  I  I  Days a f t e r  1st  I  I  2nd  3rd  I I  inoculation  .4th  |  104  about  twice as much CP-SBMV was s y n t h e s i z e d in doubly  in s i n g l y  infected  leaves a f t e r preceded  tissue.  i n o c u l a t i o n w i t h both v i r u s e s than when CP-SBMV  in the i n o c u l a t e d primary  leaves (Table IX).  hand, CP-TMV s y n t h e s i s in the 3 r d t r i f o l i a t e appreciably affected of  t i s s u e , as  More CP-SBMV was s y n t h e s i z e d i n the t r i f o l i a t e  simultaneous  CP-TMV  infected  by double  On the other  l e a f d i d not seem t o be  i n f e c t i o n or by the sequence of i n o c u l a t i o n  the primary l e a v e s . In a l l doubly  50% o f t h e : t o t a l (c)  infected  3rd t r i f o l i a t e  n u c l e o p r o t e i n produced  leaves CP-SBMV was l e s s  in such  than  tissue.  E l e c t r o n miscroscopy o f s i n g l y and s i m u l t a n e o u s l y i n o c u l a t e d primary  leaves and s y s t e m i c a l l y  infected  trifoliate  leaves.  When t h i n s e c t i o n s were examined w i t h an e l e c t r o n microscope, CP-SBMV-like p a r t i c l e s and CP-TMV p a r t i c l e s were.observed and parenchyma c e l l s and o f s y s t e m i c a l l y a few c e l l s trifoliate  o f s i m u l t a n e o u s l y doubly infected  o f doubly  trifoliate  i n o c u l a t e d primary  both  in the same v e i n a l  i n o c u l a t e d primary leaves  leaves ( F i g u r e 1 1 ) .  Occasionally,  leaves and s y s t e m i c a l l y  infected  leaves c o n t a i n e d o n l y CP-SBMV-like p a r t i c l e s and CP-TMV  particles  separately. The  CP-SBMV c r y s t a l s  this virus  reported t o occur  (Weintraub and R a g e t l i ,  s i n g l y or doubly r a t e attempt  infected  plants  in cowpea c e l l s  i n f e c t e d by  1 9 7 0 ) were, s u r p r i s i n g l y , not found in  in the present study, even when a d e l i b e -  was made to search f o r them a t the same sampling  i n o c u l a t i o n , as used  by these workers.  This is interesting  time,  after  because the  Table  IX.  Concentration of CP-SBMV and o f CP-TMV in 3rd t r i f o l i a t e leaves ( u n d i f f e r e n t i a t e d at time o f i n o c u l a t i o n ) o f C a l i f o r n i a blackeye cowpea a f t e r s i n g l e and s e q u e n t i a l i n o c u l a t i o n s o f primary leaves  Interva1 between i n o c u l a t ions (hr)  Concentration (mg/g f r . wt) o f CP-SBMV and CP-TMV in t i s s u e harvested 20 days a f t e r i n o c u l a t i o n .  I nocu1um Inital  Challenge  CP-SBMV-Sl  b  CP-SBMV-DI  0. k 0  C  CP-TMV-Sl  b  CP-TMV-D|  2.02 .80  CP-TMV  CP-SBMV  1  2k  CP-TMV  CP-SBMV  1.17  1.91  12  CP-TMV  CP-SBMV  1.86  2.19  2k  CP-SBMV  CP-TMV  0.87  2.08  12  CP-SBMV  CP-TMV  0.85  1.95  Mean o f 5  experiments.  From t i s s u e s i n g l y From doubly  infected  infected  tissue.  by CP-SBMV or CP-TMV.  .Ok  1  C  106  Figure  11.  E l e c t r o n micrographs o f c e l l s o f primary and t r i f o l i a t e leaves o f C a l i f o r n i a blackeye cowpea doubly i n f e c t e d by CP-SBMV and CP-TMV. T i s s u e was s e c t i o n e d 10 days after inoculation.  A.  V a s c u l a r c e l l o f primary l e a f showing p a r t i c l e s (S) and CP-TMV rods ( T ) .  B.  Parenchyma c e l l o f t r i f o l i a t e l e a f showing both CP-SBMVl i k e p a r t i c l e s (S) and CP-TMV rods ( T ) .  C.  V a s c u l a r c e l l o f t r i f o l i a t e l e a f showing both CP-SBMVl i k e p a r t i c l e s (S) and CP-TMV rods ( T ) .  D.  Parenchyma  cell  of healthy t r i f o l i a t e  both  CP-SBMV-like  leaf-control.  107  108  109  same CP-SBMV study.  i s o l a t e used by Weintraub and R a g e t l i (1970) was used  The o n l y major d i f f e r e n c e in the s t u d i e s reported  Hoagland's s o l u t i o n was used k.  i n s t e a d of s o i l  at the time o f simultaneous primary  V45-Bots that were not formed  double and s i n g l e  made from t r i f o l i a t e  be d e t e c t e d  leaves o f p l a n t s that were s i n g l y  o f 70 p l a n t s , s i n g l y  checked by i n f e c t i v i t y and s e r o l o g y .  by CP-SBMV based on assays  o f the f i r s t  i n o c u l a t i o n , assays  o f the t h i r d  content.  simultaneous  trifoliates  by CP-SBMV.  i n t o these  same leaves were not r e - t e s t e d 20 days a f t e r foliate  2 0 - f o l d by 10% PEG  and second t r i f o l i a t e s ;  when 1st and 2nd t r i f o l i a t e  mean that CP-SBMV d i d not move r e a d i l y  i n o c u l a t e d with .  25% o f the p l a n t s were  that 75 t o 100% o f the p l a n t s were i n f e c t e d i n f e c t i o n obtained  infec-  i n o c u l a t e d with CP-SBMV, were  At 10 days a f t e r  l a t i o n w i t h CP-SBMV and CP-TMV, l e s s than  of  by s e r o l o g y ,  sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n in p r e p a r a t i o n s  A total  simultaneous  until  -  CP-SBMV, even when such p r e p a r a t i o n s were c o n c e n t r a t e d  after  i n o c u l a t i o n s of the  leaves o f V45 Bots were t e s t e d at a l l stages  i n o c u l a t i o n , no CP-SBMV c o u l d  and a n a l y t i c a l  precipitation.  in t r i f o l i a t e  leaves.  When t r i f o l i a t e 30 days a f t e r  i s that  as the growth medium.  D i s t r i b u t i o n and c o n c e n t r a t i o n o f CP-SBMV and CP-TMV leaves o f Botswana cowpea v a r i e t y  tivity  here  in t h i s  inocu-  infected a t 20 days indicated  The lower percentage  leaves were t e s t e d could l e a v e s , but s i n c e the  i n o c u l a t i o n when the 3rd t r i -  leaves were t e s t e d , no c o n c l u s i o n can be reached  about t h e i r v i r u s  110  More o f each v i r u s , e s p e c i a l l y leaves o f V 4 5 B o t s cowpea than _  values obtained dinately  f o r CP-TMV  CP-TMV, was s y n t h e s i z e d  in t r i f o l i a t e  leaves  (Table V I I ) .  in the 3 r d and 4 t h t r i f o l i a t e  low f o r t h i s v i r u s (Table V I I ) .  in the primary  Aggregation  The  leaves were  was suspected  inorduring  centrifugation  through sucrose columns, even though e x t r a c t s of primary  and  leaves were prepared  trifoliate  However, fuged  infectivity  with  t e s t s with v i r u s p r e p a r a t i o n s  the t r i f o l i a t e  leaves.  CP-TMV seems to be s y n t h e s i z e d e q u a l l y the t r i f o l i a t e D.  1.  trifoliate  Arrival  Movement  these  in s i n g l e and double  leaves of C a l i f o r n i a  blackeye  of CP-SBMV and CP-TMV  leaves b e f o r e CP-SBMV.  leaves were c l o s e l y  cowpea.  i n t o the 3 r d t r i f o l i a t e  leaves  after  timed,  On the other  i n which the i n t e r -  leaves and removal of the 3 r d t r i -  CP-TMV a r r i v e d inoculation.  as the time r e q u i r e d by CP-TMV to reach  leaves was  that CP-TMV always a r r i v e d  In two experiments  leaves between 62 and 64 hours a f t e r  (Table X).  i n preformed  leaves.  in s e v e r a l experiments, and i t was found  inoculation  cowpea,  infection of  o f CP-SBMV and CP-TMV at the 3 r d t r i f o l i a t e  between i n o c u l a t i o n o f the primary  foliate  blackeye  contained  leaves.  i n o c u l a t i o n o f primary  studied  As i n C a l i f o r n i a  leaves  D i s t r i b u t i o n and c o n c e n t r a t i o n o f CP-SBMV and CP-TMV 3rd  val  that were not c e n t r i -  through sucrose columns-, i n d i c a t e d that the primary  more v i r u s than  at  the same b u f f e r a t the same time.  at the 3 r d t r i f o l i a t e S i x t y two hours was taken  the 3 r d t r i f o l i a t e  leaves  after  hand, CP-SBMV r e q u i r e d 84 hours to  111  a r r i v e a t the 3rd t r i f o l i a t e  leaves  i t s own r a t e t o the 3rd t r i f o l i a t e  (Table X).  Each v i r u s moved at  leaves, whether  in s i n g l e or double  i n f e c t i o n s , as long as the v i r u s e s were not mixed but were inoculated had  separately to opposite  i n d i c a t e d that CP-SBMV was delayed  in a mixed to  h a l f leaves.  reach  leaves  Preliminary  leaves.  before  (Table X) was based on data  i n o c u l a t e d to o p p o s i t e  Thus CP-TMV a r r i v e d at the 3rd t r i f o l i a t e  Time-course s y n t h e s i s o f CP-SBMV and CP-TMV tr i f o l i a t e  1 eaves.  sucrose d e n s i t y g r a d i e n t  Both v i r u s e s could  l e a f 22 hours  in preformed 3rd  be d e t e c t e d  by a n a l y t i c a l  c e n t r i f u g a t i o n in both s i n g l y and doubly  t i s s u e e x t r a c t s 5 days a f t e r  inoculation.  ments CP-SBMV was too low t o be detected  the peak o f s y n t h e s i s  leaves.  5 days a f t e r i n o c u l a t i o n .  10 t o 12 days a f t e r  The peak s y n t h e s i s p e r i o d  infec-  However, in some e x p e r i -  It would appear that both v i r u s e s , in s i n g l e and double reach  obtained  CP-SBMV. 2.  ted  inoculated  The time r e q u i r e d f o r each v i r u s  when each v i r u s was s e p a r a t e l y and s i m u l t a n e o u s l y half  observations  i f i t was s i m u l t a n e o u s l y  inoculum c o n t a i n i n g CP-TMV.  the 3rd t r i f o l i a t e  simultaneously  infections,  i n o c u l a t i o n o f the primary  i s then f o l l o w e d  by a p e r i o d o f  apparent d e c l i n e ( F i g u r e 12). 3.  E f f e c t o f sequence of a r r i v a l of the other  in the preformed 3rd t r i f o l i a t e  s i n g l e and s e q u e n t i a l It can be seen preceded  o f each v i r u s on the c o n c e n t r a t i o n  i n o c u l a t i o n s o f the primary  (Table XI) that CP-SBMV was s y n t h e s i z e d  i t by 22 hours;  leaves  after leaves.  most when CP-TMV  the r a t i o o f CP-SBMV c o n c e n t r a t i o n  in doubly  112  T a b l e X.  Movement o f CP-SBMV "and o f CP-TMV i n t o 3 r d t r i f o l i a t e leaves of C a l i f o r n i a blackeye cowpea which were formed i ( 0 . 5 - l - 0 cm long) at the time o f i n o c u l a t i o n  I n t e r v a l between inocul a t i o n o f primary leaves and t r i f o l i a t e removal (hr)  Number o f 3 r d t r i f o l i a t e leaves that became i n f e c t e d w i t h e i t h e r CP-SBMV or CP-TMV over number o f leaves t e s t e d CP-SBMV-SI  CP-SBMV-DI  CP-TMV-SI  CP-TMV-DI  Experiment 1  0/3 0/2 0/3 0/3 0/3 0/3 3/3 3/3 3/3  0/3 0 /5 0/3 0/3 3/3 3/3 3/3  0/3 0/3 0/3 2/3 2/3 3/3 3/3 3/3  54 56 60 62  -  64  -  0/4 0/4 1/4 3/4 4/4 4/4 4/4 4/4 -  0/4 3/4 4/4 4/4 4/4 4/4 4/4 4/4 4/4 4/4  -  4/4  48  50 60 66 72 78 84  86 88  b  0/3 3/3 3/3 3/3 3/3 3/3 3/3  Experiment 2  66 68 70 72 74 78 80  0/4 0/4 0/4 0/4  0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4  82  1/4  2/4  -  113  T a b l e X.  Cont. :  I n t e r v a l between inocul a t i o n o f primary leaves and t r i f o l i a t e removal (hr)  Number o f 3rd t r i f o l i a t e leaves that became i n f e c t e d with e i t h e r CP-SBMV or CP-TMV over number o f leaves t e s t e d CP-SBMVSI  CP-SBMV-DI  CP-TMV-SI  CP-TMV-DI  k/k k/k  k/k k/k  -  k/k k/k  Experiment 2  8k 86  " I n o c u l a in each experiment were a p p l i e d s e p a r a t e l y , i . e . CP-SBMV on the r i g h t h a l f l e a f and CP-TMV on the l e f t h a l f when double inoc u l a t i o n s were made o r a p p l i e d as above and the l e f t h a l f (CP-SBMV) was i n o c u l a t e d w i t h b u f f e r when s i n g l e i n o c u l a t i o n s were made. b  Not  tested.  114  F i g u r e 12.  Time-course r e p l i c a t i o n o f CP-SBMV and CP-TMV as i n d i c a t e d by t h e i r r e s p e c t i v e n u c l e o p r o t e i n c o n t e n t s (mg/g. f r . wt.) in preformed 3rd t r i f o l i a t e leaves o f C a l i f o r n i a blackeye cowpea a f t e r s i n g l e and simultaneous double i n o c u l a t i o n s of the primary l e a v e s .  A.  CP-SBMV.  B.  CP-TMV.  Third  trifoliate  leaves doubly  infected  ( A  A )  Third  trifoliate  leaves s i n g l y  infected  (O  O)  Days a f t e r i nocu1 at ion  116  infected  t i s s u e to that  in s i n g l y  neous i n o c u l a t i o n s were made w i t h theoretically,  t h i s should  i n f e c t e d t i s s u e was 2 . 7 -  When s i m u l t a -  the two v i r u s e s on the primary  have r e s u l t e d  in the a r r i v a l  a f t e r CP-TMV had a r r i v e d at the 3 r d t r i f o l i a t e  leaves.  o f CP-SBMV 22 hours S i m i l a r l y when  CP-SBMV was i n o c u l a t e d 22 hours b e f o r e CP-TMV, t h i s should  have r e s u l t e d  simu1tanous1y at the 3 r d t r i f o l i a t e  in both v i r u s e s a r r i v i n g  When CP-SBMV was i n o c u l a t e d so that  leaves,  leaves.  i t a r r i v e d a t the 3 r d t r i f o l i a t e  leaves  72  hours before CP-TMV, enhancement was apparent, but to a l e s s e r e x t e n t .  On  the other  72  hours a f t e r the o t h e r ,  i t s s y n t h e s i s was g r e a t l y r e t a r d e d ,  to  that reached  infected plants  reductions on  hand, when e i t h e r v i r u s a r r i v e d a t the 3 r d t r i f o l i a t e  in s i n g l y  (Table X I ) .  compared  Although  in CP-TMV a r e apparent when i t was introduced  leaves  after  slight CP-SBMV,  the whole t h i s v i r u s was s l i g h t l y a f f e c t e d , i f i t reached the 3 r d  trifoliate E.  leaves w i t h i n 22 hours o f CP-SBMV a r r i v a l  Concentration  o f CP-SBMV and CP-TMV  leaves o f C a l i f o r n i a differential The  blackeye  temperature  cowpea synchronously  infection  between CP-SBMV and CP-TMV and c o n t r a s t  1.  i t with  The hypothesis  in t h i s  s e c t i o n was to  system on the i n t e r a c t i o n the i n t e r a c t i o n  obtained  was, under synchronous  that s y n t h e s i s o f each v i r u s would proceed  E f f e c t o f synchronous  i n f e c t e d by  manipulation.  i n v e s t i g a t e the e f f e c t o f synchronous  infection,  leaves.  in the preformed 3 r d t r i f o l i a t e  o b j e c t i v e o f the experiments reported  under asynchronous c o n d i t i o n s .  at these  independently.  i n f e c t i o n on accumulation  o f v i r u s in the  3rd  trifoliate  The  i n f e c t i v i t y o f CP-SBMV and CP-TMV from t r i f o l i a t e  leaves. leaves o f  T a b l e XI.  