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Detection of nepoviruses by ELISA in tissue-cultured and field-grown grapevines Johnson, Raymond Camille Joseph 1988

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DETECTION  OF  NEPOVIRUSES AND  BY  ELISA  FIELD-GROWN  IN  TISSUE-CULTURED  GRAPEVINES  by RAYMOND CAM I L L E B.S.A.,  A THESIS  University  SUBMITTED  THE  JOSEPH  IN  REQUIREMENTS MASTER  of  JOHNSON  Manitoba,  1975  PARTIAL  FULFILLMENT  FOR  DEGREE  OF  THE  OF  OF  SCIENCE  in THE  F A C U L T Y OF G R A D U A T E  (Department  We a c c e p t to  THE  this  the  of  Plant  thesis  required  UNIVERSITY  OF  STUDIES  Science)  as  conforming  standard  BRITISH  COLUMBIA  October,1988  c)  Raymond  Cam I I Ie  Joseph  Johnson,  1988  In  presenting  degree  this  at the  thesis  in  University of  partial  fulfilment  British Columbia,  freely available for reference and study. copying  of  department  this or  thesis by  for scholarly  his  or  publication of this thesis  her  Plant Science  DE-6 (2/88)  O c t o b e r 11. 1988  requirements  for  may It  be is  advanced  that the Library shall make it  I further agree that permission  purposes  an  granted  for extensive  by the head  understood  that  for financial gain shall not be allowed without  The University of British Columbia Vancouver, Canada  Date  the  I agree  representatives.  permission.  Department of  of  of  my  copying  or  my written  ii ABSTRACT  The  detection  polyhedral  viruses  unreliable.  field-grown  (ELISA)  grapevines.  (plantlets)  growth  room  was  Nepovirus affected  temperature.  buffers  Arabis  were  mosaic  The  first  and  screenhouse-grown  virus  was  plantlets AMV  time,  i n in  and  parts  by  and  room  for  from  d e t e c t e d i n in  fieldThe vitro  (GFLV) d i s t r i b u t i o n This  The  detection  in plantlets.  found  in plants  growing  at  reduced  had  an  The  15°C. virus  The  viruses  of  sometimes  tissues.  highest  a l l plantlets  was  plant  the zones  were  virus  Temperature  Growth a t 30°C  best  important e f f e c t  titre.  was  distribution  came from  t a k e n from o t h e r  virus  in nearly  virus  detection  temperature  as 30 d a y s .  ELISA,  the summer.  temperature.  shoots.  d e t e c t e d i n samples  little  detection  when m o d i f i e d  throughout  the p l a n t l e t s .  for virus  proliferating  undetectable  of v i r u s  and  25°C.  the c u l t u r e  s t e p s t o 30°C  vitro  distribution  d e t e c t e d by  repeatedly  Growth room  5°C  was  grapevine fanleaf  to sample  rapidly  improved  i n in  grapevines i n i t i a t e d  plants  uniform throughout  affected  by v i r u s  reliability  (AMV)  vitro  not r e l i a b l y grown a t  detection  and  used.  virus  the  not  in tissue-cultured  f i e l d - g r o w n g r a p e v i n e s was  grinding  of nematode-transmit ted  (nepoviruses) in grapevines is often  assay  plants  not  serology  N e p o v i r u s e s were d e t e c t e d by enzyme-1 i n k e d  immunosorbent  in  by  AMV  growing  on  titres  increases  were in  became a t 30°C  r e d u c e d ELISA  f o r as  absorbance  i ii values at  4%  below  by of  76%  a  detectable in  30°C  repeatedly  the  (0.01  phosphate  was  summer the  detection  of  by  in  samples  throughout  f o l i a r the  by  increasing  or  0.15  results  M  pH  phosphate  with  AMV  summer the  GFLV  explants  detected  after  plants  by  could being  or  at  GFLV.  from the  growing  were  not  be  placed  8.2  saline.  obtained  with  was  or  20°C. at buffer  Tween-20,  essential  r e l i a b l y  buffer adding  The  the was  most  for  detected  screenhouse  and  the  at  0.05%  was  grinding  virus  grinding  7.4,  AMV  for  experienced  ELISA  pH  f i e l d  least  ELISA, The  conditions  standard  buffered  were  values dropped  AMV  plants.  saline,  when to  the  t i t r e  plantlets.  polyvinylpyrrolidone)  r e l i a b l e ELISA  virus  in vitro  in  days  months.  buffered  2%  The  grapevine  modifying  ovalbumin,  21  most  virus  typical  B.C.,  after  rooted 2  in vitro  in  Sidney,  0.2%  for  detected  Under  in  or  ringspot  and  levels.  levels  treatment  time,  M  days  plantlets  Tomato f i r s t  8  pre-treatment  detected in  after  plants  was  modified  1%  nicotine  r e l i a b l e  nicotine  enhanced  buffers. In  comparison,  associated  with  because  growing  detection  of  to  nepoviruses  detect  viruses  in in  of  plants these f i e l d  the  increased  in vitro  and  workload the  unreliable  plants,  it  remained  plants  by  ELISA.  preferable  i v  TABLE OF CONTENTS PAGE T I T L E PAGE ABSTRACT  i i  TABLE OF CONTENTS  iv  L I S T OF TABLES  v i i  L I S T OF FIGURES  ix  ACKNOWLEDGEMENTS  xi  INTRODUCTION  1  LITERATURE REVIEW  7  ELISA d e t e c t i o n  of viruses i n  differentiated in  plant  t i s s u e grown  vitro  7  T e m p e r a t u r e e f f e c t on v i r u s concentration in  i n grape p l a n t l e t s growing  vitro  Cytokinin  8 effect on.virus  i n p l a n t l e t s grown Serological  in vitro  detection  in field-grown  10  of nepoviruses  grapevines  MATERIALS AND METHODS Tissue  concentrations  culture  12 21 21  Media  21  Plant material  21  Initiation  22  Reculturing  23  Serology Virus p u r i f i c a t i o n  23 23  TABLE OF CONTENTS ( c o n t ' d ) Antiserum ^globulin  Virus  25 purification  Conjugation  27  The E L I S A t e s t  27  ELISA s e n s i t i v i t y  29  detection  plantlets  i n tissue-cultured  initiated  f r o m f i e l d and  greenhouse p l a n t s  29  AMV and GFLV d e t e c t i o n  29  TomRV d e t e c t i o n  31  Virus  32  Effect  distribution o f t e m p e r a t u r e and  BAP c o n c e n t r a t i o n  Virus  Virus  recovery  Virus  inhibition  detection  33 34  a t 30°C  in field  plants  RESULTS  35 36 39  ELISA s e n s i t i v i t y Virus  26  detection  39  i n tissue-cultured  plantlets  39  AMV and GFLV d e t e c t i o n  39  TomRV d e t e c t i o n  42  Virus Effect  in vitro  distribution  43  o f t e m p e r a t u r e and  BAP c o n c e n t r a t i o n  50  vi TABLE OF CONTENTS Virus Effect Virus  (cont'd)  recovery  63  o f 30°C  63  detection  in field  plants  DISCUSSION Virus  72 detection  in tissue-cultured  plantlets Virus  64  72  detection  in field  plants  79  Comparing the r e s u l t s o f ELISA t e s t s on in vitro and  field  plants  plantlets 90  BIBLIOGRAPHY  92  APPENDIX  99  vi i L I S T OF TABLES TABLE I II  III  IV  V  PAGE ELISA g r i n d i n g b u f f e r s used nepoviruses i n grapevines  to detect  13  D e t e c t i o n by E L I S A o f a r a b i s m o s a i c (AMV) and g r a p e v i n e f a n l e a f v i r u s (GFLV) i n p l a n t l e t s i n i t i a t e d from f i e l d p l a n t s and grown a t 25°C  40  D e t e c t i o n o f a r a b i s m o s a i c v i r u s (AMV) and g r a p e v i n e f a n l e a f v i r u s (GFLV) by E L I S A i n p l a n t l e t s grown a t v a r i o u s t e m p e r a t u r e s f r o m J a n u a r y 3, 1986 t o S e p t e m b e r 17, 1986.  41  D e t e c t i o n o f tomato r i n g s p o t v i r u s i n c u l t u r e s grown a t 20°C f o l l o w i n g i n i t i a t i o n f r o m v i r u s - i n f e c t e d mother p l a n t s  43  A r a b i s m o s a i c v i r u s d e t e c t i o n by ELISA v a r i o u s p l a n t p a r t s t a k e n from f o u r p l a n t l e t s grown a t 25°C  45  in  VI  D e t e c t i o n o f a r a b i s m o s a i c v i r u s (AMV) and g r a p e v i n e f a n l e a f v i r u s (GFLV) by E L I S A i n v a r i o u s p a r t s o f f i v e p l a n t l e t s grown a t 25°C  48  VII  D e t e c t i o n o f a r a b i s m o s a i c v i r u s (AMV) and g r a p e v i n e f a n l e a f ' v i r u s (GFLV) by ELISA i n v a r i o u s p a r t s o f 10 p l a n t l e t s grown at 20°C  49  Mean E L I S A a b s o r b a n c e v a l u e s and a n a l y s i s o f v a r i a n c e o f t e m p e r a t u r e and N - 6 - b e n z y l a m i n o p u r i n e (BAP) e f f e c t on a r a b i s mosaic v i r u s d e t e c t i o n (first experiment)  54  R e g r e s s i o n a n a l y s e s on t h e e f f e c t s o f t e m p e r a t u r e and N - 6 - b e n z y l a m i n o p u r i n e (BAP) c o n c e n t r a t i o n s on a r a b i s m o s a i c v i r u s (AMV) and g r a p e v i n e f a n l e a f v i r u s (GFLV) d e t e c t i o n by E L I S A i n p l a n t l e t s  56  VIII  IX  X  Mean a b s o r b a n c e v a l u e s o f ELISA t e s t s 58 c a r r i e d o u t on a r a b i s m o s a i c v i r u s - i n f e c t e d p l a n t l e t s 1 and 2 months i n t o t h e s e c o n d experiment to t e s t the e f f e c t of temperature and N - 6 - b e n z y l a m i n o p u r i n e (BAP) c o n c e n t r a t i o n on AMV t i t r e  vi ii LIST OF  TABLES  (cont'd)  TABLE XI  XII  PAGE R e s u l t s o f ELISA t e s t s on a r a b i s v i r u s - i n f e c t e d p l a n t l e t s exposed f o r t h r e e weeks  mosaic t o 30°C  64  A n a l y s i s o f v a r i a n c e o f ELISA a b s o r b a n c e v a l u e s o b t a i n e d by u s i n g d i f f e r e n t g r i n d i n g b u f f e r s to d e t e c t g r a p e v i n e f a n l e a f v i r u s (GFLV) and a r a b i s m o s a i c v i r u s (AMV) i n i n f e c t e d f i e l d p l a n t s throughout the summer  65  XIII  E f f e c t o f d i f f e r e n t g r i n d i n g b u f f e r s on the ELISA d e t e c t i o n o f a r a b i s m o s a i c v i r u s (AMV) and g r a p e v i n e f a n l e a f v i r u s (GFLV) i n infected f i e l d plants  66  XIV  D e t e c t i o n o f a r a b i s m o s a i c v i r u s by ELISA t h r o u g h o u t the summer i n f i e l d and screenhouse plants using d i f f e r e n t g r i n d i n g buffers  68  XV  D e t e c t i o n of g r a p e v i n e f a n l e a f v i r u s by ELISA t h r o u g h o u t the summer i n f i e l d and screenhouse plants using d i f f e r e n t g r i n d i n g buffers  69  Mean ELISA a b s o r b a n c e v a l u e s o f h e a l t h y c o n t r o l p l a n t s ground i n v a r i o u s b u f f e r s t h r o u g h o u t the summer  71  A c o m p a r i s o n o f f a l s e n e g a t i v e s c r e a t e d by u s i n g t h r e e d i f f e r e n t methods o f determining ELISA t h r e s h o l d v a l u e s f o r a r a b i s m o s a i c v i r u s (AMV) and g r a p e v i n e f a n l e a f v i r u s (GFLV) d e t e c t i o n d u r i n g the summer  86  A c o m p a r i s o n o f f a l s e p o s i t i v e s c r e a t e d by u s i n g t h r e e methods o f d e t e r m i n i n g ELISA t h r e s h o l d values f o r a r a b i s mosaic v i r u s (AMV) and g r a p e v i n e f a n l e a f v i r u s d e t e c t i o n d u r i n g the summer  88  XVI  XVII  XVIII  ix LIST OF  FIGURES  FIGURE  PAGE  1. F r e q u e n c y h i s t o g r a m o f the v i r u s d i s t r i b u t i o n as d e t e r m i n e d by ELISA i n a l l samples c u t from each of four d i s s e c t e d a r a b i s mosaic v i r u s infected plantlets  44  2. F r e q u e n c y h i s t o g r a m o f a r a b i s m o s a i c v i r u s d i s t r i b u t i o n as d e t e c t e d by ELISA i n d i f f e r e n t p l a n t p a r t s d i s s e c t e d from f o u r infected plant lets  46  3. Mean ELISA a b s o r b a n c e v a l u e s w i t h one s t a n d a r d d e v i a t i o n f o r t e s t s on a r a b i s m o s a i c v i r u s (AMV) and g r a p e v i n e f a n l e a f v i r u s (GFLV)i n f e c t e d p l a n t l e t s f o l l o w i n g growth a t d i f f e r e n t a i r t e m p e r a t u r e s and 2 mg/l N - 6 - b e n z y l a m i n o purine  51  4. Mean ELISA a b s o r b a n c e r e a d i n g s w i t h one s t a n d a r d d e v i a t i o n o f t e s t s on a r a b i s m o s a i c v i r u s - i n f e c t e d p l a n t l e t s grown a t t h r e e N - 6 - b e n z y l a m i n o p u r i n e c o n c e n t r a t i o n s f o r 2 months at 20°C  52  5. F r e q u e n c y h i s t o g r a m o f ELISA a b s o r b a n c e r e a d i n g s from t e s t s on a r a b i s m o s a i c v i r u s - i n f e c t e d p l a n t l e t s grown f o r 2 months a t t h r e e N - 6 - b e n z y l a m i n o p u r i n e (BAP) c o n c e n t r a t i o n s (2, 4, and 8 mg/l) w i t h i n each o f t h r e e t e m p e r a t u r e s (20°C, 25°C, and 30°C)  53  6. Mean ELISA a b s o r b a n c e v a l u e s from t h e f i r s t e x p e r i m e n t t o d e t e r m i n e the e f f e c t s o f g r o w i n g a r a b i s mosaic v i r u s - i n f e c t e d p l a n t l e t s at three t e m p e r a t u r e s f o r 2 months on a medium c o n t a i n i n g various N-6-benzylaminopurine concentrations  57  7. F r e q u e n c y h i s t o g r a m o f a r a b i s m o s a i c v i r u s d e t e c t i o n by ELISA t e s t s on p l a n t l e t s grown f o r 1 month a t 20°C, 25°C, and 30°C on medium c o n t a i n i n g 2 mg/l o f N - 6 - b e n z y l a m i n o p u r i n e  59  8. F r e q u e n c y h i s t o g r a m o f a r a b i s m o s a i c v i r u s d e t e c t i o n by ELISA i n p l a n t l e t s grown f o r 2 months a t 15°C, 20°C, 25°C, and 30°C on a medium c o n t a i n i n g a N - 6 - b e n z y l a m i n o p u r i n e c o n c e n t r a t i o n o f 2 mg/l - s e c o n d e x p e r i m e n t  60  L I S T OF FIGURES  (cont'd)  FIGURE 9. F r e q u e n c y h i s t o g r a m o f g r a p e v i n e f a n l e a f v i r u s d e t e c t i o n by ELISA i n p l a n t l e t s grown f o r 1 month at 20°C, 25°C, and 30°C on a medium containing a N-6-benzylaminopurine concentration o f 2 mg/1  ACKNOWLEDGEMENTS  The  author  committee:  Canada R e s e a r c h  (Faculty  Department and  t h e members o f h i s  (Research  Station,  Columbia  Department  fortheir  of British  o f Botany,  guidance,  Dr.R . J .  Runeckles,  Science, University  Taylor,  Supervisor),  Vancouver;  A d v i s o r ) a n d D r . V.C.  of Plant  D r . I.E.P.  British  t o thank  D r . R. S t a c e - S m i t h  Agriculture Copeman  wishes  Columbia;  University of  assistance,  and  pat ience. The  author  Inspection support  also  Branch,  wishes  t o thank  Agriculture  and o p p o r t u n i t y  t h e Food  Canada  to return  P r o d u c t i o n and  f o rthe f i n a n c i a l  to university  and c a r r y D  out  these  studies.  Also, the  staff  Canada this  thanks  of the Vancouver  fortheir  research. But  Kathryn  support  Research  support.and Their  this  Ann, w i t h o u t thesis  Station,  h e l p has been  to h i swife,  would  D r . M. W e i n t r a u b ,  providing  most o f a l l t h e a u t h o r  appreciation and  go t o t h e d i r e c t o r ,  whose  Agriculture  the f a c i l i t i e s f o r  immeasurable.  would  Susan,  like  to express h i s  and h i s c h i l d r e n ,  love,  n o t have  and  Matthew  u n d e r s t a n d i n g , and  been  possible.  1 INTRODUCTION  The  Saanichton  Plant Quarantine  post-entry  quarantine  grapevines  i n Canada.  authority and  the  Plant  of  the  interpretation  countries  Prunus,  these  fruit  The  plant  the  and  from  corresponding well  established  as many o t h e r  import  Chaenomeles, and  of  in order  Malus,  Vitis  these d i s e a s e s  p r o d u c t i o n or not  found  integral  limit  into  access  to  part  the  Canada. impact  Many on  export  i n Canada o r not  widespread.  in protecting  p r e s e r v i n g i t s envious  in British  wine v a r i e t i e s . importation  Pyrus,  without  to prevent  position  production is r e l a t i v e l y  Canada r e s t r i c t  Columbia.  the use  Canadian of  Pressure  o f new,  has  more c o l d  i n c o u n t r i e s where v i r u s  Virus-indexed  sources  of  The  recent  relative  climatic  i n Canada, conditions in  o f many l o n g e s t a b l i s h e d  wine-making c h a r a c t e r i s t i c s . found  i n the  disease.  Grapevine especially  the  1968-69, c.35, s . l .  i m p o r t a t i o n of  been done  p l a y s an  agriculture  and  a g a i n s t a number o f d i s e a s e - c a u s i n g  spread  are e i t h e r  Station  freedom  Act,  national  fruit  under  d i s e a s e s , some o f which have a d i r e c t  and  markets,  and  operates  act  has  Crataegus,  T h i s has  introduction  this  prohibit  certification  organisms.  of  the w o r l d ,  which  Cydonia,  program  R e g u l a t i o n s . Canada, as  around  regulations  of  The  i s the  for indexing tree  Plant Quarantine  Quarantine  proper  facility  Station  been hardy  increasing varieties  These v a r i e t i e s d i s e a s e s are  these v a r i e t i e s  European  to allow with  better  are o f t e n widespread.  a r e not  the  always  2 available, because  e i t h e r because of inadequate  the v a r i e t i e s  have o n l y  breeding  programs p r i o r  resulted  i n increased  r e c e n t l y been  to indexing.  demand  f o r indexing  This  resources  a t t h e S t a t i o n and p r o b l e m s w i t h a r e not e a s i l y  with  the indexing  viruses  i n grapevine.  several  viruses.  woody  plants  detected  techniques  used  reliability, complete  (nepoviruses); transmitted  viruses  known v e c t o r s ,  as  Saanichton limited  virus detection.  The S t a t i o n i s a v a i l a b l e to d e t e c t  be i n f e c t e d w i t h  hosts.  to d e t e c t The  viruses in  indexing  c a n be i m p r o v e d ,  accuracy,  one o r  i n terms o f  and time r e q u i r e d t o  viruses  with  distributed reservoir originally  transmitted  broad  by s o i l  v i r u s e s without  particles  categories viruses  fungi; viruses  known v e c t o r s b u t  not c h a r a c t e r i s e d .  l o s s e s are caused  without  The  by t h e v i r u s e s  i n the  v i r u s e s having  nematodes  groups.  nepoviruses  vectors.  into five  have been c h a r a c t e r i s e d ; and v i r u s e s  economic  and l a s t  fall  the nematode-borne p o l y h e d r a l  by a p h i d s ;  whose p a r t i c l e s  The  plants  t h i s has  them.  (Bovey e t a l , 1980b):  first  may  i n herbaceous  sensitivity,  Grapevine  greatest  procedures  A vine  on t h e s e  a t the  from  i n any o f t h e seven  I t i s more d i f f i c u l t  than  released  i s compounded b e c a u s e o f  genera which a r e t e s t e d at S a a n i c h t o n . concerned  programs o r  Consequently,  Station.  Viruses  situation  indexing  are polyhedral  In many c a s e s , around  the world  i n the s o i l  these  vectors  and a r e a b l e  f o r many y e a r s  i n f e c t e d vines  are widely to serve  as a  e v e n a f t e r the  have been d e s t r o y e d .  It is  3  therefore  important  introduced at  or  to i n s u r e that  spread  w i t h i n Canada any  viruses  further  are  than  by  s e r o l o g y , by  by  grafting  The  a l l grapevines inoculating  onto  traditional  are  labour  intensive  for  woody  for  greenhouse  caused with  by  they  are  much e x p e r i e n c e are  and  Under  titre,  the  are  i n the  field  sometimes  insects  training  inoculation These  plot  sap  ideal  (ELISA)  to 3 y e a r s t o the  maintenance  faint,  to i n t e r p r e t .  symptoms  can  be  require  These d e t e c t i o n  in plants,  fluctuation  and  serology is a fast enzyme-1 i n k e d  (Clark with  Adams,  precise  immunosorbent 1976)  is  and  associated  with u s i n g s e r o l o g y to d e t e c t nepoviruses  quantifiable.  materials.  o f the y e a r  results  and  promise.  objective,  1979;  to perform  and  the most  easy  al.,  the  confused  relatively  a l l times  and  The  seasonal  s e r o l o g y shows  w i d e l y used  technique  woody g r a p e v i n e  need  components.  conditions, The  onto procedures  up  due  and  field.  o r o t h e r d i s e a s e s , and  uneven d i s t r i b u t i o n  t h r e e methods,  procedure. assay  indicators  s u b j e c t to p r o b l e m s w i t h  virus-inhibiting Of  indicators,  indicators.  and  nepoviruses  herbaceous  s u b j e c t to m i s i n t e r p r e t a t i o n .  by  for  lengthy, r e q u i r i n g  facilities  damage c a u s e d  virus  indexed  They a r e more c o s t l y  the v i r u s e s  techniques  woody and  indexing.  are  onto  methods r e q u i r e the  p l a n t s and  results  are  woody g r a p e v i n e  herbaceous  at  not  present. Currently,  in  these  There  The  being r e p r o d u c i b l e ,  a r e , however,  viruses  cannot  be  (Uyemoto e t a l . , 1976;  Shanmuganathan and  Fletcher,  1982)  problems in  detected  Ramsdell  o r may  be  et  4  unevenly  distributed  within a plant  Uyemoto e t a l . , 1976). substances al., ELISA  Furthermore,  w h i c h may i n t e r f e r e  1977; V e t t e n ,  1981).  i n field-grown  modifying  heat  therapy.  viruses  little  serologically  (plantlets). potato  Very  virus  Before  i n shoot  1983)  and prune dwarf v i r u s  testing (AMV)  plantlets  growing  Grapevines Murashige  and Skoog  (Barlass  and Skene,  Goussard,1982;  et  mg/l,  with  and A l l e n ,  plants Since  in  vitro  then,  a r a b i s mosaic  ELISA  virus  (GFLV) i n g r a p e v i n e  (Monette,  1985).  in vitro  mainly  on t h e b a s i c  (1962) medium c o n t a i n i n g t h e c y t o k i n i n (BAP) t o enhance c e l l  1978, 1980a,  1984; C o r t e  used with  2-3 mg/l b e i n g  division  1980b; Chee and P o o l , 1982; 1982; Chee e t a l . ,  and de Mendoca,  a l . , 1985; and Z a t y k o and M o l n a r ,  concentrations  vitro  p r o j e c t , o n l y the  (McMorran  1984).  H a r r i s and S t e v e n s o n ,  L i and E a t o n ,  to detect  had been d e t e c t e d by  cherry  to d e t e c t  have been grown  N-6-benzylaminopurine  1984;  of this  i n sour  fanleaf virus  in vitro  1980).  i n p l a n t s c u l t u r e d in  and Kornkamhaeng,  has a l s o been u s e d  material  e l i m i n a t i o n by c o n v e n t i o n a l  t i p c u l t u r e s of potato  and g r a p e v i n e  i m p r o v e d by  have been a p p l i e d m a i n l y t o  the b e g i n n i n g  ELISA  d e t e c t i o n by  1980; E n g e l b r e c h t ,  v i r u s e s X.Y.S.M.A, and l e a f r o l l  (Bauman, C a s p e r ,  (Pierpont et  work has been done  by ELISA  sap c o n t a i n s  nepovirus  to g r i n d the l e a f  c u l t u r e techniques  p r o d u c e new p l a n t s a f t e r  serology  has been  1980; Tanne,  e t a l . , 1970;  the grape  Recently,  the b u f f e r used  Tissue  with  grapevines  ( J i m e n e z and Goheen,  (Gilmer  grapevines  1985).  1985, M o r i n i  The BAP  have v a r i e d from  2 t o 10  t h e most common c o n c e n t r a t i o n .  5  Previous  reports  replication clear,  on the e f f e c t s o f c y t o k i n i n s  i n p l a n t l e t s growing  in vitro  w i t h many r e s u l t s a p p a r e n t l y  (Fraser effect  and Whenham, 1982).  in  The  being  on v i r u s e s  purpose  of t h i s  nepovirus  grapevines  i n both  field-grown  aspects  by ELISA  B.C.and first  a)  part  grown  one  in  from  grown  thesis  detection  was t o i n v e s t i g a t e  detection in g r a p e v i n e s at  in vitro  .  i a a report  on  various  i n d i f f e r e n t i a t e d grapevine  vitro:  shoot  o f ELISA tips  to detect  obtained  AMV  from  in plantlets  the f i e l d  during  summer, the e f f e c t of v a r i o u s  primary  c y t o k i n i n used  detection c)  of t h i s  the r e l i a b i l i t y  started  b)  i n grapevines  of nepovirus  plantlets  i n grapevines  thesis project  improve  The  contradictory  vitro.  methods w h i c h would  Sidney,  have n o t been  No one has y e t i n v e s t i g a t e d t h e  o f BAP c o n c e n t r a t i o n s  growing  on v i r u s  o f AMV  concentrations  i n grapevine  o f BAP, t h e  c u l t u r e , on t h e  in plantlets,  t h e e f f e c t o f growth room  temperature  GFLV d e t e c t i o n  by t h e ELISA  d)  the a b i l i t y  to detect  by  ELISA  e)  and, t h e d i s t r i b u t i o n o f AMV  on AMV and  technique,  tomato r i n g s p o t  in plantlets started  from  virus  infected  and GFLV  (TomRV)  plants,  within  tissue-cultured plantlets. The  second  effects  of four  and  in field  AMV  aspect  of this  grinding  thesis  buffers  i s a comparison  on t h e d e t e c t i o n  and s c r e e n h o u s e - g r o w n p l a n t s  of the  o f GFLV  under one  summer's e n v i r o n m e n t a l Saanichton.  and  cultural  conditions  7  LITERATURE  ELISA d e t e c t i o n grown  ia  of viruses  Virus  taken  that  performed  grown  15-27°C levels found  than  to v a r y  in vitro  to detect  among  plants.  28°C o r 35°C.  found  were more  The  relative  X,S,M, and  parts,  detection  and M a d r i g a l - V a r g a s  either  i n potato  stems, and r o o t s  Viruses  in a l l plant  l e a f t i s s u e gave c o n s i s t e n t  potato v i r u s  months.  p l a n t l e t s growing at  the l e a v e s ,  each v i r u s .  detect  for several  and l e a f r o l l  vitro.  c u l t u r e are  potato viruses  in tissue culture  with  in  In 1983, McMorran and A l l e n  X,S,Y,A,M  detected  Lozoyo-Saldana  in  virus detection in  to s o i l  i n greenhouse-grown  of antigen  were e a s i l y and  in vitro.  detected  from  after transfer  potato viruses  reliably  of plant  derived  ELISA has been u s e d plants  tissue  from d i f f e r e n t i a t e d p l a n t l e t s g r o w i n g  t e s t s on p l a n t s  usually  i n d i f f e r e n t i a t e d plant  vitro  T h e r e a r e few r e p o r t s tissue  REVIEW  while  were leafroll  only  stems  of p o t a t o  virus  (1985) a l s o  Y.  u s e d ELISA t o  X i n whole p o t a t o p l a n t l e t s g r o w i n g a t They  a l l p l a n t l e t s kept  found  that  t h e v i r u s was  a t 28°C b u t was m i s s e d  detected  i n some h e l d a t  35°C. Bauman e t a l . virus-infected they  detected  initiated  from  temperature  sour  (1984) e s t a b l i s h e d cherry  the v i r u s  plantlets  prune  in vitro.  dwarf Using  ELISA,  i n some b u t n o t a l l p l a n t l e t s  infected plants.  was n o t g i v e n .  The c u l t u r e  room  The v i r u s was d e t e c t a b l e  only  8 after  the t h i r d Snir  necrotic sweet  and S t e i n ringspot  cherry Kuo  virus-A potato  et a l .  v i r u s by ELISA  plantlets  summer.  being  ELISA  growing  from  i n leaves  shoot  tips.  interval.  GFLV and AMV  from  i n grapevine  throughout the  between  Even  t h e age o f t h e  though  there  and p l a n t l e t  short  term  was no c o r r e l a t i o n age w i t h i n a 35 day  In 1986, Monette used ELISA  to detect  i n 9-week o l d c u l t u r e s i n i t i a t e d  greenhouse-grown v i n e s  sweet  at 22°C-32°C.  initiated  f l u c t u a t i o n s were o b s e r v e d ,  reculture  taken  potato  The v i r u s was  no c o r r e l a t i o n  concentration  sweet  GFLV, by ELISA,  and v i r u s c o n c e n t r a t i o n .  between v i r a l  and s h o o t s o f  to detect  in vitro  i n 27 o f 29 p l a n t l e t s found  i n leaves  prunus  a t 25°C.  virus-N  (1985) d e t e c t e d  Monette  plantlets antigen  potato  initiated  detectable  growing  (1985) used  and sweet  plantlets  (1985) c o n s i s t e n t l y d e t e c t e d  plantlets  Monette  both  subculture.  i n . which o n l y AMV  from  b u t n o t GFLV was  detectable.  Temperature growing  Hot years  in  on v i r u s c o n c e n t r a t i o n  i n grape  plantlets  vitro  a i r temperatures  above 35°C have been u s e d  t o e l i m i n a t e v i r u s e s from  grapevines Luhn,  effect  f o r many  conventionally-grown  (Goheen e t a l . , 1965; Bovey,  1972; Goheen and  1973). Similarly,  concentrations  h o t a i r has been shown t o a f f e c t in differentiated  grapevines  grown  virus in  vitro.  9  Galzy that 90  (1961,1963,1964) and  Galzy  GFLV-infected grapevine  days c e a s e d  to develop  normal  l e a v e s , and  normal  temperatures.  symptoms  months  being  transplanted into  (1966,1969,1972) d e t e r m i n e d required  virus-induced initiation increased a 26°C both  above  h e a l t h y and  30°C, w i t h  leafroll vitro  virus-infected poor  shoot  35°C b e i n g  (1976) s u c c e s s f u l l y  virus  and  grapevine  fleck  necrosis  grapevine  from  treatment  vitro  plantlets  treatments.  increased with  However, he  also  were e l i m i n a t e d from  found 6 of  of root  t h e growth  of  adversely  greatly  grapevines Mur  enhanced  38°C.  grown  a 70 The  and  both  number o f  fleck  14 p l a n t l e t s  vein  day  the number o f 70 that  in  (1979)  leafroll,  following  o f 35°C o r  days  e l i m i n a t e d grapevine  from  grapevine  o f 21  temperature.  temperature.  plant lets  at temperatures  virus-free  necrosis  in  fleck,  Galzy  i n c r e a s e d over  was  g r o w t h was  the optimum  at a c o n t i n u o u s l y h i g h  by  after  plantlets  was  plantlets  Mur  healthy  r e p o r t e d that  temperature  and  eliminated  at  as  grown a t  appeared  elimination Galzy  more  a t 20°C and  v a l u e t o 37°C, above w h i c h  Similarly,  Valat  still  growth of G F L V - i n f e c t e d grape  threshold  affected.  plantlets  a minimum p e r i o d  In 1969,  proportionately  had  Further studies  that  found  s u b j e c t e d to 35°C f o r  in culture  f o r permanent  symptoms.  and  than  fields.  (1968)  in culture,  These p l a n t l e t s  three a d d i t i o n a l  35°C was  Compan  plantlets  rooted better  after  at  and  day and  vein  maintained  20°C. Further  affecting  evidence  of v i r u s - t e m p e r a t u r e  the e l i m i n a t i o n  interactions  of g r a p e v i n e v i r u s e s  were f o u n d  by  Barlass  e t a l . i n 1982.  