Concentration o f CP-SBMV when CP-TMV a r r i v e d b e f o r e (+) and a f t e r (-) CP-SBMV and o f CP-TMV when CP-SBMV a r r i v e d before (+) and a f t e r (-) CP-TMV a t the preformed 3 r d t r i f o l i a t e leaves o f C a l i f o r n i a blackeye cowpea  Predicted interval between a r r i v a l o f CP-SBMV and CP-TMV at 3 r d t r i f o l i a t e s  Concentration (mg/g f r . wt) of CP-SBMV and CP-TMV in t i s s u e harvested 15 days a f t e r i n i t i a l i nocu1 at ion  (hr)  CP-SBMV-D|  b  +72 +22 + 12 0 -12 -22 -72  Mean o f 4  0.53 2.30 1.70 1.57 1.08  CP-SBMV-SI  0.85  1.14  1 .02  experiments.  b  Conc. o f CP-SBMV  C  in r e l a t i o n to a r r i v a l o f CP-TMV.  Conc. of CP-TMV in r e l a t i o n to a r r i v a l o f CP-SBMV.  CP-TMV-DI  C  CP-TMV-SI  R a t i o o f DI/SI CP-SBMV  0.64  0.6  2.50 2.78 3.00 3.20 3.00 3.10  2.7 2.0 1.9 1.3 1.3 1.2  3.1  CP-TMV  0.2 0.8 0.9 1.0 1.0 1.1 1.0  118  p l a n t s that had been were s h i f t e d  inocu1ated "and incubated f o r 42 hours b e f o r e they  t o the styrofoam chamber was n e g a t i v e f o r e i t h e r v i r u s when  e x t r a c t s were assayed on t h e i r  respective  t i o n o f p l a n t s f o r 42 hours a f t e r because  i t had been found  could not s u r v i v e for  long p e r i o d s o f c o l d  primary  Thus,  ino'culation was r o u t i n e l y  in p r e l i m i n a r y experiments  42 hours, b e f o r e the s h i f t ,  the i n o c u l a t e d  indicator hosts.  treatment  used,  that cowpea  (10°C).  incubamainly  plants  An i n c u b a t i o n  allowed the v i r u s e s t o be e s t a b l i s h e d in  l e a v e s , long b e f o r e they c o u l d move i n t o the 3rd  t r i f o l i ate 1 eaves. P l a n t s were used u n e x c i s e d , because Schlegel,  1976c),under  excised  plants  the c o n d i t i o n s o f these experiments,  p o o r l y and u s u a l l y d i d not grow f o r some time a f t e r I n f e c t i v i t y o f t i s s u e harvested at z e r o time for  (Dawson and performed  excision.  (5 days) was n e g a t i v e  e i t h e r v i r u s , thus s u g g e s t i n g that the 3rd t r i f o l i a t e leaves were not  infected  at the time p l a n t s were moved from the styrofoam chamber.  infectivity after  o f CP-TMV was r o u t i n e l y d e t e c t e d in t i s s u e a n a l y s e d 8 hours  the s h i f t  occasionally infected  t o the p e r m i s s i v e temperature,  ( i n 3 experiments  t i s s u e and not at a l l in s i n g l y  t i v i t y was always  lower than that  the s h i f t  infected  infected  ones) d e t e c t e d in doubly tissue  (Figures  t i s s u e at 24 hours,  in e x t r a c t s from doubly  r a t e of s y n t h e s i s o f each v i r u s  96 hours a f t e r  but that o f CP-SBMV was  out of 7 s u c c e s s f u l  When CP-SBMV was d e t e c t e d in s i n g l y  The  However,  i t s infec-  infected  increased r a p i d l y  to the p e r m i s s i v e temperature.  13 and 14).  tissue.  between 24 and  These  results  119  F i g u r e 13-  R e l a t i v e i n f e c t i v i t y o f CP-SBMV in 3 r d t r i f o l i a t e leaves of C a l i f o r n i a blackeye cowpea i n f e c t e d synchronously s i n g l y by CP-SBMV and doubly by CP-SBMV and CP-TMV a t 10 C and s h i f t e d t o 27 C. I n f e c t i v i t y i s expressed as mean number o f l o c a l l e s i o n s per h a l f l e a f o f GA 2 1 . Zero time i s when p l a n t s were moved from the styrofoam chamber t o the p e r m i s s i v e temperature o f 27 C.  T h i r d t r i f o l i a t e leaves doubly i n f e c t e d expressed as mean number o f l o c a l l e s i o n s ( A A) T h i r d t r i f o l i a t e leaves s i n g l y i n f e c t e d expressed as mean number o f l o c a l l e s i o n s ( O O).  120  121  F i g u r e 14.  R e l a t i v e i n f e c t i v i t y o f CP-TMV in 3rd t r i f o l i a t e leaves of C a l i f o r n i a blackeye cowpea i n f e c t e d synchronously s i n g l y by CP-TMV and doubly by CP-SBMV and CP-TMV at 10°C and s h i f t e d t o 27 C. I n f e c t i v i t y i s expressed as mean number of l o c a l l e s i o n s per h a l f l e a f o f N_. g l u t ! n o s a . Zero time is when p l a n t s were moved from the styrofoam chamber to the p e r m i s s i v e temperature o f 27 C.  T h i r d t r i f o l i a t e leaves doubly i n f e c t e d expressed as mean number o f l o c a l l e s i o n s ( A — A ). ;  T h i r d t r i f o l i a t e leaves s i n g l y i n f e c t e d expressed as mean number of l o c a l l e s i o n s ( O O).  Mean no.  o  N>  o  -t"  o  of  lesions/half  ON  o  leaf x di1. x  CO  o  O  o  10  M  o  4=-  o  ro  123  (Figures  13 and \k)  ted 3rd t r i f o l i a t e  i n d i c a t e that both v i r u s e s  t i s s u e r e p l i c a t e d synchronously.  asynchronous c o n d i t i o n s , CP-TMV was s y n t h e s i z e d doubly synchronously  infected tissue.  c o n d i t i o n s was enhanced singly  in doubly  infected t i s s u e (Figures  It  (Figure  in s i n g l y and  S i m i l a r y , CP-SBMV under synchronous  13 and 15).  the high  (up to k mg/g f r . wt)  of s y n t h e s i s of CP-SBMV d i d not appear  by synchronous  infection  (Figure  r a t e o f s y n t h e s i s of CP-TMV, under synchronous  CP-SBMV s y n t h e s i s  The  in both s i n g l y and doubly i n f e c t e d  15) than under asynchronous c o n d i t i o n s  to be a l t e r e d s i g n i f i c a n t l y  affect  equally  under  i s i n t e r e s t i n g t h a t under synchronous i n f e c t i o n more (up t o 11  ( F i g u r e 12). However, the l e v e l  did  As b e f o r e ,  infec-  i n f e c t e d t i s s u e , compared t o that in  mg/g f r . wt) CP-TMV was s y n t h e s i z e d tissue  in s i n g l y and doubly  in doubly  s i z e o f the t r i f o l i a t e  15).  Neither  infection,  infected tissue.  l e a f at the time of i n o c u l a t i o n o f the  primary leaves a f f e c t e d the d i s t r i b u t i o n o f CP-SBMV t o the t r i f o l i a t e leaves. of  When t r i f o l i a t e  leaves were 2.5 to 5-0 cm in length a t the time  i n o c u l a t i o n , no CP-SBMV could  longed able  incubation  to enter  liate  be d e t e c t e d  under d i f f e r e n t i a l  these leaves  readily.  in these leaves d e s p i t e  temperature. The optimal  pro-  However, CP-TMV was length o f the 3rd t r i f o -  leaves f o r e n t r y of both v i r u s e s was 0.5 to 1.5 cm.  III.  A n a l y s i s f o r s t r u c t u r a l i n t e r a c t i o n s between CP-SBMV and CP-TMV in C a l i f o r n i a  1.  blackeye cowpea.  A n a l y t i c a l sucrose  density gradient centrifugation.  I n f e c t i v i t y o f CP-SBMV was a s s o c i a t e d o n l y w i t h  the top f r a c t i o n and  124  F i g u r e 15-  N u c l e o p r o t e i n accumulation o f CP-SBMV and CP-TMV in C a l i f o r n i a blackeye cowpea 3rd t r i f o l i a t e leaves synchronously i n f e c t e d by the two v i r u s e s together and s e p a r a t e l y a t 10 C and s h i f t e d t o 27 C. Zero time i s when p l a n t s were moved from the styrofoam chamber t o the p e r m i s s i v e temperature o f 27 C. Leaves were harvested at v a r i o u s i n t e r v a l s a f t e r s h i f t t o 27 C.  Third  trifoliate  leaves doubly  infected  (A  A ).  Third  trifoliate  leaves s i n g l y  infected  ( O-  O).  A.  CP-SBMV n u c l e o p r o t e i n .  B.  CP-TMV n u c l e o p r o t e i n .  126  that o f CP-TMV w i t h o n l y the bottom f r a c t i o n .  F u r t h e r , when f r a c t i o n s  between CP-SBMV and CP-TMV bands from a r t i f i c i a l assayed of  on GA 21 and X a n t h i , about  these h o s t s , thus  CP-SBMV and CP-TMV. CP-SBMV on C a l i f o r n i a California local  2 local  and n a t u r a l  l e s i o n s were induced on each  i n d i c a t i n g contamination o f the middle The same f r a c t i o n s a l s o caused  blackeye cowpea and o f CP-TMV on B o u n t i f u l  blackeye cowpea.  l e s i o n s on P i n t o bean.  CP-SBMV alone  Surprisingly,  they a l s o caused  T h i s was unexpected  i s not known t o i n f e c t  a c t i o n o f CP-SBMV and CP-TMV analytical further  liability  infects  primary  T h i s unusual  in P i n t o i s d e s c r i b e d in d e t a i l  structural  bean and  reddish necrotic  s i n c e CP-TMV  t h i s host.  sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n  in subsequent  f r a c t i o n s by  systemic symptoms of  P i n t o s y s t e m i c a l l y without any symptoms on the i n o c u l a t e d and  mixtures were  leaves, inter-  later.  The  technique was not used  i n t e r a c t i o n experiments,  because i t s r e -  in s e p a r a t i n g i n f e c t i v i t y o f these v i r u s e s from a mixture was  doubtfu1. 2.  Infectivity neutralization.  artificial  The i n f e c t i v i t y of each v i r u s in  and n a t u r a l mixtures was a b o l i s h e d by treatment w i t h  logous antiserum, serum or b u f f e r  but not by treatment with heterologous a n t i b o d y , normal  (PBS) (Table X I I ) .  nor CP-TMV-RNA was e n c a p s i d a t e d  This indicated  e n c a p s i d a t e d RNA c o u l d have l o s t  cluded  that n e i t h e r CP-SBMV-RNA  in the coat p r o t e i n o f the o t h e r v i r u s .  However, because o f the remote p o s s i b i l i t y  California  i t s homo-  blackeye cowpea, which  t h a t a p u t a t i v e heterologous 1y  recognition  sites for its specific  host,  i s s u s c e p t i b l e t o both v i r u s e s , was  in the t e s t p l a n t s t o exclude t h i s p o s s i b i l i t y .  in-  However, when the  127  Table X I I .  R e l a t i v e amounts o f CP-SBMV and CP-TMV on GA 21 and N. g l u t i n o s a r e s p e c t i v l y recovered a f t e r n e u t r a l i z a t i o n o f i n f e c t i v i t y o f a r t i f i c i a l and n a t u r a l mixtures  I n f e c t i v i t y o f CP-SBMV and CP-TMV after neutralization C a l i f o r n ia blackeye  Inocu1um A.  Treatment  CP-SBMV/ CP-TMV  GA 21 CP-SBMV  N. g1ut i nosa CP-TMV  Natural m ixtures  CP-SBMV/CP-TMV  CP-TMV-antibody  + +  CP-SBMV/CP-TMV  CP-SBMV-antibody  + +  CP-SBMV/CP-TMV  Normal  + +  60  7  CP-SBMV/CP-TMV  Buffer  + +  139  113  117  B.  serum d  82  c  6  Artificial mixtures  CP-SBMV/CP-TMV  CP-TMV-antibody  + +  CP-SBMV/CP-TMV  CP-SBMV-antibody  + +  CP-SBMV/CP-TMV  Normal  + +  41  14  CP-SBMV/CP-TMV  Buffer  + +  200  190  a  serum  12  C h l o r o t i c spots on primary leaves f o r CP-SBMV or m o t t l i n g and b l i s t e r s on t r i f o l i a t e leaves f o r CP-TMV  ^  Average no. of l o c a l l e s i o n s per h a l f l e a f o f each host.  ••:  No l o c a l  ^  l e s i o n s observed. .  PBS (phosphate b u f f e r e d - s a 1 i n e ) .  128  trifoliate  leaves o f C a l i f o r n i a  i n o c u l a t i o n with e i t h e r v i r u s the  inoculum that  blackeye cowpea p l a n t s were assayed  i n f e c t i v i t y c o u l d . o n l y be a s s o c i a t e d  had been t r e a t e d  after with  with a heterologous antiserum, normal  serum or PBS. It can a l s o be seen that and  CP-SBMV a n t i b o d y , whereas CP-SBMV  by normal as  serum and CP-TMV antibody  i t has been observed  reaction  Interaction  Artificial t i o n s ) always to primary culated  it  leaves o f Pi;nto.  local  bean, the causal  Interaction of P i n t o  bean.  ( p u r i f i e d or crude p r e p a r a inoculated  l e s i o n s were i s o l a t e d and ino-  agent  inducing  l e s i o n s on P i n t o  o f t r i f o l i a t e s o f the i n o c u l a t e d  be recovered from such e x t r a c t s .  l e s i o n causal  i s not s u r p r i s i n g  l e s i o n s when they were  agent to go systemic  could  Bountiful;  The f a i l u r e o f  in B o u n t i f u l  Investigations  to determine which v i r u s was r e s p o n s i b l e  1.  in P i n t o  When the l o c a l  i s not the bean s t r a i n o f SBMV.  A.  This  affected  sera.  of CP-SBMV and CP-TMV  be recovered from e x t r a c t s  local  XII).  serum  (Rappaport and S i e g e l , 1955) that a n o n - s p e c i f i c  induced n e c r o t i c  however; CP-TMV could the  (Table  mixtures o f CP-SBMV and CP-TMV  to Bountiful  by normal  i n f e c t i v i t y was s l i g h t l y  can occur between TMV and other  IV.  not  CP-TMV was very much a f f e c t e d  suggests  were t h e r e f o r e  f o r the i n d u c t i o n  of CP-SBMV and CP-TMV a f t e r s e q u e n t i a l  that  made  of local  lesions.  inoculation  bean.  Inoculation  with  5 t o 7 days a f t e r the l a s t t r a c t i o n of CP-SBMV  intact viruses.  When l o c a l  l e s i o n s were counted,  i n o c u l a t i o n , t h e i r numbers v a r i e d with the concen-r  in the inoculum that was cha11enge-inocu1ated  (Table  XIII).  129  Table  XIII.  R e l a t i o n s h i p between number o f l o c a l l e s i o n s and v a r y i n g c o n c e n t r a t i o n s o f e i t h e r CP-SBMV or CP-TMV c h a l l e n g e i n o c u l a t e d on P i n t o primary leaves p r e - i n o c u l a t e d with a constant amount o f e i t h e r v i r u s 72 hours e a r l i e r  Inocu1um Initial  Concentrat ion (yg/ml)  Cha11enge  Concentrat ion (jjg/ml)  Mean no. o f local lesions per h a l f leaf 3  CP-TMV  50  CP-SBMV  50  75  CP-TMV  50  CP-SBMV  5  27  CP-TMV  50  CP-SBMV  0.5  16  Buffer  CP-SBMV  50  0  CP-SBMV  50  CP-TMV  50  k  CP-SBMV  50  CP-TMV  5  0  CP-SBMV  50  CP-TMV  0.5  0  Buffer  Mean o f two experiments  CP-TMV  50  0  130  The  primary  l e a v e s , p r e v i o u s l y i n o c u l a t e d w i t h a constant c o n c e n t r a t i o n  of CP-SBMV, developed  o n l y a few or no l o c a l  l e s i o n s when cha11enge-inocu-  l a t e d with v a r y i n g c o n c e n t r a t i o n s o f CP-TMV (Table X I I I ) . developed  when CP-SBMV or CP-TMV-inocu1ated  on P i n t o leaves s i m u l t a n e o u s l y doubly  local  When P i n t o primary  with a constant amount o f CP-SBMV-RNA and then  cha11enge-inocu1ated with CP-TMV at v a r i o u s i n t e r v a l s ,  culation  lesions  that the n e c r o t i c  I n o c u l a t i o n w i t h CP-SBMV-RNA and i n t a c t CP-TMV.  lesions  local  by CP-SBMV in the presence o f CP-TMV.  leaves were preinocu1ated  local  induced  i n o c u l a t e d with CP-SBMV and CP-TMV.  ( F i g u r e 16B and C). These r e s u l t s s t r o n g l y suggest  2.  lesions  i n o c u l a t e d by e i t h e r v i r u s alone d i d not develop  l e s i o n s were induced  lesions  leaves were i n o c u l a t e d with  F i g u r e 1 6 A shows the type o f l o c a l  phosphate b u f f e r o n l y .  Leaves s i n g l y  No l o c a l  that formed decreased  increased (Table XIV).  the number o f  s h a r p l y as the i n t e r v a l  from  preino-  However, on leaves preinocu1ated  with  i n t a c t CP-TMV (0.05 mg/ml), when they were c h a l l e n g e - i n o c u l a t e d 2b hours l a t e r w i t h CP-SBMV-RNA, the l o c a l same in number as those taneously  l e s i o n s that developed  induced when i n t a c t CP-TMV was i n o c u l a t e d s i m u l -  (0 hr) with the same amount o f CP-SBMV-RNA  l e s i o n s were induced a f t e r  RNase treatment  30 minutes 3.  No l o c a l  (Table XIV).  Inocula were  o f p a n c r e a t i c RNase and incubated f o r a t l e a s t ,  (room temperature) b e f o r e  inoculation.  Starch lesions. A p o s s i b i l i t y  induced m i c r o - 1 e s i o n s  (Table XIV).  o f an inoculum c o n t a i n i n g  CP-SBMV-RNA and CP-TMV, o r e i t h e r v i r u s a l o n e t r e a t e d with 50_jjg/ml  were about the  existed  that CP-TMV could have  i n v i s i b l e t o the naked eye. To t e s t  this  possibility,  F i g u r e 16.  Primary leaves o f P i n t o s i n g l y and doubly w i t h i n t a c t CP-SBMV and i n t a c t CP-TMV.  inoculated  A.  Primary  leaf  i n o c u l a t e d w i t h CP-SBMV and CP-TMV  B.  Primary  leaf  i n o c u l a t e d w i t h CP-TMV  C.  Primary  leaf  i n o c u l a t e d w i t h CP-SBMV.  132  T a b l e XIV.  E f f e c t o f pre-inocu1 at ion with CP-TMV on i n f e c t i v i t y o f CP-SBMVRNA ( p u r i f i e d ) on P i n t o primary leaves i n o c u l a t e d s i m u l t a n e o u s l y and sequent i a l ly  Initial 1 noc.  Cha11enge i noc.  CP-SBMV-RNA  CP-TMV  CP-SBMV-RNA  Mean no. o f l o c a l l e s i o n s produced per h a l f l e a f o f P i n t o by CP-SBMV a f t e r c h a l l e n ge i n o c u l a t i o n at v a r i o u s i n t e r v a l s (hr)  0.0  6.0  2k  k8  36.0  2.0  0.0  0.0  buffer  0.0  0.0  0.0  0.0  i n t a c t CP-TMV + RNAse  0.0  0.0  0.0  0.0  CP-SBMV-RNA  RNase  0.0  0.0  0.0  0.0  CP-SBMV  CP-TMV  68.5  ND  ND  ND  CP-TMV  CP-SBMV-RNA  ND  ND  30.0  ND  buffer  0.0  0.0  CP-SBMV-RNA  b  +  b  CP-TMV  +  Local  lesions  read 4 - 5  0.05 mg/ml o f e i t h e r Not  done  C  0.0  days a f t e r the l a s t c h a l l e n g e  0.0  inoculation.  i n t a c t CP-SBMV or i n t a c t CP-TMV used.  1 3 3  P i n t o primary  leaves were s i m u l t a n e o u s l y  i n o c u l a t e d with  i n t a c t CP-TMV or s i n g l y with e i t h e r v i r u s . primary allow  leaves were harvested  bleaching  by  soaking the leaves  et  a l , 1959) f o r , at l e a s t , 15 minutes.  lesions  B.  in the dark t o  in 70% e t h a n o l .  The s t a r c h  l e s i o n s were developed  in a potassium-1 a c t i c a c i d mixture  (1:20)  (Lindner  Leaves were examined f o r s t a r c h  a d i s s e c t i n g microscope with a low power o b j e c t i v e .  that c o u l d  simultaneously  at k°Z  The leaves were then t e s t e d f o r s t a r c h  by  l e s i o n s using  them a t 80°C  S i x days a f t e r i n o c u l a t i o n the  and s t o r e d o v e r n i g h t  removal o f excess s t a r c h .  i n t a c t CP-SBMV and  be seen were those on the primary leaves  The only  that had been  i n o c u l a t e d wi:th both v i r u s e s .  Time-course s y n t h e s i s  o f CP-SBMV  in t i s s u e a l s o  inoculated  with  CP-TMV. I n f e c t i v i t y o f CP-SBMV, as i n d i c a t e d by the number o f l o c a l increased  with time from  i n o c u l a t i o n to ]bb  Simi1arly,CP-SBMV s y n t h e s i s P i n t o primary  leaves,  hours a f t e r i n o c u l a t i o n  p r e v i o u s l y doubly  18). No i n c r e a s e  in CP-SBMV  primary leaves  titre  t h a t had pre-  been s i n g l y i n o c u l a t e d w i t h e i t h e r i n t a c t CP-SBMV or CP-SBMV-RNA  were assayed on GA 21 ( F i g u r e s tivity  from  i n o c u l a t e d w i t h CP-SBMV-RNA and i n t a c t  apparent when e x t r a c t s prepared from P i n t o  viously  ( F i g u r e 17).  increased w i t h time when e x t r a c t s prepared  CP-TMV, were assayed on GA 21 ( F i g u r e was  lesions,  17 and 18). However, there was some i n f e c -  in e x t r a c t s of t i s s u e that  had been s i n g l y i n o c u l a t e d with e i t h e r  i n t a c t CP-SBMV or i t s RNA, thus suggesting Pinto s u b l i m i n a l l y .  that t h i s v i r u s could  have i n f e c t e d  134  Figure  17-  Time-course r e p l i c a t i o n of CP-SBMV in P i n t o primary leaves s i n g l y and doubly i n o c u l a t e d with i n t a c t CP-SBMV and i n t a c t CP-TMV, as shown by r e l a t i v e i n f e c t i v i t y o f P i n t o primary l e a f e x t r a c t s harvested at v a r i o u s i n t e r v a l s a f t e r i n o c u l a t i o n and assayed on GA 21.  E x t r a c t s o f doubly  inoculated  leaves  ( A-  E x t r a c t s of s i n g l y  inoculated  leaves  (  O  O  ).  136  Figure  18.  Time-course r e p l i c a t i o n o f CP-SBMV i n P i n t o primary leaves s i n g l y and doubly i n o c u l a t e d w i t h CP-SBMV-RNA and i n t a c t CP-TMV, as shown by r e l a t i v e i n f e c t i v i t y o f P i n t o primary l e a f e x t r a c t s harvested a t v a r i o u s i n t e r v a l s a f t e r i n o c u l a t i o n and assayed on GA 21.  E x t r a c t s of doubly  inoculated  leaves  ( A-  •A  ).  Extracts of singly  inoculated  leaves  ( O  O  ).  2k 48  72  96 Hours a f t e r  120 inoculation  144  156  138  C.  Confirmation  1.  o f CP-SBMV ,in l o c a l  S e r o l o g i c a l assay.  CP-SBMV antiserum as well  Sap from doubly  as with  leaves  CP-SBMV antiserum.  s i n g l y i n o c u l a t e d with  serum a g a i n s t preparations replicate  bands should  serum a g a i n s t inoculated 2. (a) microscopy  with  Purified  Pinto  CP-SBMV, primary  CP-TMV antiserum.  Anti-  crude or p u r i f i e d  These r e s u l t s i n d i c a t e that CP-SBMV was a b l e to  Residual  in P i n t o primary  l e a f , to a level  detec-  inoculum cannot account f o r the r e s u l t s because  have developed  between the w e l l s c o n t a i n i n g  CP-SBMV and those c o n t a i n i n g  s i n g l y with  CP-TMV and sap from  serum d i d not r e a c t with  in the presence o f CP-TMV  t a b l e by t h i s method. precipitin  Purified  ( F i g u r e 19).  s i n g l y i n o c u l a t e d with  CP-TMV reacted w i t h  CP-SBMV or normal o f CP-TMV.  leaves  leaves.  i n f e c t e d t i s s u e reacted  CP-TMV antiserum  CP-SBMV, but not sap from P i n t o primary reacted with  l e s i o n s of P i n t o primary  sap from primary  leaves  the a n t i previously  this virus.  E l e c t r o n microscopy. Thin  sections.  CP-SBMV v i r i o n s could not be d e t e c t e d  in s e c t i o n s o f l o c a l  lesions.  T h i s does not,  the s e r o l o g i c a l evidence and the time-course s y n t h e s i s that CP-SBMV d i d m u l t i p l y  in P i n t o .  by e l e c t r o n microscopy could  by e l e c t r o n  however, i n v a l i d a t e  r e s u l t s , which show  The f a i l u r e t o d e t e c t  CP-SBMV v i r i o n s  be e i t h e r due to the preponderance o f CP-TMV  p a r t i c l e s which were s y n t h e s i z e d  and thus masked the presence o f the sphe-  rical  d i f f i c u l t i e s o f proper f i x a t i o n  tic  CP-SBMV or t o the inherent  t i s s u e . Indeed o t h e r s  TMV p a r t i c l e s  (Weintraub and R a g e t l i , 1964) f a i l e d  in n e c r o t i c l e s i o n s on N. g l u t i n o s a .  in necro-  to detect  139  Figure  19-  Immunodiffusion r e a c t i o n s o f crude sap from P i n t o primary leaves s i n g l y and doubly i n o c u l a t e d w i t h CP-SBMV and CP-TMV and of p u r i f i e d ( c o n t r o l ) CP-SBMV and CP-TMV. Gels were made with 0.75% Noble agar (w/v) i n PBS p l u s 0.02% NaN . P r e c i p i t i n bands were photographed 72 hours a f t e r i n c u b a t i o n a t room temperature.  A n t i s e r a wel1s. S-A  —  T-A  -- CP-TMV  Dilution 1/128  CP-SBMV  1/128  N-S -- Normal serum  Antigen, wells  1/128  (outside).  1.  Crude sap from primary leaves o f P i n t o i n o c u l a t e d w i t h CP-SBMV.  2.  Crude sap from primary leaves o f P i n t o doubly i n o c u l a t e d w i t h CP-SBMV and CP-TMV.  3.  Crude sap from primary leaves of P i n t o i n o c u l a t e d with CP-TMV.  singly  singly  b. P u r i f i e d  ( 0 . 2 5 mg/ml) CP-TMV -- c o n t r o l .  5.  Purified  (0.10  6.  Crude sap from primary leaves of P i n t o doubly i n o c u l a t e d w i t h CP-SBMV and CP-TMV (same as 2 above).  mg/ml) CP-SBMV -- c o n t r o l .  141  (b)  Purified virus.  When the CP-SBMV-antibody p r e c i p i t i n  band was  cut and mounted on e l e c t r o n microscope g r i d s and examined, a few p a r t i c l e s which resembled CP-SBMV were observed D.  Influence  of Cornell  ( F i g u r e 20).  i s o l a t e o f CP-TMV on CP-SBMV  i n f e c t i o n of  Pinto. When CP-SBMV and CP-TMV ted  t o P i n t o primary  (Cornell  leaves, n e c r o t i c l o c a l  w i t h a mixture o f CP-SBMV and CP-TMV l e s i o n s developed on leaves isolate)  (Figure 2 1 ) .  the C o r n e l l  with CP-TMV  Contrary  in several  (Cornell  (V.R.S. i s o l a t e ) , developed.  to the r e p o r t induces l o c a l  trials  by the w r i t e r when the l a t t e r was  lesions  inoculated  isolate).  D i r e c t p l a n t i n g o f seed produced on C a l i f o r n i a  o f CP-SBMV and CP-TMV  s i n g l y and doubly  F r e s h l y harvested  n i a blackeye cowpea p l a n t s  in cowpea. blackeye cowpea  i n f e c t e d by CP-SBMV and CP-TMV.  vine-dry  to seedlings  the two v i r u s e s .  No l o c a l  l e s i o n s on P i n t o , no l o c a l  A.  was t r a n s m i t t e d  obtained  (Bruening e t a l , 1976) that  Seed t r a n s m i s s i o n  1.  by  l e s i o n s , s i m i l a r t o those  V.  plants  inocula-  s i n g l y i n o c u l a t e d w i t h CP-SBMV o r CP-TMV ( C o r n e l l  i s o l a t e o f CP-TMV  were observed  i s o l a t e ) were s i m u l t a n e o u s l y  derived  mature seed. from planted  CP-SBMV, but no CP-TMV, seeds produced on C a l i f o r -  that were i n f e c t e d s i n g l y by CP-SBMV o r doubly  CP-SBMV was t r a n s m i t t e d  up t o 3 8 % (x = 13.5%) in seed-  l i n g s from s i n g l y i n f e c t e d p l a n t s and up t o 3 5 % (x = 7-6%) i n seeds produced on  doubly  planted  infected plants  seeds o f cowpea.  (Table XV).  CP-TMV was never t r a n s m i t t e d  through  When s e e d l i n g s were i n f e c t e d by CP-SBMV,  symptoms  142  F i g u r e 20.  E l e c t r o n micrographs of CP-SBMV-like p a r t i c l e s (arrows) p u r i f i e d from P i n t o primary l e a f l e s i o n s . The p u r i f i e d v i r u s suspension was s u b j e c t e d to immunod i f f u s i o n and the p r e c i p i t i n band was crushed on an e l e c t r o n microscope g r i d f o r o b s e r v a t i o n .  143  F i g u r e 21.  P i n t o primary leaves i n o c u l a t e d s i n g l y w i t h i n t a c t CP-TMV ( C o r n e l l i s o l a t e ) and doubly w i t h i n t a c t CP-SBMV and i n t a c t CP-TMV ( C o r n e l l i s o l a t e ) .  A.  Primary  leaf  inoculated  B.  Primary l e a f i n o c u l a t e d (Cornell i s o l a t e ) .  with CP-TMV  (Cornell  isolate)  with CP-SBMV and CP-TMV  144  T a b l e XV.  T r a n s m i s s i o n o f CP-SBMV and CP-TMV from seed produced or doubly i n f e c t e d C a l i f o r n i a blackeye cowpea  Total  by s i n g l y  seed 1 i n g s / t o t a 1 number o f germinated seed 1 i ngs CP-TMV Mean Mean Percent Percent No. range Percent  number o f i n f e c t e d  3  Seed  source  Healthy  seeds  No. 0/444'  CP-SBMV Percent range 0  0  0/444  0  0  Singly  infected  71/528  0-38  13.5  0/480  0  0  Doubly  infected  45/594  0-35  7.6  0/594  0  0  T o t a l number of s e e d l i n g s d e r i v e d at d i f f e r e n t times.  from d i f f e r e n t seed  lots  produced  145  of vein  clearing  always appeared  first  on t h e 1st  and 2nd  l e a v e s , b u t no symptoms w e r e o b s e r v e d on t h e p r i m a r y 2.  E f f e c t o f s e e d s t o r a g e d u r a t i o n on v i r u s  S e e d s w e r e sown f r o m t h e same s e e d temperature  (25°)  f o rvarious  CP-SBMV  lot after  affect  s t o r a g e a t room harvesting.  Storage  seed t r a n s m i s s i b i 1 i t y o f  in the s e e d l i n g s • ( T a b l e XVI).  3.  E f f e c t o f s e e d m o t t l e on t r a n s m i s s i o n o f CP-SBMV a n d CP-TMV through planted  CP-SBMV was t r a n s m i t t e d ( T a b l e XVI I ) .  transmission  t o 4.5  When n o r m a l  a n d m o t t l e d s e e d s w e r e sown,  and ~l,k% o f t h e s e e d l i n g s ,  i f t h i s was s o o n e w o u l d  from  p l a n t s ' . s i ngl:y i n f e c t e d  3 Botswana v a r i e t i e s infectivity  were used  plants  seeds d e r i v e d  is likely  infected  through seeds o f t h e  from  inoculated  plants.  seedlings  S i n c e fewer  the assays of C a l i f o r n i a  t h a t CP-SBMV c o u l d  be t r a n s m i t t e d  b l a c k e y e cowpea  seed.  seeds  black-  i n Botswana  b l a c k e y e and V26-Bots c o w p e a , b u t p r o b a b l y t o a l e s s e r e x t e n t t h a n in t h e C a l i f o r n i a  than  by. CP-SBMV.  a s s a y s o f l e a f e x t r a c t s made f r o m  i n t h e s e a s s a y s , compared w i t h  eye c o w p e a . i t  transmission  ( T a b l e X V I I I) a n d n e i t h e r v i r u s was d e t e c t e d  or serological  by p l a n t e d  seed  varieties.  N e i t h e r CP-SBMV n o r CP-TMV was t r a n s m i t t e d  produced  infected  D i r e c t p l a n t i n g o f seeds from s i n g l y and d o u b l y B o t s w a n a cowpea  respectively  have e x p e c t e d h i g h e r seed  l e v e l s f r o m s e e d d e r i v e d ;from d o u b l y  from those d e r i v e d B.  seed.  