m o t t l e were e l i m i n a t e d fragmented  shoot  temperature day/  20°C  only  a t the  applied  night.  necessary  hours days up  t o 113  in  plantlets  by  taking  temperatures  temperatures  o f 39°C  A l l plantlets  eliminate 66  GFLV  days.  plantlets  from  from  96  tips.  and  (35°C)  culture  22°C  AMV  days  was  at the  shoot  little  produced  as  tips  was  However,  He  found  at  daily by  after  28  from a l l  periods  d i d not  f o r as  long  from a l l  fluctuating  temperatures.  Cytokinin growing  The has  effect  in  on v i r u s  in plantlets  vitro  effects  of c y t o k i n i n s  not y e t been c l e a r l y  studies  concentrations  have y i e l d e d  on p l a n t  established  controversial  virus  because results  18  fluctuating  when a p p l i e d  lower  from  treatments f o r  that  not e l i m i n a t e d  from  followed  virus  f o r 12 hour  AMV  17 days  t h i s way  leafroll  a l l plantlets  Likewise,  after  vitro  27°C  eliminated  therapy  f o r 6 hours  d i d not e l i m i n a t e  o f 35°C  was  of  b o t h GFLV and  2 mm  grown f o r as  initiated  constant  GFLV.  (1983,1986) e l i m i n a t e d  days  a  from  temperature  w h i l e heat the  summer  initiated  speckle  o f t r e a t m e n t were v i r u s - f r e e .  plantlets  as  yellow  b e f o r e or d u r i n g  a t 22°C.  and  grown a t e i t h e r  temperature  previously  fluctuating  and  or a f l u c t u a t i n g  to e l i m i n a t e  some i n f e c t e d plantlets  leafroll  plantlets  Grapevine  lower  Monette  from  apices  o f 35°C  either  Grapevine  replication some o f  (Fraser  the  and  Whenham, 1982). stimulation  or  Most  the  type  local  and  local  has  lesions  the  and  and  to  and  cytokinins  on  and  done  apply  the  local  l e s i o n f o r m a t i o n but  using that in  N.  BAP  reduced  assays.  assays  200  Simpkins concentrations  tissues  reduce  that  as  cytokinin  Virus  work  growing  in  on in  vitro.  four  of  five  leaves  inhibited  production.  local  shown  by (1969b),  vulgaris,  also  strongly  grown f o r  28  of  the  infectivity  concentrations  The  lesion  Srivastava  Phaseolus  cultures  no  glutinosa  r e p l i c a t i o n as and  been  i t is  r e p l i c a t i o n was  BAP.  regulator,  work done  found  on  inoculation  Almost  show t h a t  or  Depending  growth  d i f f e r e n t concentrations  including  proportionately  plant  Nicotiana  not  onto  normal  v i r u s , r e s u l t s have  However, M i l o  tabacum p i t h t i s s u e  2 to  time o f the  the  presence  leaves.  enhanced v i r u s  virus  tobacco mosaic v i r u s  media c o n t a i n i n g  from  the  tobacco mosaic v i r u s  which d i d  infectivity  cytokinins,  of  with  under  t o p l a n t l e t s grown  to e x c i s e d  i n o c u l a t i o n with  infectivity  the  (1969a),  to  f o r m a t i o n but  of  r e s u l t s of  plants  Srivastava  applied  was  the  to d a t e  prior  exception  activity  indicator  p l a n t l e t s or  conventionally-grown Milo  have d e a l t  sometimes c o n t r a d i c t o r y .  studies  inappropriate  virus  concentration  been done u s i n g  vitro;  of  treatments,  l e s i o n host,  inconclusive  studies  through e v a l u a t i o n  time o f  post-treatment, the  the  inhibition  growing c o n d i t i o n s absence of  of  found  inhibited  days  on  five  dropped  were  increased  ug/l. et  al.  i n the  (1981) found medium  from  that 0 to  increasing 25.6  mg/L  kinetin  did  not  significantly N. rustics ribavirin  plantlets  in  o v e r 28 d a y s .  a t 10 and 50 mg/L, t h e y  concentration the  r e d u c e cucumber m o s a i c v i r u s c o n c e n t r a t i o n i n  of k i n e t i n  ribavirin,  slightly  When c o m b i n e d  with  found t h a t the h i g h e r  antagonised  the a n t i v i r a l  i n c r e a s i n g the v i r u s  effect of  concentration  the p l a n t l e t s . Working w i t h potato  Lozoya-Saldafia increases not  and M a d r i g a l - V a r g a s  in kinetin  eliminate potato  Serological  p l a n t s growing  concentration  ( 1985)  in  vitro,  found  from 0.3 t o 300 ppm d i d  v i r u s X from t h e in vitro  d e t e c t i o n of nepoviruses  that  plantlets.  i n field-grown  grapevines  A number o f E L I S A g r i n d i n g b u f f e r s have been u s e d t o enhance n e p o v i r u s standard  d e t e c t i o n i n grapevines  (Table  ELISA g r i n d i n g b u f f e r i s a phosphate  1 ) . The  buffered  s a l i n e b u f f e r , pH 7 . 4 , c o n t a i n i n g 0.05% Tween 2 0 , 2% polyvinylpyrrolidone 0.02%  ovalbumin  ( P B S - T - P V P ) , sometimes m o d i f i e d  (PBS-T-PVP-0).  Although  the r e s u l t s  g e n e r a l l y b e e n c o n s i s t e n t b e t w e e n s t u d i e s some have been  have  discrepancies  reported.  TomRV was d e t e c t e d by  with  Gonsalves using  expanding terminal grinding buffer.  i n 20 o f 21 i n f e c t e d v i n e s  t h e ELISA t e c h n i q u e . leaves  i n 1979  Young, r a p i d l y  were g r o u n d i n t h e s t a n d a r d  D e t e c t i o n by t h i s method was e q u a l l y  ELISA  13 Table I. ELISA g r i n d i n g buffers used t o detect nepoviruses i n grapevines  Grinding b u f f e r  1  Virus  2  Tissue Tested  Detection  Reference  PBS-T-PVP-O, pH 7.4  TomRV  leaves  seasonally unreliable  Gonsalves (1979)  PBS-T-PVP-O, pH 7.4  PRMV  leaves  unreliable  Ramsdell e t a l . (1979)  PBS-T-PVP + 1% n i c o t i n e , pH 8.2  GFLV  leaves, dormant buds  reliable  Bovey e t a l . (1980a)  PBS-T-PVP-O, pH 7.4 + 1% n i c o t i n e + 0.2% NaDIECA  GFLV  leaves  reliable  Engelbrecht (1980)  PBS-T-PVP-O, pH 7.4 + 1% n i c o t i n e  reliable  PBS-T-PVP-O, pH 7.4  unreliable  PBS-T-PVP-0, pH 7.4 + 1.25% n i c o t i n e  GFLV  leaves  reliable  Jimenez and Goheen (1980)  PBS-T-PVP  GFLV, AMV, TBRV  leaves  reliable  Tanne (1980)  PBS-T-PVP-0 • 2-mercaptoethanol  GFLV  leaves  reliable  Jankulova et a l . (1982)  PBS-T-PVP  GFLV  leaves, buds  seasonally unreliable  Shanmuganathan and F l e t c h e r (1982)  PBS-T-PVP  GFLV, AMV, TRSV  leaves  unreliable  Kearns and Mossop (1984)  GFLV, AMV  leaves  PBS-T-PVP, +2% nicotine  0.2M PBS-T-PVP, pH 7.5 • 0.5% TGA +0.1% NaDIECA PBS-T-PVP, pH 7.4 + 2.5% n i c o t i n e PBS-T-PVP, pH 7.4  variable  reliable  reliable unreliable  Walter et al.(1984)  14 Table I. (continued)  Grinding b u f f e r  1  Virus  2  Tissue Tested  Detection  Reference  PBS-T-PVP, pH 8.2 + 1% n i c o t i n e  GCMV  leaves  reliable  Lehocky e t a l . (1984)  PBS-T-PVP  GFLV, AMV,  buds, bark,  reliable  Stellmach (1985a,b)  PBS-T-PVP + n i c o t i n e  RRV, TBRV  sawdust, leaves  unreliable  PBS-T-PVP, pH 8.2 + 1% n i c o t i n e  GCMV  leaves  reliable  Kolber e t a l . (1985)  PBS-T-PVP + 1% n i c o t i n e + 1% PEG  GFLV  leaves  reliable  Bovey e t a l . (1985)  PBS-T-PVP + 1% PEG, pH increased  "  reliable  0.5 M THS-T-PVP, pH 8.2 + 1% PEG  "  reliable  2.5% carbonate, pH 7.3 + 0.5% ascorbic acid  AMV, GFLV  wood shavings  unreliable  PBS-T, pH 7.4 • 1% BSA  reliable  PBS-T-PVP, pH 7.4  reliable  PBS-PVP, pH 7.4 + 1% T r i t o n X100 • 0.2% 2-mercaptoethanol  reliable  0.5 M THS-T-PVP, pH 8.2 • 1% PEG  reliable  0.1 M THS-T-PVP, pH 8.2 • 1% BSA  reliable  2.5 % n i c o t i n e 0.2 M THS-T-PVP, pH 8.2 0.5 M THS-T-PVP, pH 8.2  "  "  less reliable  leaves  reliable reliable  Huss e t a l . (1986) and Walter and Etienne (1987)  15  Table I. (continued)  Grinding b u f f e r 2.5%  1  nicotine  Virus* AMV, GFLV  Tissue Tested  Detection  Reference  leaves  less  Huss et a l . (1986) and Walter and Etienne (1987) (continued)  reliable  PBS, pH 7.4 + 4% p o l y c l a r AT + 1% T r i t o n X100 + 0.2% 2-mercaptoethanol, PBS, pH 7.4 + 4% p o l y c l a r AT + 1% T r i t o n X100 + 0.2% 2-mercaptoethanol 2.5%  unreliable  roots  nicotine  reliable  reliable  Abbreviations f o r common buffer constituents: PBS - 0.01 M phosphate buffer s a l i n e T - 0.05% Tween 20 PVP - 2% p o l y v i n y l p y r r o l i d o n e 0 - 0.02% ovalbumin NaDIECA - sodium diethyldithiocarbamate TGA - t h i o g l y c o l l i c a c i d PEG - polyethylene g l y c o l THS - T r i s HCI s a l i n e buffer BSA - bovine serum albumin 2  TomRV - tomato ringspot v i r u s ; PRMV GFLV - grapevine fanleaf v i r u s ; AMV TBRV - tomato b l a c k r i n g v i r u s ; TRSV GCMC - grapevine chrome mosaic v i r u s ;  peach r o s e t t e mosaic v i r u s ; arabis mosaic v i r u s ; tobacco ringspot v i r u s ; RRV - raspberry ringspot v i r u s  16 sensitive  to  Using  i n o c u l a t i o n onto h e r b a c e o u s  ELISA,  Ramsdell  detect  peach r o s e t t e  though  the  ELISA  of  purified  in  plants  also The  reduced ELISA  They  GFLV  found  1.25%  J i m e n e z and  nicotine  to  to  mercaptoethanol  nicotine  to  consistent  consistently buffer.  using  a final buffer  the  10  Detection to  ng  detected was  mid-June. to  buffer  had  Engelbrecht  no  found  or w i t h  technique.  to  of  prevent The  The  buffer  pH  addition  e f f e c t on  was  of  0.01%  results.  i t e s s e n t i a l to  add  sodium d i e t h y l -  (NaDIECA) to  GFLV d e t e c t i o n  ELISA  concentration  appearing.  after grinding.  (0.02%)  as  (1980) s u c c e s s f u l l y  sap  to add  (1%), e i t h e r alone  little  from mid-May  Goheen  from  the  i n 1980,  dithiocarbamate get  7.4  not  as  even  indicators.  grinding  reactions  increased  Also  the  detect  grinding  plant  i t necessary  non-specific also  i n crude  reliably  s e n s i t i v e when compared  onto herbaceous  1980,  detected  less  not  (PRMV) i n g r a p e v i n e s  v i r u s was  i n t e s t s performed  indicators.  (1979) c o u l d  could  standard  r e s u l t s were  inoculation In  The  the  al.  mosaic v i r u s  method used  virus.  using  et  plant  the  throughout  grinding the  buffer  to  growing  season. Bovey e t during best  progressed.  leaf  (1980a) were a b l e  t e s t i n g from  around  leaves  al.  and  flowering Virus  decreased  grinding  to O c t o b e r .  time and  was  concentration  buds were a l s o  standard  June  from upper a good  buffer  lower  1%  GFLV by  as  the  was  summer  i n young  leaves.  virus.  ELISA  detection  greatest  source of  with  Virus  reduced  was to  to d e t e c t  Dormant  They used  n i c o t i n e added  just  the  before  grinding.  The  buffer  pH  was  adjusted  to 8.2  before  gr i n d i n g . Tanne virus  detected  (TBRV) i n f r e s h or  summer. she  ( 1980)  She  found  variable reduced  preferred  that  using  results.  GFLV, AMV,  frozen  using  Freezing  absorbance values  extracts  the  a buffer  tomato from  standard  about  blackring  spring  grinding  containing  samples  by  and  2%  nicotine  for a l l  late  buffer  after grinding 50%  to  as  gave  generally  three  viruses. Jankulova quantitative  et  reliably  grinding  buffer  3:1  (1982) d e s c r i b e d  determination  GFLV was  either  al.  or  detected  containing  10:1  (v.w)  Shanmuganathan and grinding  of  buffer,  in grapevines one  i n e a r l y summer.  tissue  gave  the  tobacco  tissue  standard  improved and  when the  either  2%  acid  (TGA)  high  molarity  a  grapevine  (1982), u s i n g  ELISA. modified  detect  in  tissue. the  GFLV  Dormant buds and  (TRSV) d e t e c t i o n to r e l i a b l y  the  using  Mossop r e p o r t e d  They were u n a b l e using  using  standard in  young  plant leaf  results.  K e a r n s and  ringspot  for  2-mercaptoethanol  to r e l i a b l y  only  1984,  drop of  Fletcher  tissue  In  in plants  d i l u t i o n s of  were a b l e  best  GFLV  a method  detect  GFLV  Best  was  0.5%  containing  leaf  to 0.2  M  thioglycol1ic  r e s u l t s were o b t a i n e d  phosphate b u f f e r  or  ELISA.  Detection  increased  NaDIECA, o r  and  using  i n shoot  buffer.  phosphate m o l a r i t y  were added.  GFLV, AMV,  in grapevines  grinding  n i c o t i n e , 0.1%  on  using  b o t h NaDIECA  a and  TGA. Lehoczky et  al.  (1984) and  Kolber  et  al.  (1985) u s e d  ELISA to d e t e c t field-grown  g r a p e v i n e chrome m o s a i c v i r u s  vines  i n Hungary.  grinding buffer  containing  8.2  use.  j u s t before  June, u n t i l  the  below d e t e c t i o n was  end  of  the  in July.  small Virus  berry  The  v i r u s was  September, w i t h  the  detected  upper  leaves  C o m p a r i n g d i r e c t and detect  GFLV and  AMV,  methods d e t e c t e d ng/ml and  A d d i n g 2.5%  Virus  stage,  to the  i n August  g i v i n g the  purified  for r e l i a b l e  best r e s u l t s .  were h i g h e r plant  parts.  summer. by  Virus  i n l e a f t i s s u e i n the  Attempts to  artificially  unsuccessful.  Virus  U s i n g the  (RRV)  standard  detected  longer  other late  in late  field  i n dormant b u d s , b a r k  and  These v i r u s e s  was  plants.  raspberry  scrapings,  summer  g r e e n h o u s e were  grinding buffer, Stellmach  GFLV, AMV,  f r o m dormant c u t t i n g s .  in  roots  plants  i n t h e s e f o r c e d buds  i n the  be  plants  in  than f o r  became u n s a t i s f a c t o r y  detection  found to  in field-grown spring  of  viruses.  greenhouse-grown  improve v i r u s d e t e c t i o n  found  both  in grapevine.  f o r c i n g d o r m a n t buds i n t h e  comparable to t h a t  reliably  concentrations  Detection  to  two  g r i n d i n g b u f f e r was  d i s t r i b u t i o n experiments with  June  and  (1984) found t h a t  GFLV d e t e c t i o n  and  the  v i r u s down t o a c o n c e n t r a t i o n  n i c o t i n e to the  leaves.  on  of  i n d i r e c t ELISA t e c h n i q u e s  Walter et a l .  pH  dropped  end  lower leaves  showed t h a t b o t h GFLV and.AMV were d e t e c t e d than  then  always d i f f e r e n t i a t e d between the  indispensable  to  p e a k e d i n May  detection  again  standard  adjusted  concentrations  s i m i l a r f o r u p p e r , m i d d l e , and  vines.  10  They u s e d a m o d i f i e d  1% n i c o t i n e and  Virus  (GCMV) i n  and  ringspot sawdust  were d e t e c t e d  (1985a) virus taken in  sawdust  infected plants  diluted  healthy  sawdust.  Also,  Stellmach  the  standard  grinding  buffer  and  TBRV r e l i a b l y  with  from  secondary heat  of  bark  taken  from  summer.  He  stated  nicotine In  produced a  study  grapevines Beres  containing no  nepoviruses.  virus,  delaying  sufficient both  viruses contain Also  before any  in  was  polyethyleneglycol eliminated  i f the  compensate  for  the  al. wood  dependent  upon  extract.  G F L V was  TRIS-HCl  that  GFLV,  buds  AMV,  plus  even  in  the  containing  nepoviruses  infections,  time  also  simultaneous  reduced  the  to  Stellmach  The  the  other  infection  with  of  waiting  buffer  two  one a  establishment  grinding  and  single  concentration  ensure  in  viruses  They recommended  testing.  