A p p a r e n t l y , seed c o a t c o l o u r d i d n o t a f f e c t  o f CP-SBMV, b e c a u s e  by  leaves.  transmission.  lengths of time a f t e r  up t o 18 m o n t h s d i d n o t s i g n i f i c a n t l y  trifoliate  146  T a b l e XVI.  E f f e c t o f storage d u r a t i o n o f seed on seed t r a n s m i s s i o n of CP-SBMV to s e e d l i n g s o f C a l i f o r n i a blackeye cowpea 3  Seed source Healthy  seeds  CP-SBMV-SI  b  CP-SBMV-DI  C  Age o f seed (months)  Number o f i n f e c t e d seedlings/out of number germinated  Percent 'Infect ion  3  0/10  0  8  0/25  0  18  0/25  0  3  1/10  10  8  0/24  0  18  8/86  9-3  3  2/10  20  8  1/26  4  18  0/24  0  A l l seeds were from the same seed l o t and were s t o r e d bags a t l a b o r a t o r y room temperature.  in p l a s t i c  b  Seed d e r i v e d  from p l a n t s s i n g l y  i n f e c t e d w i t h CP-SBMV.  C  Seed derived  from p l a n t s doubly  i n f e c t e d w i t h CP-SBMV and CP-TMV.  147  Table XVII.  E f f e c t o f s e e d m o t t l e on t r a n s m i s s i o n o f CP-SBMV and CP-TMV t h r o u g h p l a n t e d s e e d s o f C a l i f o r n i a b l a c k e y e cowpea t h a t w e r e s i n g l y ( S I ) and d o u b l y (Dl) i n f e c t e d  Proportion of seedlings  Seed s o u r c e  Seed coat colour  Healthy  Normal  seed  No. I n f e c t e d / no. a s s a y e d  0/90  CP-SBMV-SI  CP-SBMV-DI  CP-TMV-SI  CP-TMV-DI  No s e e d was  Percent transmi ss ion  0. .0  a  Mottled Norma 1  19/96  Mottled  -  Norma 1  3/67  4. • 5  Mottled  10/135  7. ,4  Normal  0/51  0. .0  Mottled  0/59  0. ,0  Norma 1  0/67  0. .0  Mottled  0/135  0. .0  mottled  infected  19. .8  148  Table XVIII.  Seed source  Seed t r a n s m i s s i o n o f CP-SBMV and CP-TMV in planted of three Botswana l o c a l cowpea v a r i e t i e s  T o t a l number o f s e e d l i n g s s e e d l i n g s germinated  Variety  i n f e c t e d / t o t a l of  CP-SBMV No.  seed  CP-TMV %'  0  No.  %  0  0  Heal, thy  Blackeye  0/20  s.  b  Blackeye  0/47  0  d  Blackeye  0/11  0  0/1 1  0  SI  V26-Bots  0/116  0  -  -  Dl  V26-Bots  0/38  0  0/38  0  SI  V45-Bots  -  0/28  0  Dl  -  a  a  -  A l 1 seedl ings were a l s o t e s t e d by s e r o l o g y . Singly Not  infected  tested  Doubly  plants.  - seed  infected  unavailable.  plants.  c  -  149  C.  Distribution  o f CP-SBMV and CP-TMV  Evidence of seed-borne distribution 1.  developmental  o f CP-SBMV and CP-TMV  When seed o f C a l i f o r n i a  of  in d i f f e r e n t  stages a f t e r  seed  indicator  hosts.  blackeye cowpea was harvested a t d i f f e r e n t  f l o w e r i n g , CP-SBMV was recovered from  ted  p l a n t s , b u t not from e x t r a c t s o f those produced  ted  by CP-SBMV.  infec-  on p l a n t s s i n g l y  infec-  No CP-TMV was recovered from any of the embryos.  o f T a b l e XIX a r e based on one experiment  of o n l y 5 seeds  extracts  on doubly  v i r u s e s c o u l d be recovered from a l l seed c o a t s o f a l l stages results  parts.  blackeye cowpea  pooled embryos of a l l stages o f seed m a t u r i t y produced  The  that  in seed p a r t s o f d i f f e r e n t  stages o f seed o f C a l i f r o n i a  d i r e c t l y assayed on the r e s p e c t i v e  developmental  parts.  t r a n s m i s s i o n o f CP-SBMV made i t necessary  of both v i r u s e s be i n v e s t i g a t e d  Distribution  in seed  Both  (Table XIX).  which employed a t o t a l  f o r each assay, so the data may not be r e p r e s e n t a t i v e of  a l a r g e r p o p u l a t i o n o f seeds. More e x t e n s i v e experiments  were performed  stages o f development, but instead o f u t i l i z i n g p a r t s were assayed ments CP-SBMV ted  plants,  ted  However, because  i t was necessary to t e s t procedure.  10 jjg/ml o f CP-SBMV. by washing  pooled seed p a r t s the seed in pooled embryo e x p e r i -  i n f e c t i v i t y c o u l d be recovered from embryos of doubly  decontamination with  individually.  w i t h embryos o f d i f f e r e n t  the e f f i c a c y  o f the tap water wash  Healthy embryos were a r t i f i c i a l l y The contaminated  in tap water f o r 30 minutes  infec-  embryos were then  contaminated decontamina-  and 24 hours or by soaking in  150  T a b l e XIX.  D i s t r i b u t i o n of CP-SBMV and CP-TMV in pooled seed p a r t s o f C a l i f o r n i a blackeye cowpea of d i f f e r e n t development stages of m a t u r i t y whose e x t r a c t s were d i r e c t l y assayed on i n d i c a t o r hosts a f t e r decontamination by an 8-hour tap water wash  I nocu1um  Seed part°  Age o f Pgds (days)  Mean number of l o c a l per h a l f l e a f o f : GA 21 CP-SBMV  lesion  N^. gl ut inosa CP-TMV  SI  embryo  15-20  0  N/A  SI  seed coat  15-20  229  N/A  Dl  embryo  15-20  2  0  Dl  seed coat  15-20  138  72  SI  embryo  45-50  0  0  SI  seed coat  45-50  27  65  Dl  embryo  45-50  3  0  Dl  seed coat  45-50  102  0  Healthy  embryo  15-20  0  0  Heal thy  seed coat  15-20  0  0  Heal thy  embryo  45-50  0  0  Heal thy  seed coat  45-50  0  0  Each seed p a r t s were pooled ground t o g e t h e r . 3  Age  9  s  i n t o e i t h e r 5 embryos or 5 seed c o a t s and  in days s i n c e f l o w e r i n g .  'Seeds produced Green  on p l a n t s s i n g l y  immature seed.  Vine-dry mature seed.  infected  'Not ava i 1 a b l e .  by e i t h e r CP-SBMV or CP-TMV. Seeds produced on p l a n t s doubly i n f e c t e d by CP-SBMV and CP-TMV  151 5% Na^PO^ f o r 10 minutes,  f o l l o w e d by r i n s i n g  E x t r a c t s from a r t i f i c i a l l y by washing  contaminated  in tap water  f o r 8 to 12 hours.  embryos that had been  decontaminated  f o r 30 minutes were i n f e c t i o u s , but not those decontaminated f o r  2k hours or in StNa^PO^ was abandoned a f t e r Thus a l l experiments  (Table XX).  i t was found  Initially  that  lO^Na^PO^ was used, but i t  i taffected  g e r m i n a t i o n o f embryos.  that f o l l o w used e i t h e r 2k hour tap water wash or  5% Na^PO^. The  r a d i c l e - p l u m u l e shoots were separated from c o t y l e d o n s w i t h flamed  tweezers or sharp  forceps.  When e x t r a c t s o f i n d i v i d u a l whole embryos o r r a d i c l e - p l u m u l e shoots and  seed c o a t s were assayed, CP-SBMV was t r a n s m i t t e d  plumule  shoots o f green  immature whole embryos  immature embryos  (Table XXI), 6.0% o f dough stage  (Table XXII) and 18.0% o f r a d i c l e - p l u m u l e shoots of  v i n e - d r y mature embryos transmitted  (Table X X I I I ) .  N e i t h e r CP-SBMV nor CP-TMV was  by a r t i f i c i a l l y - c o n t a m i n a t e d - d e c o n t a m i n a t e d embryos.  CP-TMV was not d e t e c t e d in embryos o f n a t u r a l l y decontaminated. tamination,  t o 10.3% o f r a d i c l e -  When embryos were shaken  infectivity  infected  in d i s t i l l e d  f o r e i t h e r v i r u s could  seeds  water  Further,  that were  b e f o r e decon-  be recovered from  such  washings when assayed,but not from washings made a f t e r decontamination. I n f e c t i v i t y of e i t h e r virus of  in the seed coat was not e l i m i n a t e d  by any  the decontamination procedures, except that at times Na^PO^ reduced  their  infectivity.  experiment  When 10% Na^PO^ was used  no.l) CP-SBMV  in one experiment ' (Tab!e XX I,  i n f e c t i v i t y was not recovered.  In a s i n g l e experiment, when i n f e c t i v i t y  of e x t r a c t s was determined  a f t e r c o t y l e d o n s and r a d i c l e - p l u m u l e shoots were s e p a r a t e d , i t was found  152  T a b l e XX.  E f f i c a c y o f decontaminating CP-SBMV and CP-TMV from a r t i f i c i a l l y contaminated embryos w i t h tap water and 5% Na PO, a f t e r which t h e i r e x t r a c t s were assayed d i r e c t l y on e i t h e r GA 21 or N. g l u t inosa  Inoculum  Decontamination procedure  P r o p o r t i o n of i n f e c t i v e embryos No. i n f e c t e d / Mean no. assayed lesions Percent  Healthy embryos  unwashed  0/10  CP-SBMVcontaminated  unwashed  10/10  100  CP-SBMVcontami nated  30 min.-wash  6/20  30  CP-SBMVcontaminated  2k  0/20  hr.-wash  5% Na PO^ +  CP-SBMVcontam i nated  12 hr-wash  CP-SBMVcontam i nated  untreated  0/50  0  10/10  k  CP-SBMV standard (10 jug/ml) CP-TMVcontam i nated  127  5% Na PO^ +  b  CP-TMVcontam i nated  12 hr-wash  0/40  untreated  3/10  CP-TMV standard (10 jig/ml)  soaked  in v i r u s  30  146  Mean number o f l o c a l l e s i o n s 'Seed  100  per h a l f  (lOjug/ml)  l e a f o f e i t h e r GA 21 o r N. g1ut i nosa,  Table XXI.  D i s t r i b u t i o n o f CP-SBMV and CP-TMV In 15~20 day o l d i n f e c t e d and a r t i f i c i a l l y c o n t a m i n a t e d g r e e e n immature seed p a r t s o f s i n g l y i n o c u l a t e d C a l i f o r n i a b l a c k e y e cowpea w h i c h were d i r e c t l y a s s a y e d i n d i v i d u a l l y on i n d i c a t o r h o s t s a f t e r d e c o n t a m i n a t i o n w i t h 5% and \0% Na.PO. f o r 10 and 30 m i n u t e s , r e s p e c t i v e l y 3  Proport ion of half leaf of:  infected  N.  GA 21  Exper iment number E x p t . no. 1 W i t h 10%  Seed p a r t  Healthy  embryo '  N  T r e a t e d (T) or untreated(U)  g l u t i nosa  CP-TMV  CP-SBMV  Natural i n f e c t i o n (N) or a r t i f i c i a l c o n t a m i n a t i o n (A)  l e s i o n s per  s e e d s and number o f l o c a l  No. o f seeds i nfected/ no. a s s a y e d  Range  Mean  T  0  0  0/10  0  0  0/10  embryo  T  0  0  0/52  0  0  0/52  N  embryo  U  65  20/20  1-4  2  11/20  A  embryo  T  0  0  0/52  0  0  0/52  N  Seed c o a t  T  15-267  107  10/10  2-208  63  10/10  Heal t h y  radicle-plumule  T  0  0/10  T  rad i c l e - p l u m u l e  U  5-159  6/57 28/52  0 0-28  0  N  0 2 10  0  radicle-plumule  0 1-2  0/10  N  0/52 12/52  0  Range**  1-195  Mean  No. o f s e e d s infected/ no. a s s a y e d  c  E x p t . no. 2 W i t h 5%  U  Table XXI.  continued:  P r o p o r t i o n o f i n f e c t e d s e e d s and number o f l o c a l l e s i o n s p e r half leaf o f : N^. g 1 u t i n o s a GA 21  E x p e r iment number  CP-SBMV  Natural i n f e c t i o n (N) or a r t i f i c i a l c o n t a m i n a t i o n (A)  Seed  A  T r e a t e d (T) or u n t r e a t e d (U) Range  CP-TMV  t  part  Mean  No. o f s e e d s i nfected/ no. a s s a y e d  No. o f s e e d s i nfected/ no. a s s a y e d  Range  Mean  0  0  0/52  E x p t . no. 2 w i t h 5% rad i c l e - p l u m u l e  T  0  0  N  seed  coat  T  23-2*i5  120  10/10  3^-101  93  10/10  N  seed  coat  U  30-300  166  10/10  3-109  32  10/10  10/10  136-168  156  10/10  Standard  Since  flowering.  'Local  l e s i o n range.  Local  l e s i o n mean,  'whole embryo  (10  ug/ml)  33-198  0/52  T a b l e XXII.  D i s t r i b u t i o n of CP-SBMV and CP-TMV in 30-35 day- o l d i n f e c t e d and a r t i f i c i a l l y contaminated immature (dough stage) seed p a r t s of C a l i f o r n i a blackeye cowpea d i r e c t l y assayed i n d i v i d u a l l y on i n d i c a t o r hosts a f t e r decontamination with 5% Na-P0, f o r 10 minutes 3  Natural i n f e c t i o n (N) or a r t i f i c i a l contamination (A)  Seed part  N  embryo  N  P r o p o r t i o n of i n f e c t e d seeds and number o f l o c a l l e s i o n s per h a l f l e a f o f : . GA 21 R. -g 1 lit innsa Treated,!. (T) CP-SBMV-SI CP-TMV-SI ~ or No. of seeds No. of seeds Untreated (U) , i n f e c t e d / no. infected/no. Range Mean assayed assayed Range Mean L  2  3/5D  0  0  0/50  61  48/48  1-1  1  2/50  0  0/50  0  0  0/40  1-11  4  10/10  0  0  0/40  T  1-8  3  9/10  3-34  15  7/10  U  24-402  182  10/10  5-116  46  10/10  U  82-170  119  19-59  44  67-216  127  42-274  146  T  1-2  embryo  U  3-260  "A  embryo  T  0  A  embryo  U  N  seed  coat  N  seed  coat  N  embryo washings  Standa rd  (10 ug/ml) ;•  6  Since flowering. Singly  infected  Local  l e s i o n ; range  Local  l e s i o n mean  Whole embryo Washings obtained by shaking 5 embryos in 2.5 ml of phosphate b u f f e r before  decontamination.  T a b l e XXIII.  D i s t r i b u t i o n o f CP-SBMV and CP-TMV in n a t u r a l l y i n f e c t e d (N) and a r t i f i c i a l l y contaminated (A) seed p a r t s o f v i n e dry mature seed of C a l i f o r n i a blackeye cowpea whose seed p a r t s e x t r a c t s were d i r e c t l y assayed i n d i v i d u a l l y on i n d i c a tor hosts a f t e r decontamination w i t h 5% Na,P0^ f o r 10 minutes f o l l o w e d by 12 hour tap water wash  P r o p o r t i o n o f i n f e c t e d seed p a r t s and mean number of l o c a l l e s i o n s per h a l f l e a f o f : N. g l u t i n o s a GA 21 Natural(N) or A r t i f i c i a l (A)  Seed part  N  R-P  N  Treated(T) or untreated(U)  CP-SBMV - s i Range  3  b  Mean  No. i n f e c t e d / no. assayed  1-2  2  4/20  0  0/50  0  0  0/50  13  10/10  1-1  1  4/10  2-8  5  3/10  3-182  86  10/10  2-60  14  10/10  5-471  120  10/10  R-P  U  1-15  4  A  R-P  T  0  A  R-P  U  3-30  N  s.c  T  N  s.c  U  76-189 129  CP-SBMV - D l Range  1 12-248  180  CP-TMV - D l  9  Mean  •  No. i n f e c t e d / noc assayed  10/13  9/50  (lOjug/m11)  Mean  0/50  2  Standard  Range  0  1-5  f  b  0  T  6  CP-TMV- s .  No. i n f e c t e d / no. assayed  Range  9  Mean  No. i n f e c t e d / no. assayed  T  1-11  4  2/64  0  0  0/64  E  U  1-1  1  2/64  0  0  0/64  N  R-P  T  1-1  1  2/40  0  0  0/40  N  S.C  T  1-1  1  1/10  1-10  3  8/10  N  E  N  h  T a b l e XXI I I.  Continued:  P r o p o r t i o n of i n f e c t e d seed p a r t s and mean number of l o c a l l e s i o n s per h a l f l e a f o f : N. g l u t i n o s a GA 21 Natural (N) or Artificial(A)  Seed part  Treated or untreated  S.C  N  Range o f l o c a l 'Mean l o c a l Rad Seed  singly  l e s i o n s per h a l f  l e s i o n s per h a l f  2-22  infected leaf.  leaf.  coat.  'Doubly i n f e c t e d with CP-SBMV and CP-TMV  CP-TMV-DI9  9  No. i n f e c t e d / , Mean no. assayed 9  flowering.  icle-plumule.  'whole embryo.  