Bovey  buffer  or  of  having  The  leaves,  found  buffer  in plants  in grapevines  grape  mixtures  of  used  did  additives.  1985,  Huss et  vines,  in detecting  detected best  dormant  a grinding  virus  detection. of  and  detection  However,  period  in  results.  mixed  sometimes  1%  detect  difficulties  infections  nepoviruses  not  ELISA  to  field-grown that  to  (1985b)  able  leaves  erratic  on  (1985) had  nepovirus than  i n young  was  down  the  based  the  et  a l . found  using one  (PEG)  upper  acidity  leaves  containing  (MW  grinding  that  6000).  1%  pH  of  grapevine  the  was  that  nature  the  buffer  were used,  and  could  1%  to  sap.  was  used  July.  be  GFLV d e t e c t i o n  rootlets  detected  early  adjusted  and  of  best  nicotine  Nicotine  shavings,  buffers  before  buffer  (1986) confirmed  reliably  G F L V was  in  highly to  prepare  in a l l tissues  when  providing  had  they  the PBS a  20 molarity above 0.1 M, a pH around 8, and contained PVP.  2% or 5%  Virus detection in rootlets and wood shavings was also  good i f these buffers were used. Walter and Etienne  (1987) systematically studied AMV  and GFLV detection in f i e l d and greenhouse-grown grapevines throughout the year using various buffers.  Samples ground  using a mortar and pestle gave better results than those obtained  by crushing  leaves in buffer within a p l a s t i c bag.  A 0.2 M Tris HCl buffer was s l i g h t l y better than a 0.5 M solution for detecting viruses in buds and a l l the leaves along the shoots throughout the summer.  Detection  using  both buffers surpassed that of a 2.5% nicotine solution in water, which f a i l e d to detect the viruses in some leaves. 0.01 M PBS buffer was unsuitable  A  for GFLV detection in grape  leaves but worked better than the nicotine solution when wood shavings were used. acceptable  Both buffers were equally  when roots were used as a virus source.  A low  molarity Tris-HCl buffer (0.01 M) r e l i a b l y detected both GFLV and AMV in wood shavings from dormant canes a l l winter long, even when the canes had been stored up to 7 months.  21 MATERIALS AND  Tissue  culture.  Media.  The  proliferation, are  based  with  on  a few  added  minutes  those d e s c r i b e d  at 20  l b s (102  culture  Plant  tubes  with white  material.  grapevines  o f the c u l t i v a r  culture  Plant  virus-infected by  field.  or  plants  and  0.7%  AMV  Dr.  and  and  121°C  screw  (130  Bacto-agar  f o r 15  ml)  Kaps'  glass  jars  shoot  tips  for  from f i e l d - g r o w n  indexing  Station  1613  x  plots  Vitis at  the  i n S i d n e y , B.C.  inoculated i n 1983  inocula  mother p l a n t s  'Kim  i n 25  caps.  (Couderc  virus  been  GFLV  (1983)  rooting  Difco  AMV-infected  LN-33  chip-bud g r a f t i n g The  Monette  1)  Tissue-culture  i n 4 f l oz  were t a k e n  had  and  volumes o f media, e i t h e r  plastic  Quarantine  screenhouse-grown by  5.7  (Appendix  covered with p l a s t i c  L . ) , o b t a i n e d from  Saanichton  sent  Stevenson  proliferation  GFLV and  into  cuttings  plantlets  pressure.  10 ml  initiation  the  The  kPa)  with parafilm,  tightly  vinifera  by  a d j u s t e d t o pH  were grown on  sealed  closed  initiation,  of grape  modifications.  diameter  and  rooting  f o r the  b e f o r e t h e y were a u t o c l a v e d at  plantlets mm  media used  and  m e d i a were a l s o was  METHODS  and came  as  dormant  then p l a n t e d  in  from  m a i n t a i n e d from  S t e l l m a c h , o f the Weinbau  These  Institute,  cuttings West  Germany. 2. ( VJtis  TomRV-infected  cuttings  h y b r i d ) were o b t a i n e d from  o f the v a r i e t y Dr.  De  Chaunac  L. S t o b b s , V i n e l a n d  Research  Station,  cuttings  o f the same v a r i e t y  repository rooting, ambient tips  Vineland, Ontario.  at the S a a n i c h t o n  the c u t t i n g s temperature  were  of  household  bleach  (Protecto  Concentrated  All  the subsequent  by  shoots  rinse  immersion  microscope  long, terminal  5.25%  plant  sections  from  i n a 1:5 d i l u t i o n  A similar  field  of  sodium  shoots  solution from  s t e p s were c a r r i e d  using a Burnell  o f 1:10  greenhouse  bleach  plants.  o u t under a  laminar  techniques.  water w i t h  until  into  use.  slight  Hand  agitation  and k e p t i n  i n s t r u m e n t s were  disinfested  70% e t h a n o l f o l l o w e d by f l a m i n g  and o t h e r  rinsing,  under  a dissecting  shoot  tips  culture  shoot  . The  s u r f a c e s were wiped w i t h a p a p e r  towel  i n 70% e t h a n o l .  After  placed  before  were g i v e n t h r e e , 3- t o 5-minute r i n s e s i n  distilled  immersed  lighting  The s h o o t s  ingredient  f o r to d i s i n f e s t  hood u s i n g a s e p t i c  last  After  i n the greenhouse at  Dishwasher Detergent)  shaker.  used  the  2 t o 4 cm  for initiation.  (active  was  sterile  B.C.  when p a c k e d ) c o n t a i n i n g 0.1% d e t e r g e n t  action'  The  the  l e a v e s and u n n e c e s s a r y  surface-disinfested  hypochlorite  flow  from  i n Sidney,  w i t h no a d d i t i o n a l  A l l expanded  selected  were  'wrist  Station  were m a i n t a i n e d  were removed from  shoots  plants  were o b t a i n e d  initiated.  Initiation. tissues  H e a l t h y dormant  a l l remaining microscope.  measuring  lightly tubes.  into  from  1.0  excess  Both  tissue  apical  t o 2.0 mm  and  were  the s u r f a c e o f i n i t i a t i n g  was  removed  axillary  isolated  and  medium i n  Reculturing. medium  i n 25  have expanded they  diameter  suitably  c o u l d adapt  medium.  The  the  25 mm  the  larger  maintained in  mm  jars  well  early  tubes  until  4 oz  jars.  i n these  to 3 or  Necrotic  tubes  and and  until  survive  plantlet  on  down t o h a l f contain  their  on  size  masses o f r a p i d l y  transfer  within growing  Plantlets  i n the  longer than After  tubes.  2 cm,  Wherever  and  adventitious  rather  than  were c u t  i n order  the v e s s e l .  s h o r t stems were r e c u l t u r e d ,  and  becoming  transfer  dividing  to  intervals,  f o r those  subsequent  in  were  noted.  a t 6 week  out  p r o l i f e r a t i o n medium, p l a n t l e t s  a t each  size  the  plantlets  were removed when r e c u l t u r i n g .  where  proliferation  forced  shoots  to  t o the p o i n t  unless otherwise  callus,  initiation  s t a g e s were c a r r i e d  size  transferred  tissue,  the  A l l subsequent  jars,  on  t h e y were j u d g e d  differentiated  4 week i n t e r v a l s  well-established  with  were m a i n t a i n e d  proliferation  were u s u a l l y  compared  roots  Plantlets  to  possible, buds  single  shoots.  Serology.  Virus from to  purification.  Chenopodium  21  leaves  received  taken  a t the P l a n t  maintained confirmed  been  AMV  and  GFLV were  quinoa W i l d , w h i c h had  days p r e v i o u s l y had  Both  with grapevine from  infected  Quarantine  i n screenhouses.  AMV  i n the mother p l a n t s  by  been  foliar  and  inoculated tissue.  grapevine  Station GFLV  purified  from  The  mother  plants  France  and  infection  serology.  14  The  had  been  original  inoculation phosphate  to  C.  quinoa was  buffer,  hydrochloride  and  pH  8.0  3%  nicotine.  between h e r b a c e o u s potassium The  phosphate,  with  containing  phosphate b u f f e r through  jersey  12,000 g  of  (w/v)  0.05  both of  at  acid  4°C  1%  and  adjusted  at  to pH  centrifuged  4°C.  (w/v)  to  NaCl  the  and  pH  8.0  for  20  to  the  squeezed  minutes  at  a S o r v a l l SS-34 r o t o r low  5.0  in  speed and  with  centrifuge.  HCI  and  left  at  12,000  and  stirred  or  near  4°C  minutes  at  12,000 g (10,000 rpm)  NaCl.  After  12,000  g (10,000 rpm)  to  supernatant.  . The  volume o f 30  minutes, for The  followed  buffer  minutes  supernatant  done a t  i n 1/10  20  6,000) were added  temperature.  gently  for (MW  room  volume o f  Waring  PEG  at  precipitation  in a  M  this rotor  minutes  the  M  4°C.  m i x t u r e was  8%  0.1  m a c e r a t e was  A l l other  was  (10,000 rpm)  using  phosphate, 0.02  The  using  The  The  inoculations  which were f r e s h l y added  were done w i t h  at  and  potassium  cysteine  homogenized  centrifugations  overnight  M  M  44,000).  M sodium  centrifuge.  supernatant  (MW  then c e n t r i f u g e d  (10,000 rpm)  S o r v a l l SS-3  PVP  p r i o r to use.  cloth  0.01  were c a r r i e d out  M ascorbic  2-mercaptoethanol,  a  1%  a 0.05  Subsequent  t i s s u e was  2 ml/g  0.02  containing  plants  herbaceous  blender  made u s i n g  by  A l l the  emulsion  0.05 the 20  M Na  following  was  and  the  pellets  citrate  minutes.  pH  was  alternating cycles resuspension until  of a  of  the  30  were  for  20  resuspended  7.0  with  centrifuged  PEG  at  for  steps  centrifuged  s o l u t i o n was  were c a r r i e d out  gently  g  added  1% at  at  8%  PEG pellet  in  concentrated  1/10  volume o f l e s s purified minutes  than  1 ml r e m a i n e d .  by c e n t r i f u g a t i o n through  Beckman SW-41 centrifuge.  rotor  in either  Absorbance/Fluorescence  254 in  using  was p a s s e d  t h r o u g h an ISCO UA-5  D e t e c t o r a t 254 nm and t h e The v i r u s the absorbance at  nm. P u r i f i e d n e p o v i r u s e s , a t a c o n c e n t r a t i o n  units  p a t h , have an a b s o r b a n c e  (Stace-Smith, Antiserum.  tests  polyclonal diluted  gel  used. double  Station.  antisera  10 o p t i c a l  i n a l l the s e r o l o g i c a l  The AMV,  and k e p t  antiserum against The t i t r e  Canada,  GFLV, and TomRV  from  rabbits  a t 4°C u n t i l  TomRV p r o d u c e d  had been p r e v i o u s l y  and had been used.  A  from mice was  determined  by a g a r  d i f f u s i o n t o be 1:1,280 f o r t h e GFLV and TomRV antisera.  pure  virus  diluted  NaN  in distilled  diffusion  used  were p r o d u c e d  polyclonal  3  o f about  by R. S t a c e - S m i t h , A g r i c u l t u r e  1:1 w i t h g l y c e r o l  monoclonal also  The a n t i s e r a  Research  o f 1 mg/ml  1985).  were s u p p l i e d  Vancouver  a  a Beckman L8-80 o r L8-70  was e s t i m a t e d by m e a s u r i n g  a 1 cm l i g h t  further  gradient  a b s o r b i n g band was c o l l e c t e d .  concentration  was  a t 180,000 g (38,000 rpm) f o r 90  a 10-40% s u c r o s e d e n s i t y  The g r a d i e n t  ultraviolet  The v i r u s  during  The t i t r e  i n saline  f o r AMV  solution  100 uq/m\ o f  (0.85 % NaCl  w a t e r ) was d e t e r m i n e d these studies  against  and 0.2%  by g e l d o u b l e  t o be 1:1,024.  The g e l s  were  made by p o u r i n g 2 ml o f a m e l t e d m i x t u r e o f 1.0% B a c t o - a g a r , 0.2%  NaN , and 0.85% NaCl 3  collodion-coated into 22  glass  the s o l i d i f i e d  in distilled  microscope  a g a r by u s i n g  c a l i b r e brass c a r t r i d g e  shells  water  slides.  onto  W e l l s were c u t  a t e m p l a t e made from  seven  ( W r i g h t and S t a c e - S m i t h ,  1966)  and  the  dilutions filled  excess  agar  of antiserum  with  the  was  incubated  with  were p l a c e d  purification.  diluted 10  ml  with of  at  12,000 g (10,000 rpm),  s t r e n g t h PBS 7 to  and  first  peak  10  ml  model  250  i n 1/2  effluent  PBS,  o r by  saturated  Centricon-30  times then  to  against added  (£  s t r e n g t h PBS  at  at  was  nm  and  sulphate  using or  centrifuge f i l t e r  10  500  either  allowed  nm  PBS  to and  (ISCO  a  The  Gilford  adjusted  to  dilution  with  precipitation  c e n t r i f u g a t i o n through (Amicon) at  of  which  1/2  using  by  of  ml  collected.  was  =1.4), e i t h e r  z a o  280  determined  280  60  to a column  and  washed w i t h  Detector)  concentration  ammonium  f o r 30  4  d i s s o l v e d i n 2 ml  absorbing  c o n c e n t r a t i o n was  1 mg/ml  and  centrifuged for  then  column was  spectrophotometer  approximately  was  2  water  o f DEAE-22 Sephadex c e l l u l o s e  . The  i n the  4  I t was  Absorbance/Fluorescence  immunoglobulin  well  of p o l y c l o n a l  (NH ) S0  d i a l y s e d three  f o r 1 hour.  f o r 5 minutes  UA-5  1/2  precipitate  been p r e - e q u i 1 i b r a t e d  bind the  PBS  ml  saturated  minutes  containing  Serial  a central  of d i s t i l l e d  4°C.  had  The  9 ml  at  1/2  around  One  minutes  half-strength  aspiration.  virus.  yglobulin antiserum  removed by  5,000 rpm  with  a in a  SS-34 r o t o r . The  monoclonal  immunoglobulin commercial  G of  antibody  to TomRV, which was  class  (determined  immunoglobulin  2a  t y p i n g k i t ) , was  affinity  chromatography using  column.  Up  2 ml  to 2 ml  of p r o t e i n - A  of  the  by  a protein-A  immunoglobulin  using  an a  purified sepharose was  sepharose p r e - e q u i 1 i b r a t e d  by CL-4B  mixed w i t h against  PBS.  to  27 After the  30 m i n u t e s ,  absorbance  eluted  with  absorbing stored  t h e non-bound components were e l u t e d  a t 280 nm was  0.2  M glycine  a t 280 nm  a t -20°C  was  i n 0.2  Conjugation. TomRV m o n o c l o n a l  One  half  grade  The bound IgG was  + 0.5 M Na C l , pH 2.5.  aliquots  until  antiserum  dissolved  i n 0.2  polyclonal  were c o n j u g a t e d  mg  (electron  microscope grade) d i s s o l v e d  was at  diluted  1:10  4°C u n t i l  buffer  conjugations antiserum hours kept  wells  a t 4°C u n t i l ELISA  500 ml o f 0.05  diluted  PVP  by C l a r k  96 w e l l s  was  ^-globulin caoted  tubes  obtained i n antiserum  solutions.  samples w h i l e AMV  were  and GFLV  then  buffer. sandwich  technique  used with  filled  minor  plates  with  IA  200  used  0.2% o v a l b u m i n and 2%  same b u f f e r four  was  The c o n j u g a t e b u f f e r  by a d d i n g  This  for 2  The c o n j u g a t e  antibody  The w e l l s  modified  to detect  pH 8.0  II ( D y n o t e c h ) p o l y s t y r e n e  were u s e d .  t o t h e PBS-Tween.  tissue-culture  M Tris-HCl  and Adams (1976) was  Immulon  a l l t e s t s was  were u s e d  in silicon  i n the c o n j u g a t e  The d o u b l e  volumes o f the v a r i o u s in  PBS. The  some GFLV  t h e PBS d i a l y s i s .  test.  modifications. with  dialysed  glutaraldehyde  readings  with  and  were overcome by d i a l y s i n g the c o n j u g a t e d  against  described  and s t o r e d  on p l a t e s  and  follows.  