Range  U  Seeds harvested k5 to 55 days s i n c e 'Seeds produced on p l a n t s  CP-SBMV-DI  by CP-SBMV or CP-TMV.  9/10  Range 3-23  Mean  No. i n f e c t e d / no. assayed  13  10/10  158  that CP-SBMV was t r a n s m i t t e d e q u a l l y by c o t y l e d o n s as by r a d i c l e - p l u m u l e shoots and the v i r u s was t r a n s m i t t e d t o about seed  the same extent  (Table XXIV). Although the t i t r e o f each v i r u s was always  than of  in p l a n t e d  in the embryos,  higher in the seed c o a t s  i t i s not p o s s i b l e to s t a t e c a t e g o r i c a l l y which stage  seed coat development c o n t a i n e d more v i r u s , because  were used at d i f f e r e n t 2.  assay  plants  times o f assay.  D i s t r i b u t i o n o f CP-SBMV and CP-TMV cowpea v a r i e t i e s .  cowpea v a r i e t i e s ,  different  in seed p a r t s o f Botswana  When embryos o f seeds  previously  produced  on Botswana  i n o c u l a t e d w i t h CP-SBMV and CP-TMV, were  assayed, n e i t h e r v i r u s could be recovered from the embryos of v a r i e t i e s tested.  However, each v i r u s was recovered from the seed c o a t s o f v a r i e t i e s  t e s t e d , except that CP-SBMV could not be recovered from the seed c o a t s of  seed d e r i v e d from doubly  i n o c u l a t e d V45~Bots p l a n t s  3. D i s t r i b u t i o n o f CP-SBMV and CP-TMV blackeye cowpea. doubly  infected  (Table XXV).  in p l a n t e d embryos o f C a l i f o r n i a  When embryos from seeds produced  p l a n t s were decontaminated  on s i n g l y o r  by a 2k hour tap wateriwash  or in  5% Na^PO^ and then p l a n t e d , C P - S B M V was t r a n s m i t t e d to the s e e d l i n g s . No v i r u s was t r a n s m i t t e d to the s e e d l i n g s d e r i v e d from h e a l t h y embryos that had been a r t i f i c i a l l y derived  from n a t u r a l l y  contaminated  contaminated  infected  and decontaminated.  In embryos  seeds and in those that had been  and p l a n t e d without decontamination,  1.€P'TS'BMV>  ..  was' ,  however, t r a n s m i t t e d to the s e e d l i n g s (Tables XXVI and XXVII). t a b l e seeds from the same seed  artificially  l o t were used and, f o r comparison  In each with  T a b l e XXIV.  D i s t r i b u t i o n o f CP-SBMV and CP-TMV in seed p a r t s o f s i n g l y i n f e c t e d (SI) v i n e dry mature seed of C a l i f o r n i a blackeye cowpea whose e x t r a c t s were d i r e c t l y assayed i n d i v i d u a l l y on i n d i c a t o r hosts a f t e r decontamination by a 2 4 h o u r tap water wash and t r a n s m i s s i o n by p l a n t e d i n t a c t seed g  Seed part  Proport ion o f i n f e c t e d seeds and mean number o f l o c a l induced by seed part. ; e x t r a c t s per h a l f l e a f o f : Q/\ 21 Mean  Coty R-P  d  6  c  glutinosa  CP-SBMV-SI No. i n f e c t e d / Percent no. assayed  Mean  c  CP-TMV-SI" No. i n f e c t e d / Percent no. assayed  1  1/26  4  0  0/22  0  2  1/26  4  0  0/22  0  1/49  2  0  0/32  0  Planted i ntact seed  a  S e e d s and embryos from the same seed l o t that had been s t o r e d bags f o r 2 weeks a f t e r h a r v e s t . Singly  lesions  infected.  Mean number of l o c a l Cotyledons without  l e s i o n s per h a l f . l e a f .  r a d i c l e - p l u m u l e shoot.  R a d i c l e - p l u m u l e shoot without c o t y l e d o n s .  in p l a s t i c  Table XXV.  D i s t r i b u t i o n o f CP-SBMV and CP-TMV in seed p a r t s o f Botswana cowpea v a r i e t i e s s i n g l y (SI) and doubly (Dl) i n o c u l a t e d with the two v i ruses  Treated (T) or untreated (U)  P r o p o r t i o n o f transmi ss ion No. i n f e c t e d / no. assayed Percent  Sou ree  Var iety  Seed part  CP-SBMV  SI  Blackeye  s.ca  U  CP-SBMV  SI  B1ackeye  R-P  T  CP-SBMV  SI  V26-Bots  s.c.  U  20/20(  CP-SBMV  SI  V26-Bots  R-P  T  0/20(  CP-TMV  SI  V26-Bots  S.C  U  CP-SBMV  Dl  V45-Bots  S.C  U  0/20(  0)  0  CP-TMV  Dl  V45-Bots  S.C  u  10/20(  2)  50  CP-TMV  Dl  45-Bots  R-P  T  0/20(  0)  0  V i rus  6  C  10/10(100)  100  0/20 ( 0)  0  B  d  C  Rad i c l e -plumule  0  10/10(150)  100  les ions per ha 1f l e a f  shoot  ^Treated w i t h 5% NajPO^ Doubly  i n o c u l a t e d p l a n t s not  checked f o r  100  0)  a„ Seed coat ^Number in p a r e n t h e s i s r e f e r s to number o f l o c a l  53)  double i n f e c t i o n by both v i ruses.  161  T a b l e XXVI.  T r a n s m i s s i o n of CP-SBMV in embryos of n a t u r a l l y i n f e c t e d seeds and a r t i f i c i a l l y contaminated embryos of C a l i f o r n i a b l a c k e y e cowpea p l a n t e d a f t e r decontamination w i t h d e t e r gent f o l l o w e d by 24 hour tap water wash  Treatment  Natural i n f e c t ion(N) or a r t i f i c i a l contaminat ion(A)  Embryos  Heal thy  U  0/10  0.0  Embryos  A  U  1/11  9-1  W  0/40  0.0  20/20  100.0  U  12/24  50-0  W  7/24  29.2  8/34  23.5  a  Embryos Soaked seed  P r o p o r t i o n of seedlings infected Washed (W) No. i n f e c t e d / or unwashed(U) no. assayed Percent  intact  Dry i n t a c t seed  A  Embryo  N  v  Dry n a t u r a 1 1 y infected intact seed  Embryos separated from seed c o a t s and then soaked f o r 1 0 - 1 5 minutes. Intact (dry) seed soaked planted. Intact intact  in 10^|ig/ml  d i r e c t l y in 1 0 ug/ml of v i r u s  (dry) seed soaked o v e r n i g h t in d i s t i l l e d in 1 0 lig/ml of v i r u s and then p l a n t e d .  of v i r u s  f o r 8 hours and then  water and then  soaked  162  T a b l e XXVII.  T r a n s m i s s i o n CP-SBMV and CP-TMV in embryos o f n a t u r a l l y i n f e c t e d seeds and in a r t i f i c i a l l y contaminated embryos of C a l i f o r n i a blackeye cowpea p l a n t e d a f t e r decontamination in 5% Na_P0^ f o l l o w e d by r i n s i n g in running tap water f o r 8 hours and t h e i r t r a n s m i s s i o n in p l a n t e d n a t u r a l l y i n f e c t e d i n t a c t seed  Treatment V i rus Source CP-SBMV  si  CP-SBMV  SI  a  Natural i n f e c t ion(N) or a r t i f i c i a l contamination (A)  P r o p o r t i o n o f seed 1 i ngs i nfected No. i n f e c t e d / no. assayed Percent  Treated (T) untreated (U)  N  T  7/50  14,0  N  U  8/48  16.7  T  0/50  0.0  CP-SBMV  A  CP-SBMV  A  U  8/49  16.3  b  CP-TMV  SI  N  T  0/50  0.0  CP-TMV  SI  N  U  5/49  10.20  CP-TMV  A  T  0/45  0.0  CP-TMV  A  U  0/50  0.0  N  T  3/40  7.5  13/202  6.4  0/40  0.0  0/202  0.0  0/20  0.0  CP-SBMV  DI  CP-SBMV  Dl  N  CP-TMV  Dl  N  CP-TMV  Dl  N  C  planted seedd T planted seed  Heal thy embryo  Seeds produced  Seeds produced  intact  U  on s i n g l y  infected plants  ^Healthy embryos a r t i f i c i a l l y (10 >jg/ml) c  intact  on doubly  contaminated  infected  plants.  with e i t h e r CP-SBMV or CP-TMV d  Naturally infected  seeds that  were p l a n t e d u n t r e a t e d .  163  transmission  o f decontaminated p l a n t e d  embryos, data on i n t a c t  seeds a r e i n c l u d e d  in each t a b l e .  mission  in s e e d l i n g s d e r i v e d  o f CP-SBMV  seedlings derived 4.  from p l a n t e d  There i s a good agreement between t r a n s -  intact  E f f e c t o f age o f C a l i f o r n i a bility  r u l e out the p o s s i b i l i t y  don  in C a l i f o r n i a  or r a d i c l e - p l u m u l e planting.  (Table XXVIII).  either virus (I0jjg/ml;  by rubbing  with  Therefore  b e f o r e p l a n t i n g or 5 before  germination  i n f e c t e d the young  seedlings  s e e d l i n g age cannot e x p l a i n the d i f f e r e n t i a l  o f the v i r u s e s from seeds o f i n f e c t e d p l a n t s .  i t was necessary  (Schneider  and Worley, 1 9 5 9 ; Roberts and P r i c e ,  to t e s t  this.  touching  the s e e d l i n g s .  c e l l s of bean,  leaves o f 4 to 6 day-old  In one experiment  were s e p a r a t e l y a p p l i e d t o s e e d l i n g s CP-SBMV In another experiment  uninjured  Suspensions o f CP-SBMV and CP-TMV  were d e l i v e r e d by p i p e t t e on the primary  tested.  Embryos  no c e l i t e ) on the c o t y l e -  Q-tips, just  that SBMV can enter and i n f e c t a p p a r e n t l y  without  cowpea d i d  be r e f r a c t o r y to CP-TMV  Whether e i t h e r v i r u s was introduced  Because o f the r e p o r t s 1967)  blackeye  and e a r l y growth.  or 5 days a f t e r p l a n t i n g , both v i r u s e s r e a d i l y  transmission  that CP-SBMV, but  embryos and seeds were sown i n s t e a m - s t e r i l i z e d sand.  i n o c u l a t e d with  days a f t e r  The f a c t  that s e e d l i n g s c o u l d  at some stage o f germination  were then  cowpea s e e d l i n g on s u s c e p t i -  t o e i t h e r CP-SBMV or CP-TMV.  not  embryos and that in  (Tables XXVI and X X V I l ) .  blackeye  CP-TMV, was c o n s i s t e n t l y seed-borne  Healthy  from planted  seed  not  infection  planted  in which mixed  (lOjjg/ml) seedlings  in which the two v i r u s e s  i n f e c t e d one o f 14  seedlings  inoculum o f both v i r u s e s was  164  Table XXVIII.  Inocu1um  T r a n s m i s s i o n o f CP-SBMV and CP-TMV when i n o c u l a t e d t o h e a l t h y c o t y l e d o n s and o r r a d i c l e - p l u m u l e s h o o t s o f C a l i f o r n i a b l a c k e y e cowpea a t d i f f e r e n t s t a g e s a f t e r sowi ng  Ages a t : i noc. (days) a  Cotyledons or radicle-plumule  P r o p o r t i o n of p l a n t s i n f e c t e d by e i t h e r v i r u s . No. %  coty1edons  2/20  10  CP-SBMV  0  CP-SBMV  0  rad i cle-p1umu1e  4/20  20  CP-SBMV  5b  cotyledons  1/10  10  CP-TMV  0  cotyledons  4/20  20  CP-TMV  0  rad i cle-p1umu1e  3/20  15  CP-TMV  5  coty1edons  6/10  60  Buffer  0  coty1edons  0/20  0  1nocu1ated j u s t  before  ^ F i v e days a f t e r  p l a n t i n g and 2 days  3  planting, a f t e r emergence.  165  a p p l i e d on primary  leaves,  CP-SBMV  i n f e c t e d 2 o f 53 t e s t e d  CP-TMV d i d not i n f e c t any o f the s e e d l i n g s . ced,  The nature o f symptoms  however, was d i f f e r e n t from that produced by s e e d l i n g s  naturally on  seedlings.  i n f e c t e d seed.  the primary  c h l o r o t i c spot  Naturally  infected seedlings  l e a f whereas s e e d l i n g s  inoculated  symptoms on the primary  leaves.  derived  never showed  produfrom symptoms  by the drop method showed In c o n t r a s t  to natural  i n f e c t i o n s , where systemic symptoms a r e c l e a r l y obvious on the f i r s t l i a t e t o form by the  10 to 12 days a f t e r sowing, symptoms on s e e d l i n g s  the drop method developed 1st and 2nd t r i f o l i a t e  leaves  18 days a f t e r i n o c u l a t i o n .  uninjured  Clearly  entry  c e l l s does not seem t o account f o r  seed-borne nature. 5.  E f f e c t o f healthy on  The  seed e x t r a c t on i n f e c t i o n o f CP-SBMV and CP-TMV  GA 21 and H_. g l u t i n o s a r e s p e c t i v e l y .  low l e v e l  artificially ted  inoculated  systemic symptoms o f mosaic and m o t t l i n g on  of CP-SBMV through a p p a r e n t l y its  trifo-  of local  l e s i o n s obtained  when n a t u r a l l y i n f e c t e d and  contaminated embryos were assayed f o r CP-SBMV o r CP-TMV promp-  the s u s p i c i o n  that embryo e x t r a c t s may c o n t a i n  t h i s was i n v e s t i g a t e d ,  i t was found that  an i n h i b i t o r ( s ) .  the g r e a t e r  When  the c o n c e n t r a t i o n o f  the embryo e x t r a c t used to d i l u t e e i t h e r v i r u s to 10 ^ug/ml the g r e a t e r the degree o f i n h i b i t i o n . when u n d i l u t e d  I n f e c t i v i t y o f CP-SBMV was reduced by 45 and 25%  and 1/10 d i l u t i o n  e x t r a c t s were used, r e s p e c t i v e l y , and CP-TMV  i n f e c t i v i t y was reduced by 96 and 69%, r e s p e c t i v e l y , by s i m i l a r e x t r a c t s (Table XX.I.X).  166  T a b l e XXIX.  E f f e c t o f an e x t r a c t o f h e a l t h y seed o f C a l i f o r n i a blackeye cowpea on i n f e c t i v i t y o f CP-SBMV and CP-TMV on GA 21 and N. g1ut i nosa, r e s p e c t i v e l y  Mean number o f l o c a l  l e s i o n s per h a l f  H_. g l u t i n o s a  GA 21 CP-SBMV Dilution treatment  Mean lesions  Standard  227  3  b  CP-TMV  Percent reduction 0.0  Mean lesions 196  178  c  21.6  164  1/10  171  C  24.7  6l  und i1uted  124  45.4  7  3  S t a n d a r d was made o f 10^jg/ml o f e i t h e r v i r u s  Percent reduction 0.0  b  1/100  d  leaf o f :  C  d  e  16.3 68.9 96.4  in phosphate b u f f e r ,  pH 7.1  bed G Values in each column not f o l l o w e d c a n t l y at P = 0.05 by DMR t e s t .  by the same l e t t e r vary  signifi-  167  D.  E f f e c t o f g e r m i n a t i o n o'n s u r v i v a l of C a l i f o r n i a  blackeye cowpea.  T h i s study was c a r r i e d out because is not seed-borne naturally  infected  and i n f e c t i v i t y o f v i r u s on seed  because  i t was suspected that perhaps  of i t s i n a c t i v a t i o n during germination.  seeds were sown in sand,  i n f e c t i v i t y o f CP-SBMV  nated seed c o a t s was reduced o r e l i m i n a t e d compared  No s i g n i f i c a n t d i f f e r e n c e was apparent  e x t r a c t s from germinated  and ungerminated  The  i s not c l e a r .  in germi-  between CP-TMV  (Table XXX).  reason f o r the low i n f e c t i v i t y o f CP-SBMV  nated seeds  When  to that o f ungerminated  seed c o a t s ( F i g u r e 22).  seed  CP-TMV  in seed c o a t s o f germi-  168  Figure 22.  R e l a t i v e i n f e c t i v i t y o f CP-SBMV in seed coat e x t r a c t s of germinated and ungerminated seeds d e r i v e d from C a l i f o r n i a blackeye cowpea p l a n t s , s i n g l y i n f e c t e d by CP-SBMV. Seed c o a t s used in t h i s experiment were not washed before assay. I n f e c t i v i t y i s i n d i c a t e d by l o c a l l e s i o n s on primary l e a f o f GA 21 cowpea. Each assay was made up o f 5 pooled seed c o a t s (one seed coat/0.25 ml of phosphate b u f f e r pH 7 . 1 ) .  A.  Inoculated w i t h seed coat e x t r a c t s o f ungerminated seeds.  B.  Inoculated w i t h seed coat e x t r a c t s o f germinated  seeds.  169  T a b l e XXX.  E f f e c t o f germination on i n f e c t i v i t y and s u r v i v a l o f CP-SBMV and CP-TMV in the seed coats o f seed o f C a l i f o r n i a blackeye cowpea  Mean number o f l o c a l  l e s i o n s per h a l f  leaf o f :  N. g l u t i n o s a GA  Treatment Ungerminated  Mean 1es ions  21  CP-SBMV Percent reduct ion  51  Germinated  0.0 92.2  A l l seed coats were washed o v e r n i g h t separat ion. 'Percent r e d u c t i o n expressed seed c o a t s .  in running  r e l a t i v e to lesions  CP-TMV Mean 1es ions kl  Percent reduct ion 0.0  C  38  C  9-5  tap water  induced  after  by ungerminated  '^Values i n each column not f o l l o w e d by the same l e t t e r vary at P = 0 . 0 5 by S t u d e n t - t - d i s t r i b u t ion.  significantly  170  DISCUSSION  In the present  s t u d i e s evidence has been presented  r e p l i c a t i o n o f CP-SBMV varieties.  This  in C a l i f o r n i a  i s g r e a t l y enhanced by that o f CP-TMV  blackeye cowpea, under both asynchronous and synchronous Evidence a l s o  i n d i c a t e s that  in a mixture by  does not n e c e s s a r i l y r e s u l t in i n t e r a c t i o n o f t h e i r s t r u c t u r a l  components, even  i f t h e i r v i r i o n s a r e present  s t u d i e s a l s o present CP-SBMV  condi-  infection conditions  Evidence a l s o  i n f e c t i o n and consequent enhancement o f one v i r u s  the other  i s very  in the same c e l l .  These  evidence, which suggests that seed-borne nature o f  likely  a consequence o f embryo i n f e c t i o n r a t h e r than  contamination o f s e e d l i n g s  fornia  i n d i c a t e s that CP-TMV  bean t o become s u s c e p t i b l e to CP-SBMV.  double  in two cowpea  i n f l u e n c e of CP-TMV on CP-SBMV s y n t h e s i s was e f f e c t i v e ,  tions of i n f e c t i o n . Pinto  to show t h a t the  I.  Interaction  A.  E f f e c t s on p l a n t  The  fact  by the t e s t a e d u r i n g  germination.  in cowpea. growth.  that double i n f e c t i o n d i d not reduce p l a n t growth  blackeye cowpea any more than CP-TMV alone,and  symptoms, produced on t r i f o l i a t e  leaves  CP-TMV more than those o f CP-SBMV alone, i n f e c t i o n were a d d i t i v e .  of such p l a n t s , resembled suggest that  those o f  the e f f e c t s o f double  The r e s u l t s a l s o suggest that v i r u s  1972; Kuhn and Dawson,  in C a l i -  that the systemic  may not n e c e s s a r i l y be c o r r e l a t e d with s e v e r i t y o f symptoms. (Dodds and Hamilton,  surface  1973) made s i m i l a r  concentration Others observations.  171  The  i s o l a t e of CP-SBMV used  s t r a i n than the one the  used by Kuhn and  i n a b i l i t y of our  growth and by Kuhn  and  B. It  yield  in these s t u d i e s could have been a m i l d e r Dawson  i s o l a t e to cause any  components in C a l i f o r n i a  Dawson caused s i g n i f i c a n t  D i s t r i b u t i o n and is not c l e a r why  (1973).  This  significant blackeye  i s '(indicated by  reductions  in p l a n t  cowpea, whereas that used  p l a n t growth r e d u c t i o n s of 18 to 32%.  c o n c e n t r a t i o n of v i r u s e s . each of the v i r u s e s was  synthesized  less  in doubly  i n f e c t e d primary leaves than s i n g l y i n f e c t e d s i m i l a r  leaves of  blackeye  in the t r i f o l i a t e  It  cowpea and  yet CP-SBMV was  i s u n l i k e l y that CP-SBMV and  g r e a t l y enhanced  CP-TMV in the  t h e s i s of each v i r u s , e s p e c i a l l y of CP-SBMV, was between pre-inocu1 at ion by one  other was  increased.  In other  i m p l i c a t e d , i t was  l e s i o n s on with  bean was  bean pod  CP-SBMV and which may  early  (Lee and in the  be needed f o r  in i n f e c t i o n  for infectible  syninterthe  sites  found that the number of soybean mosaic v i r u s l o c a l  CP-TMV may  case or coat p r o t e i n .  retarded more as the  s t u d i e s where c o m p e t i t i o n  m o t t l e v i r u s , and  be that  ( c e n t r e s ) , because  cha11enge-inocu1 at ion by  very much reduced when i t was  i n o c u l a t i o n decreased It may  v i r u s and  leaves.  i n o c u l a t e d primary l e a f of  cowpea were competing f o r the same i n f e c t i b l e s i t e s  val  California  r e d u c t i o n was Ross,  inoculated  g r e a t e r as the  simultaneously interval  1972b).  inoculated  leaves of C a l i f o r n i a  blackeye  be competing f o r some component, such as  (Hunter et a l ,  be one  1976;  of the p r o t e i n s  S i e g e l et a l ,  1978).  cowpea,  ribosomes,  t r a n s l a t i o n of the messenger RNAs f o r t h e i r R e p l i c a s e may  between  repli-  synthesized However,  172  unless  age i s a l s o a f a c t o r , o v e r l o a d i n g  congruent with the f a c t  that doubly  o f the t r a n s l a t i o n a l system  infected t r i f o l i a t e  each of the v i r u s e s e f f i c i e n t l y and that double i n f e c t i o n was g r e a t l y enhanced.  the c o n c e n t r a t i o n  i s a very  initially  resistance of v i r u s c e l l - t o - c e l l that c h a l l e n g e - i n o c u l a t i o n  and  the second v i r u s even becomes g r e a t e r .  increase  in the y i e l d o f PVX in tobacco leaves  inoculated  1 and 2 o f b a r l e y , doubly  to age of the leaves  culated  t o CP-SBMV sing1e leaves  primary  trifoliate  than  Lack o f enhancement  i n o c u l a t e d w i t h BSMV and TMV, was  (Dodds 1972).  o f V 4 5 B o t s , which _  i s apparently  systemically  i n f e c t i o n , CP-SBMV t i t r e was g r e a t e r  in s i n g l y i n o c u l a t e d  ones.  leaves was about the same as that  leaves.  inocu-  1964; Damirdagh and Ross, 1967) and these have been  In the primary leaves resistant  doubly  (Rochow and Ross, 1955; S t o u f f e r  p a r t l y to age ( S t o u f f e r and Ross, 196lb).  in leaves  attributed  However,  i n o c u l a t e d v i r u s e s were in one mixture.  Ross, 1961b; C l o s e ,  of TMV  If one remembers  leaves was superimposed d i r e c t l y on  l a t e d with PVX and PVY have been observed  attributed  (manual) i n o c u l a t i o n ,  areas o f the same l e a f , then e x t r a wounding o f the same l e a f  simultaneously  and  could be  i n j u r y may induce some s o r t o f  spread and s y n t h e s i s .  o f primary  p r o b a b i l i t y of excluding  Slight  synthesized  i n e f f i c i e n t way o f i n t r o d u c i n g v i r u s , not many c e l l s a r e  i n f e c t e d and a l s o mechanical  pre-inoculated  by mechanical  in-  o f CP-SBMV in  Another p l a u s i b l e theory  that s i n c e the two v i r u s e s a r e introduced which  leaves  is  Although the c o n c e n t r a t i o n  CP-SBMV t i t r e  in doubly in the ino-  in the s y s t e m i c a l l y  of CP-TMV was s l i g h t l y  infected greater  173  in s i n g l y  inoculated  in the i n o c u l a t e d  than doubly -inocu1ated primary  leaves than  in the s y s t e m i c a l l y  However, the r e s u l t s o f \A5-B0ts were based only  leaves,  infected  i t was  greater  trifoliate  on two experiments, and  they may not j u s t i f y any c o n c l u s i o n  about the r e p l i c a t i o n of CP-TMV.  is c l e a r ,  o f V45~Bots by CP-SBMV  i s that  systemic  invasion  leaves.  What  i s dependent on  i n f e c t i o n o f t h i s host by CP-TMV. For marked enhancement o f PVX by PVY, i t has been suggested and  Ross, 1961b) that  the  l e a f through the v a s c u l a r  CP-TMV  interaction  PVX i s enhanced more by PVY, only system.  1st  fits  enters  the CP-SBMV/  in V 4 5 B o t s . -  syntheses o f CP-SBMV and CP-TMV were g r e a t e s t  and 2nd t r i f o l i a t e  i n d i c a t e that  in r a p i d l y  l e a f l e t s o f C a l i f o r n i a blackeye cowpea.  the s y n t h e s i s  vaded when e l o n g a t i n g for  explanation  i f the l a t t e r  in C a l i f o r n i a blackeye cowpea, but i t does not seem t e -  nable f o r the same p a i r o f v i r u s e s The  This  (Stouffer  of both v i r u s e s  and expanding r a p i d l y .  is greatest Similar  elongating  These r e s u l t s  when leaves a r e i n -  r e s u l t s were obtained  CP-SBMV and CCMV by Kuhn and Dawson ( 1 9 7 3 ) . The  r e s u l t s of sequential  inoculation  suggest that  g r e a t l y enhanced  in the 3 r d t r i f o l i a t e  c u l a t i o n , CP-TMV  i n o c u l a t i o n must precede CP-SBMV  not  i n d i c a t e which v i r u s a r r i v e s f i r s t  can  a l s o be seen that  inoculated  leaves that were not formed a t inoi n o c u l a t i o n , but they do  at these 3 r d t r i f o l i a t e  leaves.  It  f o r CP-SBMV t o be enhanced, CP-TMV must e i t h e r be  s i m u l t a n e o u s l y with CP-SBMV on the primary  precede CP-SBMV  f o r CP-SBMV to be  i n t o the preformed 3 r d t r i f o l i a t e  l e a f , or CP-TMV must  leaf.  It i s t h e r e f o r e  174  suggested  that f o r enhancement o f CP-SBMV t o occur  CP-TMV must be i n o c u l a t e d CP-SBMV.  Perhaps  time o f i n f e c t i o n  12 t o 2 2 hours before or 12 t o 2 2 hours  by CP-SBMV, represses  CP-SBMV-specific  Such an i n h i b i t o r o f CP-SBMV r e p l i c a t i o n  activated  as soon as the host c e l l s a r e i n f e c t e d be an inherent mechanism  a g a i n s t CP-SBMV by CP-SBMV. be  infection.  shortly  so that the host CP-SBMV  inhibitory  infection  system  by CP-SBMV.  The i n h i b i t o r may not n e c e s s a r i l y be coded f o r  before or a f t e r , or s i m u l t a n e o u s l y  with  When CP-SBMV  i s reduced, and CP-SBMV s y n t h e s i s infection  hr), i t s effect  infection  sistance against It  short  Simi-  i s no longer  by that time, may have developed n o n - s p e c i f i c r e -  infection.  time o f each o t h e r ,  that f o r CP-TMV t o i n f l u e n c e into  so that CP-TMV can n u l l i f y  is controlled  p r e d i s p o s i t i o n of c e l l s cell-to-cell  i s enhanced o n l y s l i g h t l y .  ( i ) the two v i r u s e s must be introduced  CP-SBMV s y n t h e s i s  such a subs-  precedes that o f CP-SBMV by a wide margin  i s t h e r e f o r e suggested  of CP-SBMV  CP-SBMV,  precedes that o f  in n u l l i f y i n g the i n h i b i t o r o f CP-SBMV  e f f e c t i v e , as the host,  i t must  i s no longer a b l e t o produce t h i s substance a g a i n s t  or s y n t h e s i s .  l a r l y when CP-TMV (72  The i n h i b i -  in the host genotype to p r o t e c t i t  CP-TMV by 7 2 hours, the i n f l u e n c e o f CP-TMV on c o u n t e r a c t i n g tance  subs-  may be produced or  For the e f f e c t o f CP-TMV, t o be maximally e f f e c t i v e ,  introduced,  after  i n f e c t i o n o f CP-TMV, w i t h i n a r e l a t i v e l y s h o r t space of  tance(s).  t o r could  1 eaves,  in the t r i f o l i a t e  and ( i i )  replication  the l e a f w i t h i n  the mechanism by which  t h a t enhancement  i s a result of  by the helper v i r u s or i t s by-product which  movement and t r a n s l o c a t i o n o f the dependent  virus.  allows  175  T h i s proposal matter,  does not take p h y s i c a l presence o f CP-TMV o r , f o r that  i t s rapid synthesis  continue  t o repress p r o d u c t i o n  CP-TMV may be t r a n s l o c a t e d by CP-TMV v i r i o n s . theory  i n t o account, except that perhaps CP-TMV must o f such a substance.  in the host without  The r e s u l t s o f synchronous  Indeed, the e f f e c t o f  n e c e s s a r i l y being  accompanied  i n f e c t i o n a l s o support the  that the magnitude o f CP-TMV s y n t h e s i s may not be a f a c t o r in the  enhancement o f CP-SBMV, f o r CP-TMV was g r e a t l y s y n t h e s i z e d was r e p l i c a t e d Additional  to about the same extent  support  and y e t CP-SBMV  as under asynchronous c o n d i t i o n s .  f o r the t h e s i s that p h y s i c a l presence o f CP-TMV does not  seem to be a f a c t o r in CP-SBMV enhancement  i s that both v i r u s e s  occurred  in the same c e l l s o f doubly  i n f e c t e d primary  leaves and t r i f o l i a t e  Both v i r u s e s could  in the same c e l l  o f primary  be found  leaves  leaves.  in which  CP-SBMV was reduced by t h e presence of CP-TMV. Hamilton and N i c h o l s  (1977)  could  find  no proof  that p h y s i c a l  presence o f BMV was r e p o n s i b l e f o r enhanced s y n t h e s i s o f TMV The  main  i n f l u e n c e o f CP-TMV, on CP-SBMV  invasion of t r i f o l i a t e  by e i t h e r removal o f i n h i b i t o r y synthesis  This  movement o f t h i s v i r u s from c e l l - t o - c e l l  observation liate  that  i t can be  translocated  i t was d i f f i c u l t  leaves o f doubly  be p r e d i s p o s i n g  substances or e n a b l i n g  in the i n o c u l a t e d l e a v e s .  system, wherefrom  i n f e c t i o n and i t s systemic  leaves of V 4 5 B o t s , could _  in b a r l e y .  increased  increased  CP-SBMV  s y n t h e s i s may lead to  and e v e n t u a l l y  i n t o the v a s c u l a r  i n t o the t r i f o l i a t e  to d e t e c t CP-SBMV  the c e l l s  leaves.  in 1 s t and 2nd t r i f o -  i n f e c t e d V45~Bots p l a n t s may i n d i c a t e that move-  ment o f CP-SBMV may be p a r t l y dependent on m e t a b o l i t e t r a n s l o c a t i o n , which should  The  have been p r e f e r e n t i a l l y  routed  to the newly expanding 3rd  176  and  subsequent t r i f o l i a t e  interaction  in b a r l e y  leaves.'  This  i s perhaps analogous to BSMV/TMV  (Dodds, 1972; Dodds and Hamilton, 1972).  a t t r i b u t e that t o m e t a b o l i t e s i s o f CP-SBMV and CP-TMV  transport only,  But to  i s not congruent with  synthe-  in the 1st and 2nd t r i f o l i a t e s of C a l i f o r n i a  blackeye cowpea, where they were s y n t h e s i z e d  more than  in the later-formed  1 eaves. The i n f l u e n c e o f CP-TMV on CP-SBMV systemic  i n v a s i o n o f V45 Bots i s -  s i m i l a r t o the i n f l u e n c e o f BSMV or BMV on systemic spread and enhancement of TMV  in b a r l e y  (Hamilton and Dodds, 1970; Dodds, 1972; Dodds and Hamilton,  1972; Hamilton and N i c h o l s , that  in some cases  1972), TMV could  infected  be detected  trifoliate  synthesized employed  in concentrated  no CP-SBMV could  sucrose d e n s i t y  invades t h i s host  gradient  however, in  extracts of barley singly be detected  ino-  by i n f e c t i v i t y or  c e n t r i f u g a t i o n in e x t r a c t s o f s i n g l y  1 eaves that were concentrated  20-fold.  