i n PBS. T h i s  against  High background  a t 4°C a f t e r  The as  w i t h PBS  used.  control  130 ml o f 0.06%  dialysis  as  of ^ g l o b u l i n  a t 4°C a g a i n s t  by e x t e n s i v e  antisera  (Boehringer-Mannheim,  overnight  followed  The peak  used.  and AMV  of a l k a l i n e phosphatase  1) was  then  c o l l e c t e d , d i l u t e d t o 1 mg/l and  The GFLV  the  mg  0.  until  was u s e d  to g r i n d  different grinding in field-grown  buffers  plants  (see  methods f o r t e s t i n g three,  plants).  The w e l l s were  10-second washes, w i t h t a p water u s i n g  plexiglass 96  field  p l a t e washer which shot  w e l l s a t once.  incubated  a homemade  water d i r e c t l y  The c o a t i n g and s a m p l e s t e p s  overnight  a t 4°C, t h e c o n j u g a t e  given  into a l l  were  f o r 4 hours a t  37°C, and t h e s u b s t r a t e a t room t e m p e r a t u r e .  A blocking  s t e p was added b e f o r e  t h e s a m p l e s were p u t on t h e p l a t e s .  This  the wells completely  involved f i l l i n g  s o l u t i o n of f e t a l dissolved and  calf  serum o r b o v i n e  w i t h a 2%  serum  albumin  i n PBS-Tween, i n c u b a t i o n f o r 30 m i n u t e s a t 37°C,  rinsing.  The s a m p l e s were a l w a y s g r o u n d  of g r i n d i n g b u f f e r . conjugation  i n 10 v o l u m e s  A t e s t p l a t e was r u n a f t e r  to-determine  the concentrations  o f c o a t i n g and  conjugated  y - g l o b u l i n which would g i v e a t l e a s t  difference  i n absorbance values  virus-inoculated  C. quinoa  after  AMV and TomRV p l a t e s were, c o a t e d <yg/ml w h i l e but  a 10:1  ( 4 0 5 nm) b e t w e e n h e a l t h y and 1 hour  . As a r e s u l t , t h e  at a concentration of 1  t h e GFLV p l a t e s were c o a t e d  u s u a l l y a t 2 ug/ml.  each  The c o n j u g a t e s  s o m e t i m e s a t 1 og/ml were d i l u t e d t o  1:1000 f o r AMV and TomRV, and 1:500 f o r GFLV. The  c o l o r r e a c t i o n i n t h e w e l l s was r o u t i n e l y  measured  q u a n t i t i v e l y w i t h a T i t e r t e k D u a l Band M u l t i s c a n p l a t e reader The  (Flow  values  obtained except very  Laboratories  Inc.), unless  otherwise  were c o r r e c t e d f o r b a c k g r o u n d b u f f e r  from w e l l s t r e a t e d i d e n t i c a l l y  (<0.01) i n s a m p l e s from  tissue-cultured plantlets,  readings,  to other w e l l s ,  f o r t h e a d d i t i o n o f the p l a n t sample.  low r e a d i n g s  mentioned.  Because o f  healthy  corrected readings  greater  than  0.05  were c o n s i d e r e d  plantlet  cultures.  field  p l a n t s was  three  standard  readings  technique  in tests  carried  The d e t e c t i o n l e v e l  o u t on  f o r ELISA  s e t a t t h e mean o f the h e a l t h y  d e v i a t i o n s , because of h i g h e r  found  ELISA  positive  in healthy  sensitivity.  field  on  samples  plus  background  plants.  The s e n s i t i v i t y  f o r d e t e c t i n g GFLV and AMV test  tests  was  on p u r i f i e d  o f t h e ELISA determined  carrying  out t h e ELISA  antigen  standard  g r i n d i n g b u f f e r , as d e s c r i b e d by C l a r k  by  diluted in and Adams  ( 1976) .  Virus  detection in tissue-cultured  field  and g r e e n h o u s e p l a n t s .  AMV  and GFLV d e t e c t i o n .  AMV-infected 1985:  June  and h e a l t h y 11, J u l y  GFLV-infected  non-inoculated  until  field  p l a n t s on t h r e e  9, and'August  from  the shoot  tips  five  initiated  S h o o t s were h a r v e s t e d  26.  p l a n t s were h a r v e s t e d  S h o o t s were t a k e n five  plantlets  Shoots  p l a n t s , on each d a t e were p l a c e d  into  from  dates i n from  o n l y on August  virus-infected  from  26, 1985.  p l a n t s and from  and k e p t  culture,  a t 4°C  within 2  days . All  in vitro  plantlets  were m a i n t a i n e d  f o r up t o 23  weeks  i n growth chambers a t 25 x 1°C and under  sec"  light.  1  L i g h t at a l l stages  experimentation fluorescent plantlets  for a l l plantlets  tubes  then  under  50 x 5 2 £ m ~  2  o f growth and was  s u p p l i e d by c o o l  a 16 hour p h o t o p e r i o d .  were moved, f o r 7 weeks,  white  The  t o a room h a v i n g  the  same  light  quality  25°C  at n i g h t  were moved  experiments  from  only  i n September, growing  plants  The p l a n t l e t s  moved  tested  initiated  5 weeks from  after  and BAP  in  plantlets.  Plantlets  tested  moved  a t 4-6  t o 20°C The  dates.  ELISA  plantlet  from  by ELISA  either  7 weeks  from  after  plants  plants were plantlets  on t h e  to determine  first  the e f f e c t  on t h e d e t e c t i o n  a l l three i n i t i a t i o n  Only  of  AMV  dates  after  being  25°C. dates c o i n c i d e d  and t h e amount  was  The  and a g a i n 24-26 weeks  plants  reculturing tested  by t h e c o n d i t i o n  of material  Only  with  were n e c e s s a r i l y  being determined  vigorously-growing  AMV-infected  field  concentration  weeks  testing  reculturing.  attention  initiated  the G F L V - i n f e c t e d p l a n t s  Not a l l p l a n t l e t s this  a t the  GFLV-infected plants  26 from  i n an.experiment  temperature  after  tested  b e i n g r e t u r n e d t o 25°C.  of  date,  to being p l a c e d  the p l a n t l e t s  were  the AMV-infected  two d a t e s were u s e d  were  h e l d at  t h e end o f t h e  were a g a i n t e s t e d  August  d a t e and 33 from  initiated  a chamber  t o t h e room w i t h the h i g h e r t e m p e r a t u r e s .  29 p l a n t l e t s harvest  the p l a n t l e t s  1986.  temperatures,  field  from  having a constant  into  o r t h r e e t i m e s , and t h o s e from  being  Then,  a chamber  a t 25°C and p r i o r  fluctuating  once.  fluctuating  t h e y were m a i n t a i n e d u n t i l  AMV-infected  twice  into  the d a y .  o f 25 ± 1°C and f i n a l l y  20 ± 1°C where  While  temperatures  t o 29°C d u r i n g  f o r 5 weeks  temperature  higher,  but w i t h  available  healthy-looking was u s e d  g i v e n t o t h e type o f t i s s u e  of each for testing  tissue  f o r ELISA  on e v e r y  from  testing.  samples  No  ground  before  November  adventitious always  moved were  selected  o f the samples  taken  t o t h e room w i t h  earlier  samples  samples  from  0.4  Only  masses o f p r o l i f e r a t i n g after  from  the h i g h e r  less  the l a t e r  test  than  adding  absorbance  virus  d e t e c t i o n , the e x c e p t i o n b e i n g  developing  colour.  The r e s u l t s  because  o f equipment  the y e l l o w  from  using  malfunction.  corresponded  were  three  weights  readings to  the f i r s t  of  rapidly dates,  fluctuating  the T i t e r t e k  visually  t o an a b s o r b a n c e  2  on GFLV  plate  on t h a t  date,  T r i a l s had e a r l i e r  interpreted  less  taken  test  a l l testing  visually  of  determine  to the h i g h e r  were e s t i m a t e d  colour  they  only  1 hour b e c a u s e  exposure  were q u a n t i f i e d  reader.  reaction  after  The r e s u l t s  t h e one f o l l o w i n g  temperatures,  that  read  fresh  temperatures  g, w h i l e  t h e s u b s t r a t e were u s e d  which was  the  before  had f r e s h  after  plantlets,  Although  Twelve o f t h e 178 0.1  dates  g. G e n e r a l l y , ELISA  buffer,  fluctuating  f o r later dates.  weighed  date.  plantlets  hours  except  that  i n 10 volumes o f g r i n d i n g  s m a l l e r than  than  1986.  buds were  ground  weights  20,  as a  shown  positive  of at l e a s t  0.1  units. TomRV d e t e c t i o n . healthy  controls  were  Shoot  tips  initiated  two d a t e s ,  May  2 and J u l y  maintained  a t 20 + 1°C under  from  from  10, 1986.  TomRV-infected  greenhouse  and  cuttings  The p l a n t l e t s  a 16 hour p h o t o p e r i o d  were a t 50-75  uEm-* s e c " . 1  Virus weeks the  after  second.  d e t e c t i o n by ELISA was the f i r s t  initiation  carried date,  o u t 18 and 24  and 15 weeks  A l l samples weighed a t l e a s t  0.1  on  after  g and most  32 c o n s i s t e d of r a p i d l y p r o l i f e r a t i n g plantlets  from the f i r s t  tissue.  initiation  Not a l l  d a t e were t e s t e d on b o t h  d a t e s b e c a u s e o f p o o r g r o w t h and m o r t a l i t y . readings  from t h e f i r s t  substrate while  d a t e were t a k e n 20 m i n u t e s f o l l o w i n g  a d d i t i o n because of r a p i d c o l o u r  those from the o t h e r  Results  Virus distribution.  the p l a t e  To d e t e r m i n e  distributed  within infected plantlets,  originating  from t h e f i r s t  initiation  i n t o segments o f v a r i o u s  shoots with  leaves  proliferating (R).  i f AMV  was  uniformly  four p l a n t l e t s completely following  V i r u s d e t e c t i o n by E L I S A individual  elongated  (LS & L V S ) , masses o f r a p i d l y  a d v e n t i t i o u s buds w i t h  shoots  ( S S ) , and  roots  The f r e s h w e i g h t s o f t h e s h o o t s and r o o t s were u s u a l l y  less  t h a n t h o s e o f t h e masses o f a d v e n t i t i o u s  available adventitious dissected varying  fresh weights.  plantlets  were t e s t e d .  A total  after  equal  o f 30 e l o n g a t e d  t i s s u e s , and r o o t s  size,  with  shoots,  39  f r o m two o f t h e  ELISA absorbance v a l u e s  m e a s u r e d 90 m i n u t e s a f t e r s u b s t r a t e plantlet  tissues. A l l  t i s s u e s f r o m e a c h p l a n t l e t was  i n t o s e c t i o n s of approximately  masses o f a d v e n t i t i o u s  were  a d d i t i o n f o r the  d i s s e c t e d a t 21 weeks, and 2 h o u r s and 20 m i n u t e s  substrate The f i r s t  f o r the other distribution  three  study  f i v e GFLV-infected  plantlets.  was e x p a n d e d u s i n g  o f p l a n t p a r t s were t a k e n f r o m f i v e and  reader.  d a t e were  plant parts:  attached  a f t e r 2 hours.  fresh weights  21 and 22 weeks o f g r o w t h a t 25°C. was c a r r i e d o u t on t h r e e  development,  t e s t s were r e a d  were q u a n t i f i e d u s i n g  dissected  Absorbance  samples  a d d i t i o n a l AMV-infected  p l a n t l e t s g r o w i n g a t 25°C.  The  elongated were and  shoot  tested  w h i c h were a l l l o n g e r  i n two s e c t i o n s , t h e stem w i t h o u t  the leaves  2 hours  samples,  (LVS).  ELISA  growing  and 10 G F L V - i n f e c t e d  a t 20°C  were once a g a i n  f o r 4 months. tested  were measured  as c o m p l e t e  values  were measured 2 h o u r s  Effect infected cabinet  entities  of temperature  with  AMV  healthy  equal-sized  parts  subculture  subjected  (LS & L V S ) .  were  subdivided  and p l a c e d  into  from e a c h o r i g i n a l  were c o n s t a n t  5 uE m  -2  tubes sec  - 1  absorbance  o f BAP.  to w i t h i n  approximately  c u l t u r e tubes,  level.  2  All  + 1°C.  The p l a n t s  and 75  1  Tissue  was  ground  i n 10 volumes o f s t a n d a r d  grinding buffer using a  Virtis  polytron  A l lsubcultures  mother p l a n t l e t Analysis out  tissue were  of variance  grinder.  t e s t e d on t h e same ELISA and r e g r e s s i o n a n a l y s e s  on b u f f e r - c o r r e c t e d a b s o r b a n c e v a l u e s  substrate wells  addition, using  the average  l o c a t e d r a n d o m l y on the p l a t e .  were  white  s e c " f o r t h e AMV  f o r the GFLV e x p e r i m e n t s .  with  being  s u p p l i e d by c o o l  a t 50 + 5 <vE m~  growth  Infected  mother p l a n t l e t factor  Plantlets  to v a r i o u s  into  25 mm  grown under a 16 hour p h o t o p e r i o d fluorescent  ELISA  were  and BAP c o n c e n t r a t i o n .  to each e x p e r i m e n t a l  temperatures  others  shoots  following substrate addition.  and c o n c e n t r a t i o n s  plantlets  w h i c h had been  Some o f t h e e l o n g a t e d  and GFLV were s u b j e c t e d  temperatures  i n 10  plantlets  i n two p a r t s , w h i l e  tested  t  mm,  leaves (LS)  absorbance values  same p l a n t p a r t s were a l s o t e s t e d  AMV-infected  a  20  following substrate addition.  The  and  than  from  each  plate. , were  2 hours  readings  carried after  from  three  34  Two plants. placed the  experiments  were c a r r i e d  In the f i r s t ,  three  groups  o f 25 p l a n t l e t s  a t 2 0 ° , 25° and 30°C,  within  which  medium a t 2, 4, and 8 mg/l.  groups  o f 25 p l a n t s  temperatures.  Each  plantlet  was  of  AMV-infected  of  the f i r s t  second. ground  after  completion  subdivided for  each  hours  Virus  was  culture.  samples  of a slowly Nine  (treated)  They  later. and 25°C  tested 0.2  after  6,  at the  was  in vitro  35 ± 5 mg  tested  measured 21  incubation  o u t 4,  which  the  a t 4°C  had  been  first  i n two and moved t o  regularly  six plantlets  the second  were  was  plantlets  were d i v i d e d  carried  were  developing reaction.  and  AMV  10, 16, 26, and 32 were  temperature  16, and 20 weeks.  g on the f i r s t  were  w i t h 2 mg/l o f BAP  following  were r e c u l t u r e d  Similarly, after  o f 25 mg  were u s e d  f o r 2 months d u r i n g  20°C  20°C  weight  samples  weighing  addition,  recovery.  by ELISA  testing  out a t t h e c o m p l e t i o n  E L I S A . c o l o u r development  experiment  weeks  carried  Half of ELISA  GFLV-infected plantlets  substrate  and 25°C.  20°C  one month.  having a fresh  temperature-BAP  detection  after  with  experiment.  Tissue  grown a t 30°C  to  experiment,  2 months.  and p u t a t 2 0 ° , 2 5 ° , and 30°C  because  added  and a t 1 and 2 months d u r i n g t h e  samples  o f twenty  after  overnight,  plantlets  lasted  1 month w h i l e 1 gram  1 month.  from  In t h e s e c o n d  a t t h e 15° and  experiment  recultured  of each  Groups  tested  experiment  Tissue  BAP was  were  were put a t 1 5 ° , 2 0 ° , 2 5 ° , and 30°C  2, 4, and 8 mg/l o f BAP  each  o u t on A M V - i n f e c t e d  t r a n s f e r r e d to experiment  Samples weighed  two d a t e s w h i l e 0.5 g samples  at  were  and least tested  on  the l a s t  plantlets had  treated  and  t h a n 2 cm were r o o t e d  control  cultures  10 and  a f t e r each e x p e r i m e n t a l These p l a n t s  tubes to a growth  room w i t h  t o the g r e e n h o u s e where  (approximately produced  from  l o t were  the s e c o n d .  plants  Virus quickly eight  moved t o the  i n stages lighting  f o r AMV,  after  by  5 1/2  ELISA  from  a t 30°C.  detection  was  plantlet  and 25°C  and p l a c e d by ELISA was  experiment  13  weeks.  elapsed  g r o u p and  leaves  ELISA,  while  and 8 months o f  newly-opened  and  plants  from c u l t u r e ,  tested  from  ambient  5 months  these  grinding  buffer  In an a t t e m p t to see  affected  by g r o w t h  and w e e k l y  weighed  in half  f o r 6 weeks, were s u b d i v i d e d  a t 30°C  i n a growth  chamber.  