If CP-SBMV  s y s t e m i c a l l y when i n o c u l a t e d alone then, i t must be  to a l e v e l  below that which could  in t h i s study.  that o f CP-TMV on CP-SBMV  be detected  The two systems a l s o d i f f e r  i n f l u e n c e o f BSMV or BMV on TMV  in V45 Bots was e f f i c i e n t _  i n v a s i o n o f V45~Bots by CP-SBMV v i r u s on c o n d i t i o n i n g c o t t o n  in that whereas the  under normal  temperature, temperature  The i n f l u e n c e o f CP-TMV on systemic  i s s i m i l a r t o the e f f e c t o f Anthocyanos i s  plants  tobacco s t r e a k v i r u s  by the methods  in b a r l e y was dependent on high  regimes, s u i t a b l e f o r p l a n t growth.  Brazilian  The two systems d i f f e r ,  (Hamilton and Dodds, 1970; Dodds, 1972; Dodds and Hamilton,  c u l a t e d with TMV, whereas analytical  1977).  t o become s y s t e m i c a l l y  (Costa,  1969)-  invaded by the  177  The  present  r a t u r e system  s t u d i e s have u t i l i z e d (Dawson and S c h l e g e l ,  the synchronous d i f f e r e n t i a l  1973), f o r the f i r s t  mixed v i r u s  i n f e c t i o n s and i t s a p p l i c a t i o n i s promising.  be extended  to i n c l u d e s e q u e n t i a l  be used with  l e s s problems  be used as v i r u s hosts. to study mixed v i r u s Harrison,  i n o c u l a t i o n s and there  i n f e c t i o n s (Otsuki  i t can  and Takebe, 1976, 1978; Barker and  The  present  that o n l y  studies f a i l e d  to reveal  any evidence o f genomic masking  However, t h i s  i s not s u r p r i s i n g when one con-  two cases o f in v i v o genomic masking have been  between s t r u c t u r a l l y d i s s i m i l a r p l a n t v i r u s e s Dodds, 1974; Peterson and Brakke, 1973). Ross, 1974c; Hamilton and N i c h o l s ,  structural  i s no doubt  1977a, 1977b).  between CP-TMV and CP-SBMV.  and  The system can  Another synchronous system has r e c e n t l y been used  In v i v o s t r u c t u r a l i n t e r a c t i o n s .  1974;  in studying  i f c o l d - t o l e r a n t p l a n t s , such as b a r l e y , can  C.  siders  time,  tempe-  reported  (Dodds and Hamilton, 1971, Others  1977) could  (Morris,  not d e t e c t  1970; Goodman  such  in vivo  interactions.  In the present  studies,  i t i s not known why s t r u c t u r a l i n t e r a c t i o n s  between CP-SBMV and CP-TMV d i d not occur.  One can o n l y  postulate,  even  then without much c e r t a i n t y , s i n c e our knowledge o f what happens in the mil l i e u o f the i n f e c t e d c e l l with the l i t e r a t u r e before  i s meagre.  review that  some o f the c o n d i t i o n s  i n t e r a c t i o n s between pools  the homologous s p e c i f i c i t y  It was s t a t e d  of v i r a l  between v i r a l  in the s e c t i o n  dealing  that must be met  components can occur a r e that  RNA and coat  p r o t e i n be broken  down and that assembly s i t e s of the v i r u s e s more or l e s s c o i n c i d e .  It  is  178  a l s o necessary that t h e r e be temporal the v i r u s e s f o r i n t e r a c t i o n that both CP-SBMV and proof that both  c o i n c i d e n c e in the l i f e c y c l e s of  to o c c u r .  Although  CP-TMV were present  replicated  in that  Assembly s i t e s of v i r u s e s ,  et  S c h l e g e l , 1969), c h l o r o p l a s t s a l , 1971), and  have a l l been  including  those of TMV,  in doubt.  The  whose l i f e nucleus  r e p l i c a t i o n , and  f o r TMV  perhaps  mediates  l i f e c y c l e , were found  of  ds-RNA.  in the nucleus and  a d d i t o n , accumulation  found  S t u d i e s on  in the phloem;  cation.  to be  inter-  w i t h these o r g a n e l l e s . discounts  mitochon-  s y n t h e s i s , because sites  The o n l y evidence a v a i l a b l e f o r SBMV i s  cytoplasm  (Weintraub  bean s t r a i n s of SBMV  and  Ragetli,  1970).  of v i r u s - l i k e p a r t i c l e s of the bean s t r a i n were  i t was  It i s d i f f i c u l t  TMV  The most p l a u -  r e p l i c a t i o n and or  in which v i r u s - l i k e p a r t i c l e s of the cowpea and  were found In  to sediment  i m p l i c a t e s c y t o p l a s m i c membranes as s i t e s o f TMV  assembly of SBMV are l a c k i n g .  that  believed  (Ni1sson-Ti11gren et a l , 1974)  they were a s s o c i a t e d w i t h TMV  However,  assembly, are m i t o c h o n d r i a  because the r e p l i c a t i v e forms,  d r i a and  1974)  in the these orga-  virions.  (Jackson et a l , 1971)  However, recent evidence  (Jackson  ( S h a l l a et a l , 1975), as s i t e s f o r  n e l l e s could have been p r e v i o u s l y mistaken  in the v i r u s  (Langenberg  assembly of TMV.  s y n t h e s i s , because the i n t r a p 1 a s t i d i a 1 p a r t i c l e s present  s i b l e s i t e s f o r TMV  cycle  (Ni1sson-Ti11gren et a l ,  in the s y n t h e s i s and  c h l o r o p l a s t s have been d i s c o u n t e d  t h e r e i s no  ( S i n g e r , 1972), m i t o c h o n d r i a  c y t o p l a s m i c membranes  implicated  in the same c e l l ,  cell.  has been e x t e n s i v e l y s t u d i e d , are s t i l l and  the present s t u d i e s show  also  implied that these may  be s i t e s f o r r e p l i -  to s p e c u l a t e whether CP-SBMV and  CP-TMV  assembled  179  at common s i t e s .  If they d i d not assemble a t common s i t e s ,  then t h i s  be one reason why t h e i r s t r u c t u r a l components d i d not i n t e r a c t . that  the v i r u s e s d i d not assemble a t the same s i t e s .  show that cance  in some c e l l s ,  i s not c l e a r .  assembly s i t e s such  CP-SBMV, was enclosed  Further,  they could  It i s l i k e l y  E l e c t r o n micrographs  by membranes whose  signifi-  have had common r e p l i c a t i o n and  but the s p e c i f i c i t y o f the RNA-protein  interaction.  could  interaction  precluded  Indeed RNA-protein s p e c i f i c i t y was found to be high  between TMV s t r a i n s (Atabekova e t a l , 1 9 7 5 ) . of CP-SBMV and CP-TMV  Time-course s y n t h e s i s  in primary and t r i f o l i a t e  leaves  of C a l i f o r n i a  even  studies blackeye  cowpea i n d i c a t e d that CP-TMV s y n t h e s i s was always more r a p i d than that of CP-SBMV. tion  T h e o r e t i c a l l y one would assume that  f o r the two v i r u s e s , as measured by i n f e c t i v i t y and spectrophometry,  represent  what happens  i n s i d e the c e l l ,  of CP-TMV would have l e f t capsidate  then the a c c e l e r a t e d r e p l i c a t i o n  p r o t e i n subunits  in excess o f that needed to en-  homologous RNA, thus f u r n i s h i n g subunits  CP-SBMV-RNA. given  i f the k i n e t i c s o f r e p l i c a -  that would  The r a p i d i t y a t which BSMV may be s y n t h e s i z e d  (Dodds, 1 9 7 4 ;  Dodds and Hamilton,  1974)  encapsidate  in barley i s  as one p o s s i b i l i t y  may have favoured TMV-RNA genomic masking by excess BSMV p r o t e i n , absence o f homologous In the present  in the  specificity.  studies,  RNA o f v i r u s by the coat  i t i s a l s o p o s s i b l e that genomic masking of  p r o t e i n o f the other  i n s t a b i l i t y o f the reassembly product, a t t a c k detection,  that  by i n f e c t i v i t y , d i f f i c u l t .  occurred,  but because o f the  by c e l l u l a r  RNase made t h e i r  A l s o because o f n o n - s p e c i f i c  reactions  180  between v i r a l  antigen  and a n t i b o d i e s as well as normal  of a low percentage o f h e t e r o l o g o u s l y  encapsidated  serum, d e t e c t i o n  product  would be d i f f i -  cult. I I. The to  I n t e r a c t i o n in P i n t o . present  a detectable  regarded  s t u d i e s a l s o show that CP-SBMV can i n f e c t and m u l t i p l y  level  in the i n o c u l a t e d  leaves of P i n t o bean, a host  as immune (Shepherd and F u l t o n ,  c l e a r why P i n t o  i s i n f e c t e d by CP-SBMV  1962;  Shepherd,  1971).  otherwi  It i s not  in the presence o f CP-TMV.  However,  t h i s type o f s i t u a t i o n does not seem to be novel  and unique; o t h e r s  1959; Weathers, 196l;Pound  i n c i d e n t s , where a v i r u s  not of  e t a l , 1962)  reported  known t o i n f e c t a host, d i d so in the presence o f another. i n t e r a c t i o n , by these v i r u s e s The  simplest explanation  of P i n t o could they  be that a f t e r  become pre-disposed  infection  production  by CP-SBMV.  o f how CP-TMV  that manner d u r i n g  i n f l u e n c e s CP-SBMV  infection  the P i n t o l e a f c e l l s a r e i n f e c t e d by CP-TMV,  t o CP-SBMV  infection.  o f substance(s)  As hypothesized  be that CP-TMV  infection  that would normally  Another p o s s i b l i t y could  or RNA molecules a r e adsorbed  The extent  was not c h a r a c t e r i z e d f u r t h e r .  cement o f CP-SBMV, in cowpea, i t could or r e p r e s s e s  (Tochiha  inoculation.  P a r t i c l e s o f raspberyy  nullifies  act against  be that CP-SBMV  to CP-TMV p a r t i c l e s and e n t e r  f o r enhan-  virions  the c e l l s in  r i n g s p o t and tobacco  r a t t l e v i r u s e s have been observed t o form i n v i v o and i n v i t r o aggregates (Barker reasons:  and H a r r i s o n ,  1977b).  However, t h i s does not seem t e n a b l e  ( i ) mere p h y s i c a l a t t r a c t i o n a t l e v e l  f o r two  o f the v i r i o n would not  181  n e c e s s a r i l y mean that a f t e r and  u n c o a t i n g , the host genotype would  accept CP-SBMV-RNA template  and ( i i ) because CP-SBMV-RNA, c h a l l e n g e -  i n o c u l a t e d 2k hours a f t e r p r e - i n o c u l a t i o n with CP-TMV, s t i l l l e s i o n s o f the same magnitude as in a simultaneous When CP-TMV d i s s o c i a t e d ted t o P i n t o , no l o c a l A third  possibility,  the host c e l l  p r o t e i n was mixed with l e s i o n s developed  otherwise  i n t a c t CP-SBMV and i n o c u l a -  (Molefe, unpublished o b s e r v a t i o n ) .  becomes a v a i l a b l e  However, u n l e s s such s t i m u l a t e d r e p l i c a s e i s  by the h o s t - v i r u s genotype t h e r e would be an i n f e c t i o n o f hosts regarded  is a t t r a c t i v e ,  as immune, so long as the "incompetent" This  i s not the case.  however, because o f recent r e p o r t s (Ikegami  1978; Romaine and Z a i t l i n ,  by i n f e c t i o n .  virus is This  hypothesis  and F r a e n k e l -  1978; White and Dawson, 1978) that  s o l u b l e RNA polymerases a r e present lated  local  i n o c u l a t i o n with CP-TMV.  n a t i v e RNA-dependent RNA polymerase which then  accompanied by a competent v i r u s .  Conrat,  induced  l e s s f a v o u r e d , could be that CP-TMV i n f e c t i o n s t i m u l a t e s  to the CP-SBMV-RNA template. determined  recognize  in h e a l t h y c e l l s and a r e stimu-  However, the s t r e n g t h of t h i s h y p o t h e s i s  i s weakened  by the r e p o r t (White and Dawson, 1978) that the a c t i v i t y o f these p o l y merases may be s t i m u l a t e d by mock-inocu1 at ion in cowpea and tobacco l e a v e s , thus s u g g e s t i n g that the i n c r e a s e in enzyme a c t i v i t y sively virus-specific. merases may occur is,  However, m o d i f i c a t i o n o f the a c t i v i t y o f such  poly-  in the i n t e r a c t i o n o f the host and v i r u s genomes, that  the dependent v i r u s must possess  infect  i s not e x c l u -  such a host.  some genes w i t h the p o t e n t i a l to  It may be t h a t host enzyme, as suggested  else-  182  where  (Romaine and Z a i t l i n ,  transcriptional and  1978), " i s the 'core' polymerase which lacks  c o n t r o l , and through  i t s a s s o c i a t i o n with o t h e r  virus-coded  host-coded p r o t e i n s on the membrane i t forms the f u l l y - c o m p e t e n t  case".  The suggestion  that a dependent v i r u s might have the . p o t e n t i a l to  i n f e c t an immune host d u r i n g a mixed  infection  i s not u n l i k e l y ,  of SBMV, because two o f i t s r e l a t i v e s have been reported when i s o l a t e d from bean  from cowpea  (Yerkes  specific  in the case  to infect  bean,  (Lamptey, 1972) and.'infect., cowpea-.when i s o l a t e d  and P a t i n o ,  I960) in s i n g l e  infection.  of CP-SBMV one would assume that the p o t e n t i a l f o r some reason  repli-  to infect  Thus in the case bean e x i s t s , but  the genome o f t h i s v i r u s i s unable t o s t i m u l a t e the v i r u s -  r e p l i c a s e to a s u f f i c i e n t  level  to d i r e c t  the s y n t h e s i s o f v i r a l '  RNA . III. The  Seed present  transmission. s t u d i e s have a l s o presented  CP-SBMV and not CP-TMV  seed  seed  o f the embryo or d e v e l o p i n g  previous  good agreement between seed p l a n t e d embryos.  that  Transmission results  It i s d o u b t f u l  (Shepherd that  s e e d l i n g w i t h v i r u s from the  c o a t s would account f o r such c o n s i s t e n c y  of CP-SBMV, and y e t not r e s u l t  and  o f cowpea confirms  F u l t o n , 1962; Gay, 1973; Kuhn and Dawson, 1973).  contamination  which suggests  i s c o n s i s t e n t l y seed-borne in cowpea.  of CP-SBMV through p l a n t e d and  evidence  in CP-TMV seed  in the seed t r a n s m i s s i b i 1 i t y transmission.  There was a  t r a n s m i s s i o n o f CP-SBMV through p l a n t e d  seeds  The f a i l u r e to recover CP-SBMV c o n s i s t e n t l y from embryo  e x t r a c t s , d i r e c t l y assayed  on GA 21, c o u l d be a t t r i b u t e d  t o the apparent  183  presence of i n h i b i t o r ( s ) o f i n f e c t i o n  in the seed o f cowpea that may  i n t e r f e r e w i t h v i r u s at the i n f e c t i o n  sites.  