c a r r i e d out at the onset thereafter.  a t l e a s t 0.2  w e i g h i n g more t h a n 0.5  how  at 30°C,  A M V - i n f e c t e d p l a n t l e t s w h i c h had been d i v i d e d  detection  date  from b o t h  treated  f o r the f i r s t  i n the s t a n d a r d  inhibition  virus  parts  the  1% n i c o t i n e .  and grown at 20°C four  Young,  were g r o u n d  containing  removed  c o r r e s p o n d e d to 6 1/2  t r e a t m e n t a t 30°C  vitro  g r o u p was  tested  from the s e c o n d g r o u p were  for  which  6 weeks,  Fifteen  plants  time post  but  t h e y were k e p t under  17 weeks a f t e r b e i n g  dates  in  fluorescent  10 and  These  plants  were moved  25°C) c o n d i t i o n s . the f i r s t  control  tested.  temperatures.  then  and h e a l t h y  to 30°C were grown a l o n g s i d e  and a l s o  longer  respectively,  the  subjected  cultures  Shoots  lower  Virus-infected  o r i g i n a t i n g from the same mother  n e v e r been  treated  the  date.  g.  Samples  g, w i t h  Virus  of t h i s  tested  samples  from e a c h  on t h e  In an a t t e m p t t o  into  last  standardize  results  over  t i m e , the same c o n j u g a t e was  testing  dates.  Pure v i r u s ,  ng/ml i n C.  100  buffer,  was  sensitivity  diluted  quinoa f o l i a g e  used  as a c o n t r o l  of each  Virus detection  test  used  on a l l  to a c o n c e n t r a t i o n of  ground  i n 10 v o l u m e s o f  t o measure  detection  date.  in field  plants  L e a f s a m p l e s were t a k e n f r o m v i r u s - i n f e c t e d f i e l d - g r o w n and during  screenhouse-grown  t h e summer o f 1986.  t a k e n from p l a n t s t h r e e AMV  The  g r a p e v i n e s on f i v e screenhouse  i n f e c t e d w i t h one  sources.  The  o f s i x GFLV s o u r c e s  vinifera  P i n o t Chardonnay  respectively; California, and  two  unknown  v e i n b a n d i n g and  Two  o f t h e AMV  V. vinifera  LN-33 ( V. vinifera Noir  ( V. vinifera  years e a r l i e r or  one  V.  Rotberger  L . ) , which  had  vinifera  the  plants.  two  sources.  L).  varieties  inoculated  f o u r GFLV v i r u s Healthy  2  sources  control  s a m p l e s were t a k e n from n o n - i n o c u l a t e d LN-33 and field  in  S t . G e o r g e , and P i n o t  been c h i p - b u d  of the f i r s t  two AMV  du L o t ) ;  ( V. vinifera  s a m p l e s were t a k e n from  w i t h one  from  w i t h the o t h e r r e c e i v e d from  L. x C o u d e r c 1 6 1 3 ) ,  of the f i r s t  strains  unknown  ( V.  L.),  G e o r g e ( V. rupestris  i n two  varieties,  field  vinifera  and  Riesling  s o u r c e s came from F r a n c e ,  West Germany, i n t h e v a r i e t y Virus-infected  ( V.  deforming  i n the v a r i e t y ' S t .  s o u r c e s from F r a n c e ,  varieties.  or  GFLV s o u r c e s c o n s i s t e d o f " m i l d "  from O n t a r i o , i n t h e v a r i e t i e s  and  dates  s a m p l e s were  "severe" strains L.)  and h e a l t h y  S t . George  37  Young,  fully  three  to f o u r  After  removal,  tested, cut  shoots  spot  plate  PBS,  pH 8.4  into  with  each g r a p e v i n e  were k e p t  into  squares with  four  with  at or near  The y o u n g e s t  aliquots  a glass  0.05% Tween-20,  A, pH 8.4  with  added.  at the s t a r t  with  was  until were  a knife, o f 0.1  mixed  g each.  porcelain  buffers:  A - 0.15 M  and 2% PVP,  added, C - s t a n d a r d  or D - standard  The n i c o t i n e  sampled.  leaves  0.2% o v a l b u m i n  pH 7.4  t i p s of  4°C  r o d on a  1% n i c o t i n e  buffer,  and 2% PVP,  1% n i c o t i n e buffers  from  2-3 mm  were g r o u n d  M PBS g r i n d i n g  ovalbumin  taken  i n 10 volumes o f each o f f o u r  - buffer  0.01  the growing  3 days.  and w e i g h e d  aliquots  from  were  the next  in half, diced  The  leaves  the l e a v e s  within  thoroughly,  B  opened  0.05% Tween-20,  buffer  C, pH 8.2  f r e s h l y added  o f e a c h day and a g a i n  0.2% with  to the  3 to 4 hours  later. The  ground  been r a n d o m l y (Moses on  samples  selected  and O a k f o r d ,  each p l a n t  each p l a t e  healthy  control  2 hours.  unless after by  by  1 hour.  significant  o f random  A l l four  grinding  had t h r e e  wells.  buffer  exceeding  2.0  of variance  difference  was  using  and  read was  before  performed,  the p r o t e c t e d  (LSD) p r o c e d u r e which was  three after 1  used  were  of the b u f f e r  was  used  f o r each  wells  absorbance u n i t s  were s t a n d a r d i s e d  mean c o m p a r i s o n  buffers  the 2 hour r e a d i n g  the absorbance values Analysis  colour  w h i c h had  permutations  As w e l l , control  The r e a c t i o n  The p l a t e s  subtracting  treatment  1963);  wells  tables  In most c a s e s ,  readings  each b u f f e r .  using  three  were put on one p l a t e .  buffer,  and  were put i n t o  observed analysis wells f o r  followed least applied  38  only if the overall F test for treatments was  significant.  RESULTS  ELISA  sensitivity  The in  most d i l u t e  standard  reading ground for  g r i n d i n g b u f f e r which  at l e a s t  twice  AMV  gave an ELISA  that of healthy  i n t h e same b u f f e r was  and GFLV  absorbance  C. quinoa  10 ng/ml f o r AMV  detection in tissue-cultured  AMV every  and GFLV d e t e c t i o n . plantlet  though background from the  of p u r i f i e d  leaves and 125 ng/ml  GFLV.  Virus  in  concentrations  healthy  absorbance values p l a n t s were v e r y  (Table  tested positive.  tested  twice  The virus  The v i r u s  tested only cumulative  increased with  eventually  Only  14  detected  first  two i n i t i a t i o n  after  being  three  plantlets  in plantlets low 2 h o u r s  even  started after  adding  plantlets  tested three  detected  times  in eight  were  other  once.  number  of p l a n t l e t s  the number  testing  o f times  tested.  i n a l l b u t one p l a n t l e t dates.  tested only from  was  date  (58%) o f a l l p l a n t l e t s  and 10 (21%) o f t h o s e  always p o s i t i v e .  test  I I ) . . ' A few A M V - i n f e c t e d  always  plantlets  and GFLV were n o t d e t e c t e d  grown a t 25°C on e v e r y  field  substrate  AMV  plantlets  This  once.  the t h i r d  plantlet  The v i r u s initiation  positive for AMV  started  was  from t h e  d i e d a t 12 weeks,  was  not d e t e c t e d i n  date.  Each o f  T a b l e I I . D e t e c t i o n by E L I S A o f a r a b i s m o s a i c v i r u s (AMV) and g r a p e v i n e f a n l e a f v i r u s (GFLV) i n p l a n t l e t s i n i t i a t e d f r o m f i e l d p l a n t s and grown a t 25°C Initiation date A)  Date t e s t e d (weeks post i n i t i a t i o n )  P o s i t i v e s per testing date  Cumulative positives  1  AMV June  12  S e p t . 4 (12) O c t . 2 (16) Nov. 20 (23)  12/21 9/24 25/25  12/21 18/27 27/27  July  12  Oct. 2 Nov.15  8/14 13/13  8/14 15/16  20/36 17/34 23/33  20/36 26/37 34/37  August  26  (12) (18)  O c t . 21 (9) Nov.10 (12) Dec. 29 (19)  76/80  Total B)  GFLV August  Nov.  26  17  (13)  13/36  13/36  'Numerator = p l a n t l e t s w i t h ELISA absorbance v a l u e s g r e a t e r t h a n 0.05. D e n o m i n a t o r = t o t a l number o f p l a n t l e t s tested. these three was  cultures  l a t e r detected  initiation,  i n two  initial 29 and  not  at  detected initial  in a cabinet  this  detection  p l a n t l e t s tested 18 o f  twice  21  detection,  while  The  with  one  times.  The  virus  weeks p o s t  a constant a i r  other p l a n t l e t  died  temperature. in plantlets following  being consistent  more f o l l o w i n g  tested  at a l l i n o n l y  three  t h e s e p l a n t l e t s 34  always detected  detection;  in only  of  20°C f o r 6 weeks.  before being placed was  been t e s t e d  a f t e r growing  t e m p e r a t u r e of  AMV  had  once more.  initial The  p l a n t l e t tested  g r o w i n g at  i n only  25°C.  11  of  detection,  v i r u s was twice  the  not  following  41  Only  13 o f 36 p l a n t l e t s  13 weeks.  Subsequent  20°C p e r m i t t e d AMV the  detection  plantlets  evaluation  o f ELISA  addition,  virus  plantlets  from  was  after  from  2 hours,  control  plants  while  plantlet  transfer  to  in a l l plantlets.  reduced  2 hours  i n only  initiation  C.  drastically  at the h i g h e r  plates  healthy  and b u f f e r  after  III).  plantlets. infected  after  (Table  detected  the three  32 G F L V - i n f e c t e d samples  test  f o r GFLV  was  had been p l a c e d f o r 7 weeks  positive  eventually  and GFLV d e t e c t i o n  temperatures  of  testing  tested  fluctuating  Based  on  after  8 o f 60 dates  AMV-infected  and i n none o f t h e  controls,  quinoa,  a l l reacted  wells  visual  substrate  Positive  plantlets  after  consisting positively  and h e r b a c e o u s  d i d not develop  any  colour.  T a b l e I I I . D e t e c t i o n o f a r a b i s m o s a i c v i r u s (AMV) and g r a p e v i n e f a n l e a f v i r u s (GFLV) by ELISA i n p l a n t l e t s grown a t v a r i o u s t e m p e r a t u r e s from J a n u a r y 3, 1986 t o September 17,1986  Growth room temperature (°C)  Tes t date  Duration at temperature (wks)  Virus  P o s i t i v e s per t e s t date  25 t o 29  7  AMV GFLV  8/60 0/32  Mar. 29  25  5  AMV GFLV  0/29 0/33  May  20  6  AMV GFLV  28/29 31/33  20  25  AMV GFLV  19/21 30/33  Feb.  20  10  Sept.  16 iit 17  N u m e r a t o r = p l a n t l e t s w i t h ELISA a b s o r b a n c e r e a d i n g s g r e a t e r t h a n 0.05 u n i t s . D e n o m i n a t o r = t o t a l number o f plantlets tested.  The  effect  detection, (Table  although  initiation returned  after  However, t h e s e  Virus  plantlets  initiation  positive the  after  becoming  positive.  of these  as a t 25°C ( T a b l e I I I ) . plantlets  from t h e  25 weeks a f t e r  being  19 o f t h e AMV and 30 o f t h e GFLV 2 hours a f t e r  negative p l a n t l e t s  by E L I S A 6 w e e k s . a f t e r  being  substrate  addition.  had p r e v i o u s l y t e s t e d p l a c e d a t 20°C.  Also,  i n w h i c h t h e v i r u s e s h a d n o t been  i n the p r e v i o u s assay  tested positive  by ELISA  25 weeks. TomRV d e t e c t i o n in vitro.  but  t o 20°C f o r 6 weeks;  were t e s t e d a g a i n  were p o s i t i v e  three p l a n t l e t s  detected  v i r u s e s became d e t e c t a b l e  and 33 G F L V - i n f e c t e d  date  moved t o 20°C, o n l y  five  being  and 31 o f t h e 33  grown a t 20°C as w e l l  When 21 A M V - i n f e c t e d  plantlets  from the t h i r d  d e t e c t i o n by ELISA was a l s o i n c o n s i s t e n t f o r  plants being  third  plantlets  moving the p l a n t l e t s  28 o f t h e 29 A M V - i n f e c t e d  reversed  i n any o f t h e 29  w h i c h were t e s t e d 5 weeks a f t e r  t o 25°C.  GFLV-infected  on v i r u s  n o t p e r m a n e n t , was n o t e a s i l y  and 33 G F L V - i n f e c t e d date  once a g a i n  All  temperature  I I I ) . V i r u s was n o t d e t e c t e d  AMV-infected  with  of the higher  one p l a n t l e t  (Table  IV).  detected  originating  The p l a n t l e t  TomRV was d e t e c t e d from  infected  sampled.  The mean E L I S A a b s o r b a n c e v a l u e  plantlets  started  testing  dates.  greenhouse p l a n t s  i n which the v i r u s  showed p o o r g r o w t h so t h a t o n l y  in a l l  was n o t  leaves  were  f o r samples  from  f r o m h e a l t h y p l a n t s was 0.00 f o r b o t h  43  T a b l e IV. D e t e c t i o n o f tomato r i n g s p o t v i r u s i n c u l t u r e s grown a t 20°C f o l l o w i n g i n i t i a t i o n from v i r u s - i n f e c t e d mother p l a n t s Duration in c u l t u r e (wks)  Initiation  1»* 2  Positives  18 25 15  n A  1  9/9 6/6 8/9  N u m e r a t o r = p l a n t l e t s w i t h ELISA a b s o r b a n c e r e a d i n g s g r e a t e r than 0.05 u n i t s . D e n o m i n a t o r = t o t a l number o f plantlets tested.  Virus  distribution.  distributed subsequent  in virus  having  Virus  elongated  tissue  plantlet optical  units.  consisted lowest found  reading  less  other  samples  The o t h e r  from  g r e a t e r than  than  others.  types  i n samples o f  (Table V).  plantlet  from  less  0.05  sample  f o r the n e c r o t i c  mean a b s o r b a n c e v a l u e  than  from  proliferating  The mean ELISA  Three  C and one  B (absorbance  than  by ELISA plantlet  negative  plantlet  f o r SS t i s s u e  low v a l u e s  p l a n t A, t h e  some  dependable  from  on two  with  absorbance values  i n a long shoot.  greatest  pronounced  of a p i e c e of n e c r o t i c  were h i g h e r the  than  D had ELISA  In one p l a n t l e t ,  4 times  d e t e c t i o n was tissues  non-uniformly  c o n c e n t r a t i o n , as i n d i c a t e d  values  elongated  t o be  four d i s s e c t e d p l a n t l e t s  (Figure 1).  a b s o r b a n c e v a l u e s , was v e r y sections  was found  w i t h i n the f i r s t days  variability  AMV  plantlet  D  tissue.  The  = 0.08) was  absorbance  f o r LS & LVS t i s s u e  values even when  sample was i n c l u d e d . was found  i n the R  also,  The  tissue.  44  F i g u r e 1. F r e q u e n c y h i s t o g r a m o f v i r u s d i s t r i b u t i o n as d e t e r m i n e d by E L I S A i n a l l s a m p l e s c u t f r o m e a c h o f t h e f i r s t f o u r d i s s e c t e d a r a b i s mosaic v i r u s - i n f e c t e d p l a n t l e t s .  Table plant  V. A r a b i s m o s a i c v i r u s d e t e c t i o n by ELISA i n p a r t s t a k e n from f o u r p l a n t l e t s grown at 25°C  Positives  Plant let  LS  per  & LVS*  plant  SS  A B C D  7/7 6/6 8/11 5/6  14/14 6/6 4/4 15/16  Totals Mean ELISA v a l u e s  26/30 0 . 59  39/40 0 .73  part  various  1  R  Totals  1/1 3/3  21/21 12/12 13/16 23/25  4/4 1.11  69/74  'Numerator = p a r t s w i t h a b s o r b a n c e r e a d i n g s g r e a t e r t h a n 0.05. D e n o m i n a t o r = t o t a l number o f p a r t s t e s t e d . = not t e s t e d a  L S & LVS = s h o o t s 20 mm or l o n g e r w i t h l e a v e s a t t a c h e d . SS = masses o f r a p i d l y p r o l i f e r a t i n g a d v e n t i t i o u s buds w i t h s h o o t s 10 mm or l e s s i n l e n g t h . R = root t i s s u e .  Virus similar  plant  Samples o f highest  concentration  SS  parts from  plantlets,  6  t i s s u e s than  both  the  being 30 had of  within'the plantlet  absorbance values  three LVS  highest  found  samples  a wider  and  i n these of  LS  & LVS  plantlet  and  A had  10  of  the  and less  taken  from  values For  7 of  37  absorbance values  of  For was  other  2).  and  the  other  observed  plant  from  parts,  in  B  a l l four  6  samples Only SS  plantlets, from  SS  5 of  than  1.0  of  tissues  30  samples  t i s s u e s , a l l from  greater  LS  with  f o r p l a n t l e t s D and  t h a n 0.10.  samples  lowest  plant.  range o f v a l u e s  tissues.  37  both  for that  lowest  & LVS  f o r samples  same p l a n t l e t s ( F i g u r e  A had  f o r samples  absorbance values LS  also varied  units.  46  20  30°C  10  0.5  1.0  1.5  2.0  ABSORBANC E(405nm)  F i g u r e 2. F r e q u e n c y h i s t o g r a m o f a r a b i s m o s a i c v i r u s d i s t r i b u t i o n as d e t e c t e d by E L I S A i n d i f f e r e n t p l a n t p a r t s d i s s e c t e d f r o m f o u r i n f e c t e d p l a n t l e t s : LS & LVS= s a m p l e s o f e l o n g a t e d s h o o t s w i t h l e a v e s a t t a c h e d , SS = s a m p l e s t a k e n from r a p i d l y p r o l i f e r a t i n g a d v e n t i t i o u s t i s s u e , R = samples of root t i s s u e .  AMV  was  ELISA was  detected  i n a l l R samples.  absorbance  values for tissue  0.015  on  the f i r s t  When compared, between  the f r e s h  same t y p e and example, range,  t h e r e were  2.0,  and  and  0.561  less  g gave  shoots.  and  and  20°C  LVS,  for  and  tested  different  B o t h AMV  and  LS & LVS  As  an  lower  i n LVS  The  The  highest was  GFLV  next  from e l o n g a t e d t i s s u e  virus Both  removed were  ELISA  the  detected where LS  and  absorbance  0.00.  a t 25°C  detected sample  growing  (Table i n LS,  o f LS &  lowest absorbance from  AMV  LS  from  in plantlets  one  g  were  t a k e n from  from p l a n t l e t s  samples and  a t 25°C  viruses  and  0.015  (Table V I ) .  i n samples  with  respectively.  f o u n d between  with only  The  the  1.0  weighing  0.125,  than f o r p l a n t l e t s  samples,  of  value.  t h r e e between  GFLV were more e a s i l y  n e g a t i v e f o r AMV.  samples  o f AMV  parts  to 0.06  growing  R samples  negative.  distribution was  and  being  i n SS  found  tissue  was  individually.  o b t a i n e d from h e a l t h y  The  VII).  was  was  of p r o l i f e r a t i n g  sampled  poorly  second.  plant  while tissue  part  plantlets  weight  plantlets  detection  tested  samples  reading  at  five  mean  gm  t h a n 2.0,  1.0,  the p l a n t  Virus  consistently LVS  the 0.05  samples  the  absorbance  v a l u e s o f 0.787 and  GFLV were d e t e c t e d  stems  within  than  on  of i n d i v i d u a l  the same r e l a t i o n s h i p  detection and  relationship  three  When the n e x t sampled,  apparent  values greater  two  from h e a l t h y  0.00  the o b s e r v e d ELISA A,  comparison,  d a t e and  weight  for plant  absorbance  no  By  AMV-infected  LVS  reading  T a b l e V I . D e t e c t i o n o f a r a b i s m o s a i c v i r u s (AMV) and g r a p e v i n e f a n l e a f v i r u s (GFLV) by ELISA i n v a r i o u s p a r t s o f f i v e p l a n t l e t s grown a t 25°C Positives Virus  Plant l e t  AMV  1 2 3 4 5  2  0/1 1/1 1/1 0/1 1/1  Totals Mean ELISA v a l u e s GFLV  LS  1 2 3 4 5  3/5 0.61 1/1 1/1 1/1 2/2 1/1  Totals Mean E L I S A v a l u e s  per plant  LVS  SS  0/1 1/1 1/1 0/1 0/1  2/2 1/1 2/2 3/3 2/2  part  1  R  Total  1/1 1/1 1/1 1/1 1/1  3/5 4/5 5/5 4/6 4/5  6/6 2/5 10/10 0 . 48 0 . 98 1 . 54 1/1 0/1 0/1 0/2 1/1  6/6 2/6 0 . 24 0 .07  2/2 2/2 2/2 1/1 3/3  21/26  1/1 1/1 1/1 1/1 1/1  5/5 4/5 4/5 4/6 6/6  10/10 5/5 0 . 46 0 . 44  23/27  N u m e r a t o r = p a r t s w i t h EL-ISA v a l u e s g r e a t e r t h a n 0.05. D e n o m i n a t o r = t o t a l number o f p a r t s t e s t e d . 2  L S = s h o o t s 20 mm o r l o n g e r w i t h l e a v e s removed. LVS = l e a v e s removed f r o m t h e above LS. SS = masses o f r a p i d l y p r o l i f e r a t i n g a d v e n t i t i o u s buds w i t h s h o o t s 10 mm or l e s s i n l e n g t h . R = root t i s s u e .  plantlets reading  was 0.15, f o u n d i n a LVS s a m p l e .  f o r any s a m p l e o f e l o n g a t e d  GFLV-infected  p l a n t l e t s was 0.21.  The  t i s s u e from  lowest  T a b l e V I I . D e t e c t i o n o f a r a b i s m o s a i c v i r u s (AMV) g r a p e v i n e f a n l e a f v i r u s (GFLV) by ELISA i n v a r i o u s 10 p l a n t l e t s grown a t 20°C Positives  Virus  AMV  Plantlet  1 2 3 4 5 6 7 8 9 10 Totals Mean ELISA  GFLV  1 2 3 4 5 6 7 8 9 10  Totals Mean E L I S A  LS  2  LVS  3/3  3/3  per p l a n t  LS &  LVS  _ _ _  part  SS  1/1  1/1  1/1 1/1  1/1 1/1  1/1 1/1 1/1  1/1  1/1  1/2 2/2  2/2 2/2 3/3 3/3 3/3 3/3 4/4 3/3 2/2 1/1  7/7 0 . 70  7/7 1 . 19  7/8 0 . 83  26/26 0.93  2/2  2/2  1/1  1/1  2/2 3/3 3/3 3/3 3/3 3/3 1/2 2/3 2/2 4/4  6/6 0 . 79  26/28 0.15  2/2 1/1 1/1 1/1 1/1 1/1 1/1 2/2 1/1  1/1 1/1 1/1 1/1. 1/1 2/2 1/1  10/10 0.35  10/10 0 . 57  1/1 1/1  and parts  of  1  R  Total!  1/1 1/1 1/1 1/1  9/9 4/4 6/6 4/4 6/6 7/7 6/6 4/4 6/7 3/3  1/1 1/1 1/1 1/1  8/8 0 . 42 1/1 1/1 1/1 1/1 1/1 1/1 1/1 0/1 1/1 1/1 9/10 0.14  55/56 7/7 6/6 5/5 6/6 6/6 6/6 5/6 5/7 7/7 8/8 61/64  'Numerator = p a r t s w i t h ELISA v a l u e s g r e a t e r t h a n 0 .05. D e n o m i n a t o r = t o t a l number o f p a r t s t e s t e d . = not t e s t e d . 2  L S = s h o o t s 20 mm o r l o n g e r w i t h l e a v e s removed. LVS = l e a v e s removed from t h e above LS. LS & LVS = c o m p l e t e l o n g s h o o t w i t h l e a v e s . SS = masses o f r a p i d l y p r o l i f e r a t i n g a d v e n t i t i o u s buds w i t h s h o o t s 10 mm o r l e s s in length. R = root t i s s u e .  Virus viruses fairly  d e t e c t i o n i n SS and R was  in plantlets easily  absorbance was  detected  value  taken  a t 20°C.  to d e t e c t  from  Whereas AMV  lowest  i n these  had a b s o r b a n c e v a l u e s  two o f t h e s e  lower  from  plantlets  0.1, w i t h nos.  5 and 8, a l s o  Increasing  less  than  room  This  o u t on AMV-  effect  temperatures  concentration  readings  readings  less  with  than  plantlets,  i n SS m a t e r i a l .  only  15°C t o 30°C o f ELISA  plantlets  w i t h i n 1 month.  i n the p r o l i f e r a t i o n  ELISA, a b s o r b a n c e  0.1  the R samples  of these  from  and G F L V - i n f e c t e d  came about  Of t h e  than  readings Two  GFLV  and BAP c o n c e n t r a t i o n .  a r e d u c t i o n i n mean a b s o r b a n c e carried  lower  had the low r e a d i n g s  of temperature growth  0.05.  ELISA  p l a n t l e t s (nos.  Also,  5, 8, and 10 had ELISA  one v a l u e  Effect  0.05.  (lowest  plant parts.  three GFLV-infected  than  was  f o r R = 0.19),  5, 7, and 8 ) , f i v e being  f o r t h e two  i n a l l SS and R samples  f o r SS = 0.37,  more d i f f i c u l t  SS samples  growing  different  caused  tests (Figure 3).  I n c r e a s i n g the BAP  medium  affected  in plantlets  t h e mean  growing  a t 20°C  (Figure 4 ) . In 0.05  the f i r s t  absorbance u n i t s ,  plantlets (Figure 25°C,  growing  5).  0.097.  Even  30°C w i t h  using a threshold value of  was e a s i l y  detected  had s u f f i c i e n t  virus  absorbance value  the v i r u s  i n a l l the  r e g a r d l e s s o f BAP c o n c e n t r a t i o n  though a b s o r b a n c e v a l u e s  The l o w e s t  However,  AMV  a t 20°C,  a l l plantlets  detection.  at  experiment,  were  titres for  found  was u n d e t e c t a b l e  a l l 75 p l a n t l e t s  having  lower a t  a t 25°C  i n any  was  plantlets  absorbance values  below  51  Figure 3 . Mean ELISA a b s o r b a n c e v a l u e s w i t h one s t a n d a r d d e v i a t i o n f o r t e s t s on a r a b i s m o s a i c v i r u s (AMV) and grapevine fanleaf virus (GFLV)-infected p l a n t l e t s following g r o w t h a t d i f f e r e n t a i r t e m p e r a t u r e s and 2 mg/1 N - 6 - b e n z y l a m i n o p u r i n e : A - f i r s t e x p e r i m e n t on A M V - i n f e c t e d p l a n t l e t s , 2 months a f t e r s t a r t ; B - s e c o n d e x p e r i m e n t on A M V - i n f e c t e d p l a n t l e t s , 1 month a f t e r s t a r t ; C - s e c o n d e x p e r i m e n t , 2 months a f t e r s t a r t ; D - G F L V - i n f e c t e d p l a n t l e t s , 1 month a f t e r s t a r t .  52  Figure 4. Mean ELISA a b s o r b a n c e r e a d i n g s w i t h one s t a n d a r d d e v i a t i o n o f t e s t s on a r a b i s m o s a i c v i r u s i n f e c t e d p l a n t l e t s grown a t t h r e e N - 6 - b e n z y l a m i n o p u r i n e (BAP) c o n c e n t r a t i o n s f o r 2 months a t 2 0 ° C : A - f i r s t e x p e r i m e n t ; B - s e c o n d experiment.  F i g u r e 5. F r e q u e n c y h i s t o g r a m o f ELISA a b s o r b a n c e r e a d i n g s f r o m t e s t s on a r a b i s m o s a i c v i r u s - i n f e c t e d p l a n t l e t s grown f o r 2 months a t t h r e e N - 6 - b e n z y l a m i n o p u r i n e (BAP) c o n c e n t r a t i o n s (2, 4, and 8 mg/l) w i t h i n each o f t h r e e t e m p e r a t u r e s (20°C, 25°C, and 3 0 ° C ) .  54  0.02 and n i n e o f t h e s e h a v i n g v a l u e s below from and with at  healthy  0.00.  Samples  plantlets  s u b m i t t e d t o the same  temperatures  BAP c o n c e n t r a t i o n s  had a mean a b s o r b a n c e  v a l u e o f 0.005  a maximum v a l u e o f 0.02.  30°C The  were a l l below analysis  experiment  (Table  temperature  ELISA  0.05 a b s o r b a n c e  of variance  VIII) indicates  effect  on v i r u s  units.  of the r e s u l t s  for this  an i n t e r a c t i o n  and BAP c o n c e n t r a t i o n .  a f a r greater  readings f o r p l a n t l e t s  between  However, t e m p e r a t u r e has  detection  t h a n BAP.  T a b l e V I I I . Mean ELISA a b s o r b a n c e v a l u e s and a n a l y s i s o f v a r i a n c e o f t e m p e r a t u r e and N - 6 - b e n z y l a m i n o p u r i n e (BAP) e f f e c t on a r a b i s m o s a i c v i r u s d e t e c t i o n ( f i r s t e x p e r i m e n t )  BAP (mg/1) Temperature  2  4  8  20 25 30  0 .80 , 0 .40 . 0 . 00  0 .. 84 0 . 42 0 . 00  0 .96 0 .. 37 0 . 00  Mean  0 . 40  0 .42  0 . 44  Sources of v a r i a t ion  df  SS  Plantlet Temperature BAP Temperature  24 2 2 4  0. 59 27.83 0 . 07 0.31  x BAP  Mean 0 . 86 0 . 40 0 .00  P>F 1.95 1112.50 2.82 6.25  0 . 0071 0.0000 0.0618 0.0001  Regression concentration absorbance %  o f 2 mg/l y i e l d e d a l i n e a r  units  reduction  the  a n a l y s i s on t h e t e m p e r a t u r e  sums o f s q u a r e  BAP c o n c e n t r a t i o n  (Figure effect  increase  6).  (Table  resulted in a  only  equation  with  a t 20°C o f BAP  absorbance =  in total  sums o f  (Table IX).  Similar temperature with  Increasing  statistically  0.738 + 0.027 BAP f o r a 15.3% r e d u c t i o n squares  ( T ) f o r a 90.2  a n a l y s i s on t h e r e s u l t s  gave a l i n e a r  a t a BAP where  IX).  in virus concentration  Regression  a t 20°C  equation  = 2.381 - 0.079 t e m p e r a t u r e  in total  significant  effect  results on ANV  the s e c o n d  was r e d u c e d  on t h e e f f e c t  concentrations  experiment  o f growth  i n p l a n t l e t s were  (Table  X).  Virus  i n p l a n t l e t s as t e m p e r a t u r e  after  only  1 month.  Analysis  after  1 month i n d i c a t e d t h a t  room  was  of variance  concentration increased,  BAP c o n c e n t r a t i o n  had no  effect  on v i r u s d e t e c t i o n a t 20°C  temperature  effect  a t 2 mg/l o f BAP was s i g n i f i c a n t  ELISA for  Regression absorbance  analysis fitted  Although  statistically  plantlets,  temperature  individual  plants  plantlets While less  in total  after  a t a BAP c o n c e n t r a t i o n  than 0.05, u n l i k e  f o r t h e group o f  d e t e c t i o n by ELISA i n for a l l  o f 2 mg/l ( F i g u r e 7 ) .  a t 30°C  the f i r s t  equation  (Table I X ) .  1 month was n o t u n i f o r m  a l l p l a n t l e t s growing  detected  on AMV  (P =  t o the d a t a  2  sums o f s q u a r e s  significant  effects  but t h a t  the q u a d r a t i c  = 8.866 - 0.597 T + 0.010 T  a 52.9% r e d u c t i o n  even  o f the r e s u l t s  significant  0.01).  obtained  had a b s o r b a n c e  experiment,  i n a l l p l a n t l e t s a t the two lower  AMV  values  was n o t  temperatures.  56  Table IX. Regression analyses on the effects of temperature and N-6-benzylaminopurine (BAP) concentrations on arabis mosaic virus (AMV) and grapevine fanleaf virus (GFLV) detection by ELISA i n p l a n t l e t s Sources of variation  d.f.  SS  % reduction i n t o t a l SS  A) AMV - experiment 1, BAP effect at 20°C Linear Residual  1 73  0.34 1.89  15.3  Regression equation: absorbance = 0.738 + 0.027 BAP, R  = 0.392  2  B) AMV - experiment 1, temperature e f f e c t , 2 months at 2 mg/1 BAP Linear Residual  1 73  7.85 0.85  90.2  Regression equation: absorbance = 2.381 - 0.07926 T, R  = 0.95  2  C) AMV - experiment 2, temperature e f f e c t , 1 month at 2 mg BAP Linear Quadratic Residual  1 1 72  11.17 1.05 8.88  52.9 57.9  Regression equation: absorbance = 8.866 - 0.59688 T + 0.01005 T , R = 0.761 2  2  D) AMV - experiment 2, temperature e f f e c t , 2 months at 2 mg/1 BAP Linear Quadratic Residual  1 1 81  58.41 2.15 28.00  66.0 68.4  Regression equation: absorbance = 7.362 - 0.437255 T + 0.006402 T , R = 0.827 2  2  E) AMV - experiment 2, BAP e f f e c t , 2 months at 20°C Linear Residual  1 70  6.22 29.30  17.5  Regression equation: absorbance = 0.923 + 0.1179 BAP, R  2  = 0.419  F) GFLV - e f f e c t of temperature after 1 month Linear Quadratic Residual  1 1 57  3.35 0.80 8.53  39.3 48.6  Regression equation: absorbance = 7.606 - 0.54658 T + 0.009774 T , R = 0.697 2  2  57  1.0  <  0.0-  •  BAP(mg/L)  F i g u r e 6. Mean ELISA a b s o r b a n c e v a l u e s from t h e f i r s t e x p e r i m e n t t o d e t e r m i n e the e f f e c t s o f g r o w i n g a r a b i s m o s a i c v i r u s - i n f e c t e d a t t h r e e t e m p e r a t u r e s f o r 2 months on a medium c o n t a i n i n g v a r i o u s N - 6 - b e n z y l a m i n o p u r i n e (BAP) c o n c e n t r a t i o n s (» — » = 20°C, + + = 25°C, • •= 3 0 ° C ) .  58 T a b l e X. Mean a b s o r b a n c e v a l u e s o f ELISA t e s t s c a r r i e d o u t on a r a b i s m o s a i c v i r u s - i n f e c t e d p l a n t l e t s 1 and 2 months i n t o the s e c o n d e x p e r i m e n t t o t e s t the e f f e c t o f t e m p e r a t u r e and N - 6 - b e n z y l a m i n o p u r i n e (BAP) c o n c e n t r a t i o n on AMV t i t r e  BAP c o n c e n t r a t i o n Temperature (°C)  (mg/l) .  2  4  8  1 month 20 25 30  0.,95 0 . 23 0, . 00  1.. 00  1 . 07  B) 2 months 15 20 25 30  2 . 24 1 .. 20 0 .44 0 .01  2.. 