t o r (s) may have been the f a c t o r recover  The presence o f t h i s  r e s p o n s i b l e f o r t h e f a i l u r e o f o t h e r s to  i n f e c t i o u s SBMV from e i t h e r  immature embryos  (McDonald, 1971;  McDonald and Hamilton,  1972) or embryos of dry mature seed  McDonald and Hamilton,  1972) o f bean  blackeye cowpea  i n f e c t e d w i t h the bean s t r a i n o f SBMV,  infected  Indeed an i n h i b i t o r o f i n f e c t i o n which reported  in bean seed e x t r a c t  o t h e r s which and  reduced  (Cheo,  implicated  with the Ghana s t r a i n o f SBMV. reduced a c t i v i t y o f SBMV has been  1955), and Crowley  (1955) r e p o r t e d  i n f e c t i v i t y o f cucumber mosaic v i r u s and TMV i n cucumber  tobacco s e e d s , r e s p e c t i v e l y .  has been  (Cheo, 1955;  (Lamptey, 1972; Lamptey and Hamilton, 1974)  and embryos o f dry mature seed of C a l i f o r n i a  The presence o f i n h i b i t o r s  i n seed  extracts  in lower seed t r a n s m i s s i o n percentages o b t a i n e d w i t h  embryo e x t r a c t s assayed d i r e c t l y on i n d i c a t o r p l a n t s than w i t h assay of bean common mosaic v i r u s (Powell  inhibi-  and S c h l e g e l ,  1970).  seedling  ( S c h i p p e r s , 1963) and o f squash  Negative r e s u l t s o f d i r e c t  mosaic  assay of embryo  e x t r a c t s on i n d i c a t o r hosts could a l s o be due to too low a c o n c e n t r a t i o n of the v i r u s  in the embryo.  SBMV has been r e p o r t e d to be seed-borne  (Zaumeyer  and H a r t e r , 1 9 ^ 3 ; Uyemoto and Grogan, 1977) and embryonica11y-transmitted (Uyemoto and Grogan, 1977) in bean.  Recent  l o g i c a l 1 y-spec i f i c e l e c t r o n microscopy particles and  r e s u l t s a t V.R.S., u s i n g s e r o -  (SSEM), i n d i c a t e evidence o f v i r u s - l i k e  in washed embryos o f bean, v a r . E a r l y G a l l a t i n ,  i n f e c t i v i t y was n i l  N i c h o l s , unpublished  i n f e c t e d w i t h SBMV,  when p a r t i c l e s observed were few (Hamilton and  results).  184  The  best evidence o f CP-SBMV  i n f e c t i o n o f embryos was the t r a n s m i -  s s i o n o f the v i r u s t o s e e d l i n g s by decontaminated c o n t a m i n a t i o n or f o r t u i t o u s e n t r a n c e o f CP-SBMV was the cause o f i t s seed-borne decontaminated Additional which  nature, then a r t i f i c i a l l y  e v i d e n c e f o r embryo i n f e c t i o n  i n d i c a t e that  contaminatedthey d i d not.  i n the seed c o a t s was reduced  These r e s u l t s suggest  d e v e l o p i n g s e e d l i n must be i n f e c t e d  embryos  by CP-SBMV comes from the r e s u l t s  i n f e c t i v i t y o f CP-SBMV  CP-SBMV t r a n s m i t t e d through p l a n t e d seed  If s u r f a c e  into u i n i n j u r e d  embryos should have t r a n s m i t t e d the v i r u s ;  by g e r m i n a t i o n o f seed.  or  embryos.  that,  i f the source o f  i s the seed c o a t , then the embryo  (contaminated)  at or before  germinat ion. It or  i s not c l e a r why CP-SBMV was not t r a n s m i t t e d through p l a n t e d seed  embryo e x t r a c t s o f two Botswana l o c a l  contamination supported  i s not a f a c t o r  That  t r a n s m i s s i o n o f CP-SBMV  seed coat  is also  by the r e s u l t s o f these v a r i e t i e s whose seed c o a t s c o n t a i n e d  both v i r u s e s . a low l e v e l  CP-SBMV may be s e e d - t r a n s m i t t e d in these v a r i e t i e s ,  not d e t e c t e d by the number o f seeds used  other p o s s i b i l i t y varieties.  in seed  cowpea v a r i e t i e s .  but t o  in these s t u d i e s . An- ii  i s that CP-SBMV may not be s e e d - t r a n s m i t t e d in these  It i s not unusual  that a v i r u s may be t r a n s m i t t e d through  seed o f one v a r i e t y or c u l t i v a r o f a p l a n t and not so seed t r a n s m i t t e d in another o f the same s p e c i e s o r genus. Thus, squash mosaic seed-transmitted et  in some v a r i e t i e s o f squash,  a l , 1959) and the M2 i s o l a t e of peanut  seed o f some c u l t i v a r s o f groundnut 1977).  v i r u s was  but not in o t h e r s  (Grogan  m o t t l e v i r u s was t r a n s m i t t e d in  but not in o t h e r s (Adams and Kuhn,  185  It  i s t h e r e f o r e proposed  through seed o f C a l i f o r n i a plays  very l i t t l e ,  that CP-SBMV i s embryonica11y t r a n s m i t t e d  blackeye cowpea and that v i r u s  i f any, r o l e  o r , f o r that matter, o f CP-TMV.  in seed t r a n s m i s s i o n o f t h i s  Some v i r u s e s occur  some h o s t s , but they a r e not seed-borne; ted  through p l a n t e d seed o f sugar beet  CP-TMV  in the present  in the seed coat o f  thus c u r l y top v i r u s  (Bennett and Esau,  i s not t r a n s m i t -  1936) and nor i s  i n o c u l a t i o n a r e suspected t o i n t e r f e r e - ; w i t h  i n f e c t i v i t y assay o f the v i r u s a b e t t e r way t o circumvent  would be t o assay, f o r presence o f v i r u s decontaminated  embryos.  immunosorbent assay t r o n microscopy avoid  virus  studies.  Where i n h i b i t o r s o f manual the  in the seed coat  this  problem  in the seed, s e e d l i n g s d e r i v e d from  The use o f techniques  as s e n s i t i v e as emzyme-1inked  ( C l a r k and Adams, 1977) o r s e r o l o g i c a 1 1 y - s p e c i f i c  elec-  ( D e r r i c k and B r l a n s k y , 1 9 7 6 ) , c o u l d a l s o be employed t o  inhibitors;  however these methods may not i n d i c a t e the v i a b i l i t y o f  the v i r u s so scanned. The mechanism o f seed  t r a n s m i s s i o n i s p o o r l y understood.  s t u d i e s CP-SBMV was t r a n s m i t t e d l e s s than was  in seeds o f s i n g l y transmitted less  infected  in seeds from doubly  plants.  in seeds o f soybean  v i r u s and bean pod m o t t l e v i r u s  infected  soybean  infected  plants  mosaic  by soybean  virus mosaic  In c o n t r a s t , CP-SBMV was  blackeye cowpea, doubly  CCMV and CP-SBMV (Kuhn and Dawson, 1 9 7 3 ) . seed  doubly  (Ross, 1963)-  t r a n s m i t t e d more in seed o f C a l i f o r n i a  perhaps  Similarly,  In t h e present  It i s tempting  t r a n s m i s s i o n o f the s e e d - t r a n s m i s s i b l e v i r u s  i n f e c t e d by  to speculate that, i s reduced  in seed o f  186  doubly  i n f e c t e d p l a n t s , because t h e i r embryos possess an inherent p r o t e c t i v e  mechanism a g a i n s t In doubly  infection  by the v i r u s which  infected plants, infection  v i r u s would  i s not normally  by e i t h e r CP-TMV or bean pod m o t t l e  induce a n o n - s p e c i f i c i n h i b i t o r y s u b s t a n c e ( s ) , which would  reduce the a b i l i t y o f the embryo-borne v i r u s t o i n f e c t hypothesis  i s very  simple  characteristics The  observation  Dawson  (1973).  (1940) e x p l a i n e d l a c k o f embryo i n f e c -  that the gametophytic g e n e r a t i o n  may be  by non-embryo-borne v i r u s e s due t o some p h y s i o l o g i c a l  inherent  duced more mottled and  Bennett  by some v i r u s e s , by suggesting  immune to i n f e c t i o n  the embryo. T h i s  and i t may not r e f l e c t what a c t u a l l y happens  between h o s t - v i r u s genotypes. tion  embryo-borne.  in the gametophytic make-up.  that double i n f e c t i o n , i n  the present  seed than s i n g l e i n f e c t i o n confirms In n e i t h e r study was t h e r e evidence  s t u d i e s , pro-  the r e s u l t s o f Kuhn to i n d i c a t e that seed  m o t t l i n g a f f e c t e d seed t r a n s m i s s i o n o f CP-SBMV. By c o n t r a s t , seed m o t t l i n g in soybean,  i n f e c t e d by soybean mosiac v i r u s , was c o r r e l a t e d w i t h  t r a n s m i s s i o n o f t h i s v i r u s than was the case in normal Kennedy and Cooper,  seed  higher  (Ross,  seed  1963;  1967).  It was a l s o not p o s s i b l e t o c o r r e l a t e seed t r a n s m i s s i o n of CP-SBMV with  i t s concentration  in the l e a v e s .  This observation  made f o r soybean mosaic v i r u s when i t was in double mottle  v i r u s . The l a t t e r  in the present ble v i r u s .  is similar  i n f e c t i o n with  i n t e r a c t i o n and that o f CP-SBMV/CP-TMV,  to that bean pod  reported  s t u d i e s , r e s u l t e d in the enhancement o f the s e e d - t r a n s m i s s i -  However, in double  v i r u s were t r a n s m i t t e d  l e s s than  i n f e c t i o n s CP-SBMV and soybean mosaic in seed d e r i v e d from s i n g l y i n f e c t e d  187  plants.  In c o n t r a s t , CCMV/CP-SBMV  CP-SBMV n u c l e o p r o t e i n y i e l d s ,  i n t e r a c t i o n , which leads to depressed  r e s u l t e d in higher  seed t r a n s m i s s i o n  of CP-SBMV than those  found  Adams and Kuhn ( 1 9 7 7 )  could not c o r r e l a t e seed t r a n s m i s s i o n o f peanut  v i r u s with The  the l e v e l  in seed d e r i v e d from s i n g l y  rates  f e c t i o n o f P i n t o bean by CP-SBMV  is very  likely  in the f i e l d .  t o CP-SBMV;  infection  i n f e c t i o n o f V 4 5 B o t s and on _  i s not without  in-  s i g n i f i c a n c e in the e p i d e -  Although no v e c t o r o f TMV  i s known, i t  that the two v i r u s e s may be adsorbed to the claws o f c r a w l i n g  i n s e c t s and d e p o s i t e d sistant  mottle  o f v i r u s in the leaves and f l o w e r s o f groundnut.  i n f l u e n c e o f CP-TMV on systemic  miology o f d i s e a s e s  infected plants.  by them on a host otherwise  regarded  as immune or r e -  in the presence of CP-TMV the two v i r u s e s may  through f o r t u i t o u s i n j u r y .  Indeed, S c o t t and F u l t o n  initiate  (1978)  reported  t h a t b e e t l e s c o u l d a c q u i r e and d e p o s i t both CP-TMV and the bean s t r a i n o f SBMV.  Although o n l y  CP-TMV could had  fed.  the l a t t e r was t r a n s m i t t e d  be recovered  The f a c t  from  leaf surfaces  source  but seeded d i r e c t l y ,  of wet seed.  in seed coats o f cowpea,  in t h i s manner t o d i s t a n t p l a c e s .  in the seed coat a l s o means that the seed coat  o f inoculum f o r e i t h e r v i r u s .  planted,  in the areas where the b e e t l e s  that both CP-SBMV and CP-TMV occur  means that these v i r u s e s can be t r a n s p o r t e d T h e i r presence  by the b e e t l e s , i n f e c t i o u s  For i n s t a n c e  can p r o v i d e  Although cowpea i s not normally  the v i r u s e s may e n t e r  the embryo d u r i n g  transhandling  in Botswana, where cowpea i s grown e x t e n s i v e l y ,  under d r y - l a n d  farming,  i t i s sometimes necessary  of time before  sowing, to supplement the low l e v e l  to soak seed f o r a p e r i o d o f m o i s t u r e a v a i l a b l e in  188  in the s o i l .  A f t e r soaking the seed  i s broadcast or sown with a p l a n t e r ,  both o f which may i n j u r e the embryos. of  introducing The  the v i r u s e s  T h i s could  be an inadvertent  i n t o embryos.  i n f l u e n c e o f CP-TMV on i n f e c t i o n o f P i n t o  lend support to the e v o l u t i o n  theory  strains  (Lamptey and Hamilton,  evolved  and l o s t  the a b i l i t y  speculated  1974).  It could  to infect  by CP-SBMV may a l s o  f o r the o r i g i n o f SBMV be t h a t , however, CP-SBMV  bean, i t and the host  still  the p o t e n t i a l f o r an i n t e r a c t i o n ( i n f e c t i o n ) , under c o n d i t i o n s to P i n t o and in t h i s case  lesions of Pinto  as we11 as cowpea.  conditions.  what would happen i f CP-SBMV, i n the  l e a f , was c o n t i n u o u s l y  CP-TMV and a f t e r s e v e r a l  possess  not f a v o u r a b l e  i n f e c t i o n by CP-TMV would be u n f a v o u r a b l e  It would a l s o be i n t e r e s t i n g t o see local  way  passaged  passages, attempt t o i n o c u l a t e  in the presence o f i t s i n g l y to P i n t o  189 SUMMARY  Double i n f e c t i o n o f C a l i f o r n i a CP-TMV r e s u l t e d leaves  i n lower y i e l d s o f each v i r u s  in the i n o c u l a t e d  and i n the enhancement o f CP-SBMV s y n t h e s i s  infected The  blackeye cowpea by CP-SBMV and  t r i f o l i a t e leaves  i n f l u e n c e o f CP-TMV  primary  in the s y s t e m i c a l l y  compared t o y i e l d s in s i n g l y i n f e c t e d t i s s u e .  i n f e c t i o n on enhanced s y n t h e s i s o f CP-SBMV was  e f f e c t i v e under both asynchronous and synchronous c o n d i t i o n s o f i n f e c t i o n . CP-TMV  i n f e c t i o n also conditioned  infected  by CP-SBMV.  the presence o f CP-TMV Pinto  bean.  Evidence  V45-Bots cowpea t o become s y s t e m i c a l l y  i s a l s o presented which suggests that in  i n f e c t i o n CP-SBMV  i n f e c t e d and m u l t i p l i e d in  Although double i n f e c t i o n enhanced CP-SBMV s y n t h e s i s  both v i r u s e s were detected  in the same c e l l s o f doubly  blackfeye cowpea, no genomic masking was detected CP-SBMV, but not CP-TMV, was t r a n s m i t t e d decontaminated embryos of C a l i f o r n i a caused CP-SBMV to be t r a n s m i t t e d plants.  It i s t h e o r i z e d  physiologically thus  increased  by a l l o w i n g  blackeye cowpea.  CP-TMV on systemic  in seed from s i n g l y i n f e c t e d  It i s a l s o concluded  i s a r e s u l t o f embryo i n f e c t i o n .  s t u d i e s on mixed v i r u s  a l s o have important e p i d e m i o l o g i c a l  aimed at o b t a i n i n g  that  that  CP-SBMV  Pinto  bean t o  the importance o f  i n f e c t i o n s i s not j u s t a l a b o r a t o r y  CP-SBMV through seed o f cowpea should  the host  The i n f l u e n c e o f  i n f e c t i o n o f V45-Bots and on rendering  become s u s c e p t i b l e to CP-SBMV emphasizes the f a c t  can  Double i n f e c t i o n ;  t o be i n f e c t e d by CP-SBMV and  s y n t h e s i s o f CP-SBMV.  seed t r a n s m i s s i o n  seed and planted  i n f e c t i o n predisposes  more c e l l s  California  between the two v i r u s e s .  through planted  l e s s than  that CP-TMV  infected  and that  c u r i o s i t y , but  s i g n i f i c a n c e . 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