49 1..35  A)  i  not  negative  ELISA  absorbance value  results  values less  found  after  at  20°C  and  1 month and t h r e e  than 0.1.  a t 30°C  tissue  was  after  gave  more had  By c o m p a r i s o n ,  growing  value of healthy  25°C  the h i g h e s t 0.01.  1 month  The was  w i t h a maximum v a l u e o f 0.01. The  detection second  inhibiting by ELISA  experiment  undetectable plantlet  effect  of higher temperatures  a t 2 mg/l o f BAP was after  2 months  20°C,  (Figure  i n 24 o f 25 p l a n t l e t s  h a v i n g an a b s o r b a n c e  however, was 15°C,  " ~  growing  i n the p l a n t l e t s  mean a b s o r b a n c e 0.00  2.33 1 .90  tested, a l l the p l a n t l e t s  TweIve o f  . - 1  also and  Mean v i r u s  still 8).  a t 30°C,  on  evident AMV  AMV i n the  was  w i t h the o t h e r  v a l u e o f 0.13.  The  u n d e t e c t a b l e i n some p l a n t l e t s  virus, growing at  25°C. concentration  was g r e a t e s t  in plantlets  20  20° C  0 J 20  El. BEal  f  ? f  |  J  in  i—  Z  <  CQ  s  D Z 0 20  ABSORBANCE (405 nm) F i g u r e 7. Frequency histogram of a r a b i s mosaic v i r u s d e t e c t i o n by ELISA t e s t s on p l a n t l e t s grown f o r 1 month 20°C, 25°C and 30°C on a medium c o n t a i n i n g 2 mg/1 o f N-6-benzylaminopurine.  60  26 15° C  20° C  Z <  u. 0 °  I  *4  I I | • I l • | • • • l | l l l T |  26 25° C  1 f t , . , 30°  i  1 1 1  * i  C  s D Z  - T" 2.0 p  r  i 1.0 1  3.0  0.0  1  • i • • •' i • • • i 2.0 1  1  3.0  A B S O R B A N C E (405nm)  F i g u r e 8. Frequency histogram o f a r a b i s mosaic v i r u s d e t e c t i o n by ELISA i n p l a n t l e t s grown f o r 2 months a t 15°C, 20°C, 25°C, and 30°C on a medium c o n t a i n i n g a N - 6 - b e n z y l a m i n o p u r i n e c o n c e n t r a t i o n o f 2 mg/l - s e c o n d e x p e r iment.  61 g r o w i n g a t 15°C ( T a b l e X ) . non-infected  plantlets  maximum v a l u e  was 0.00 a f t e r  reading  from  2 months w i t h a  o f 0.02.  Analysis indicated  The mean ELISA  o f v a r i a n c e on t h e r e s u l t s  after  2 months  t h a t BAP c o n c e n t r a t i o n had no s i g n i f i c a n t  at  15°C b u t was  significant  (P = 0.01) a t 2 0 ° C .  of  BAP on v i r u s  c o n c e n t r a t i o n a t 20°C was b e s t where ELISA  The e f f e c t fitted  linear  equation  (Table  I X ) , f o r a 17.5 % r e d u c t i o n i n t o t a l sums o f  When t e m p e r a t u r e was a n a l y s e d best  effect  by a q u a d r a t i c e q u a t i o n  In still  reducing  comparing  tested  negative  a t 1 month.  i n two o t h e r  0.05 a f t e r  1 month.  original  plantlets  observed  all  plantlets  The mean ELISA  tested  a t each tissue  with  fitted  =7.362 by 68.4 %. was  w h i c h had  o r i g i n a l values  below  w i t h an  t o 0.01.  reduced  the lowest  BA  became  one p l a n t l e t  also  While  growing  GFLV d e t e c t i o n  absorbance  value  a t 20°C was 0.12, o n l y two  above 0.1 a f t e r  absorbance  temperature having  were  in six plantlets  o f 0.76 d r o p p e d  b u t one had v a l u e s  30°C.  healthy  Conversely,  (Figure 9).  had v a l u e s  squares.  1 and 2 months, AMV  However, v i r u s  the temperature  in plantlets  plantlets  after  plantlets  absorbance value  Increasing  the r e s u l t s  where a b s o r b a n c e  a t 2 months,  detectable  = 0.923 + 0.118 BAP  t h e t o t a l sums o f s q u a r e s  the r e s u l t s  undetectable,  by a  a t a BAP c o n c e n t r a t i o n o f 2 mg/1  by r e g r e s s i o n a n a l y s i s ,  0.437 + 0.006,  in  absorbance  effect  growing  a t 25°C, and  below 0.02 a f t e r readings  growth a t  for plantlets  were 0.58, 0.05, and 0.01; w i t h  a mean a b s o r b a n c e v a l u e  o f 0.01 and a  62  F i g u r e 9. Frequency histogram of grapevine f a n l e a f v i r u s d e t e c t i o n by ELISA i n p l a n t l e t s grown f o r 1 month a t 20°C, 25°C, and 30°C on a medium c o n t a i n i n g a N - 6 - b e n z y l a m i n o p u r i n e c o n c e n t r a t i o n o f 2 mg/1.  maximum v a l u e o f 0.02.  The  7.606 - 0.547 T + 0.01 reduction  in total  Virus  T ,  quadratic fitted  2  the r e s u l t s  sums o f s q u a r e s  recovery.  AMV  was  (Table  previously  the  t e m p e r a t u r e s w h i l e i t was  each  test  date  i n the c o n t r o l  exposed  t o 30°C.  control  plants  AMV  was  produced AMV  was  with  growing  controls  i n any  and  an a v e r a g e  8 days.  v a l u e s dropped  The  growing  biggest  a t 20°C.  plantlets readings  from  of  leaves  ground  absorbance  100  aborbance and  or g r e a t e r .  By  i n 10 volumes  throughout  ELISA  e x c e p t one  day,  two  20°C  comparison,  after  previously  only  from  diluted  0.00  rapidly  in plantlets  five  from  a  of the  had  control  i n C.  quinoa  of g r i n d i n g  buffer  1.02,  1.54,  and  2.04  units  dates.  The  mean  ELISA,  test  readings for tissue  stable  and  ng/ml o f pure v i r u s  f o r the f o u r  reduced  (Table X I ) .  the t w e n t y - f i r s t group  control  v a l u e of  in a l l plantlets  r e a d i n g s o f 1.2,  respectively,  a t 30°C  was  plants However,  Samples  absorbance  drop o c c u r r e d  t h e 25°C  o f 0.05  sample  By  from  25°C.  detection  the p l a n t l e t s  plantlets.  produced  Effect  absorbance  on  previously  o f the g r e e n h o u s e  o f 0.0008.  placing  detected  25°C.  shoots of t r e a t e d  AMV  of  t h e y were moved t o  not  t h a n at  a t b o t h 20°C  o f 30°C.  X  v a l u e s were h i g h e r i n the  in a l l plants  had  i n any  repeatedly  plantlets  at 20°C  not d e t e c t e d  after  a standard deviation  after  low  ELISA  growing  detected  healthy  The  from r o o t e d  plantlets  grown a t 30°C  f o r a 48.6  IX).  never d e t e c t e d  plantlets lower  equation, absorbance  from h e a l t h y  the t e s t i n g  had  plantlets  period.  were  T a b l e X I . R e s u l t s o f ELISA t e s t s v i r u s - i n f e c t e d p l a n t l e t s exposed  on a r a b i s m o s a i c t o 30°C f o r t h r e e weeks  Mean a b s o r b a n c e (days from s t a r t ) Original temperature Plantlet  0  8  14  1 2 3 4 5 6 7 8 Healthy  0. 20 0 ,. 28 0 .. 43 0 . 49 0 .. 46 0 .91 . 0 ,. 38 0 .,48 0 ,. 00  0 , 07 0 ,. 1 5 0 ,. 04 0 .. 27 0 .. 44 0 .. 1 1 0 ,. 23 0 .. 17 0 . 00  0 . 08 0 . 08 0 .03 0 . 04 0 .03 0 . 09 0 . 12 0 . 06 0 . 00  0 .03 0 . 02 0 . 03 0 .05 0 .01 0 . 03 0 .05 0 . 03 0 .01  1 . 34 1 .01 . 1 . 49 1 .,36 2 . 02 2 . 62 0 .75 1 . 18 0 . 00  0 .21 0 ., 1 5 0 . 29 0 . 19 0 . 52 0 .29 0 . 14 0 . 36 0 .01  0 .07 0 .05 0 . 09 0 . 04 0 . 10 0 . 18 0 . 10 0 . 08 0 .01  0 . 06 0 . 06 0 . 02 0 . 04 0 .05 0 .05 0 . 03 0 . 12 0 .02  1 2 3 4 5 6 7 8 Healthy  Virus  detection  The field  in field  grinding  plants  buffers  and s c r e e n h o u s e  t h e summer  When c o m p a r i n g testing  date using  were s i m i l a r  u s e d on l e a f  o f AMV  f o r t h e two v i r u s e s  given  f o r each v i r u s  from  effect  (P  harvest  (Table X I I ) .  the p r o t e c t e d  absorbance  taken  and GFLV a t e a c h  the mean ELISA  ELISA  samples  g r a p e v i n e s had a s i g n i f i c a n t  = 0.0001) on the d e t e c t i o n throughout  21  absorbance  LSD p r o c e d u r e , t h e r e s u l t s (Table XIII).  v a l u e s and t h e mean at each  v a l u e s on e a c h  testing  The mean  standard error date.  (MSE) i s  The means  T a b l e X I I . A n a l y s i s o f v a r i a n c e o f ELISA a b s o r b a n c e v a l u e s o b t a i n e d by u s i n g d i f f e r e n t g r i n d i n g b u f f e r s t o d e t e c t g r a p e v i n e f a n l e a f v i r u s (GFLV) and a r a b i s m o s a i c v i r u s (AMV) i n i n f e c t e d f i e l d p l a n t s t h r o u g h o u t t h e summer  est  date  GFLV June 3  Source  df  SS  Plants Buffer Error  24 3 72  24 . 6 8.3 3. 1  24 . 06 65. 27  0 .0001 0 .0001  F  Pr  > F  June  24  Plants Buffer Error  31 3 93  3.9 3. 3 1. 5  7 .96 70 . 51  0 .0001 0 .0001  July  15  Plants Buffer Error  31 3 93  3.3 4. 5 1. 4  7 .42 103 . 28  0 .0001 0 .0001  Plants Buffer Error  31 3 93  24 . 6 20 . 5 6.6  1 1 .23 96. 78  0 .0001 0 .0001  Plants Buffer Error  31 3 93  24 . 3 18.5 6.6  1 1 .03 89 . 46  0 .0001 0 .0001  Plants Buffer Error  13 3 39  4.8 4.8 1.9  7 .73 33 . 14  0 .0001 0. 0001  August  5  September  2  AMV June 3  June  24  Plants Buffer Error  15 3 45  2. 7 3. 5 0.9  9 .19 59 .74  0 .0001 0 .0001  July  15  Plants Buffer Error  15 3 45  3. 0 1. 0 1. 0  8 .90 13 .96  0 .0001 0 .0001  Plants Buffer Error  15 3 45  3. 4 13.1 2.0  5. 07 97 . 14  0 .0001 0 .0001  Plants Buffer Error  15 3 45  8.3 14.4 2.7  9 .10 78. 94  0. 0001 0 .0001  August  5  September  2  66  T a b l e X I I I . E f f e c t o f d i f f e r e n t g r i n d i n g b u f f e r s on t h e E L I S A d e t e c t i o n o f a r a b i s m o s a i c v i r u s (AMV) and g r a p e v i n e f a n l e a f v i r u s (GFLV) i n i n f e c t e d f i e l d p l a n t s Mean a b s o r b a n c e v a l u e  Buffer  June  3  A) GFLV A B C D  1 .31 1 .28 0 . 59 1 ,12  a a c b  LSD MSE  0 . 12 0 .043  B)  1  3  2  per t e s t i n g  date  June 24  July  15  Aug. 15  Sept. 2  0 0 0 0  0 0 0 0  b a d c  0 .84 1 .03 0 .10 1 .11  0. 58 0 .93 0 .15 1 .13  .54 .54 .17 .56  a a b a  .52 .60 .11 . ,46  0 .06 0 ,016 .  0 .06 0 ,015 .  0 . 13 0 .071  0 , 70 b 0 .84 a 0 .32 . c 0 ,93 , a  0 .40 . b 0, . 53 a 0 .23 , c 0 ,. 52 a  0 .87 1 .13 0 .13 1 . 33  0 . 10 0 .019  0 . 11 0 . 023  0 .15 0 .045  b a c a  c b d a  0 .13 0 .069  AMV 1 .16 1 .27 0 . 52 1 .09  A B C D  ab a c b  0 .17 0 . 048  LSD MSE  c b d a  0 1 0 1  .76 .28 .21 .41  b a c a  0 .18 0 .061  ' R e f e r s t o the f o l l o w i n g g r i n d i n g b u f f e r s : A - 0.15 M p h o s p h a t e b u f f e r e d s a l i n e ( P B S ) , pH 8.4 w i t h 0.05% Tween-20, 0.2% o v a l b u m i n and 2 % p o l y v i n y l p y r r o l i d o n e ; B - b u f f e r A w i t h 1% n i c o t i n e , pH 8.4.; C - s t a n d a r d g r i n d i n g b u f f e r - 0.01 M PBS, pH 7.4, w i t h 0.05% Tween-20, 0.2% o v a l b u m i n and 2 % p o l y v i n y l p y r r o l i d o n e ; D - b u f f e r C w i t h 1% n i c o t i n e , pH 8.2. ^ A b s o r b a n c e means f o l l o w e d by t h e same l e t t e r a r e n o t s i g n i f i c a n t l y d i f f e r e n t a t P = 0.05 w i t h i n e a c h h a r v e s t u s i n g t h e LSD v a l u e f o r t h a t harvest. MSE = mean s t a n d a r d e r r o r . 3  followed using  by the same  the l e a s t  harvest  date  obtained buffer test  (P = 0.05).  (buffer  are not s i g n i f i c a n t l y  significant difference  by g r i n d i n g  date.  letter  C) was  The mean a b s o r b a n c e  foliar  samples  inferior  In t h e e a r l y  indicated  different f o r each  values  i n standard  grinding  f o r both v i r u s e s  on e v e r y  summer,  t h e mean a b s o r b a n c e  values  67  obtained  f o r t h e two 0.15 M PBS b u f f e r s (A and B) were  superior  t o the s t a n d a r d  1% n i c o t i n e  grinding buffer modified  and i n c r e a s i n g  advantage being  gained  t h e pH; w i t h  by a d d i n g  no  nicotine  by  adding  significant t o t h e 0.15 M PBS  buffer.  However, as t h e summer p r o g r e s s e d ,  nicotine  t o t h e 0.15 M PBS b u f f e r i n c r e a s e d t h e a b s o r b a n c e  values  significantly  over  buffer.  Also, adding  standard  grinding buffer,  they  were e q u a l  nicotine  nicotine,  buffer  were e q u a l l y s e n s i t i v e  plants  ( i . e . with  on not  July  three  dates  especially  the r e s u l t s those  than  i n at l e a s t  Using  the e x c e p t i o n the standard  less  later  sensitive  (Table XV).  detected  as c o n s i s t e n t l y  as AMV  buffer,  i n 141 u s i n g  using the  the s t a n d a r d  standard  was d e t e c t e d  o f one p l a n t  buffer,  AMV  was  i n t h e summer,  when t h e s t a n d a r d GFLV was, however, n o t  p l a n t s d u r i n g the  i n 134 u s i n g  t h e same b u f f e r w i t h  grinding buffer,  b u f f e r with  in infected  when t h e o t h e r b u f f e r s were  on G F L V - i n f e c t e d  the v i r u s  dates.  plants.  b u f f e r was u s e d  summer,  with the  g r e a t e r than the  to detect  i n the screenhouse  Of 153 t e s t s  that  the s t a n d a r d g r i n d i n g  grinding  used.  such  one p l a n t on e a c h o f t h e f i r s t  and was h a r d e r  GFLV d e t e c t i o n was  t h e pH o f t h e  two t e s t i n g  i n d e t e c t i n g AMV  t h r e s h o l d ) , with  0.15 M PBS  obtained  on t h e l a s t  absorbance values  15 ( T a b l e X I V ) .  detected  and i n c r e a s i n g  improved  three b u f f e r s other  pre-determined  o f the s t a n d a r d  to or b e t t e r than  0.15 M b u f f e r w i t h All  those  the a d d i t i o n of  IX n i c o t i n e  t h e 0.15 M PBS  IX n i c o t i n e ,  87  and i n 139 p l a n t s u s i n g and i n c r e a s e d pH.  68  Table XIV. Detection o f a r a b i s mosaic v i r u s by ELISA throughout the summer i n f i e l d and screenhouse p l a n t s using d i f f e r e n t g r i n d i n g b u f f e r s '  Variety  Plant  June 3 A B C D  June 24 A B C D  • • • • + + + •  • • +*• • + + . + + • * • + + + • . • •  a  J u l y IS A B C D  Aug. 12 A B C D  Sept. 2 A B C D  + + + + +• + + + • • + + + + + • + + • » • + + + + + • + • . . . .  + + + . + + + + .  • • • •.  • • • •  + • . •  . . • •  •  •  •  +  +  -  •  •  +  -  •  . • • •  . . + + •  + . • . +  _ . +  . . + . +  . . + . .  . . . . .  . . .  . . • . .  A) F i e l d p l a n t s St. George M M M  " M M  LN-33 P i n o t Noir  1 2 3 4 5 6 7 8 9  + + + + • •  . + + + + •  • • + + + + •+ • •  + + + • + + + •  • + • + • • • + •  • + + + + + + • .  - • + + + + . . -+ -+ -+ . + • +  • • • « . • + ••. + • • + • • • + + . • + • + + . + + + • + + • .  B) Screenhouse p l a n t s Unknown #1 H  M  Unknown »2 n M  Rotberger  10 \\  12 13 14 15 16  TOTALS  •• • • • +  +  -  •  • • • • • • • • + • • + 14 14 12 14  . • • +  +  • . • •  +  •  • • + + • • + +  16 16 15 16  • + • •  •  • • • +  •  . + + +  IS 15 14 15  16 16 7 16  16 16 11 16  * ( • ) • p o s i t i v e , (-)* negative. Blank spapes were untested. 'Refers to the f o l l o w i n g g r i n d i n g b u f f e r s : A - 0.1S M phosphate buffered s a l i n e <PBS). pH 8.4 with 0.05% Tween-20. 0.2X ovalbunin and 2% p o l y v i n y l p y r r o l i d o n e ; B - b u f f e r A with IX n i c o t i n e . pH 8.4.; C - 0.01 M PBS, pH 7.4, with 0.05% Tween-20, 0.2% ovalbumin and 2% p o l y v i n y l p y r r o l i d o n e ; D - b u f f e r C with 1% n i c o t i n e . pH 8.2.  69  Table XV. Detection of grapevine f a n l e a f v i r u s by ELISA throughout the suoaer i n f i e l d and screenhouse p l a n t s using d i f f e r e n t g r i n d i n g b u f f e r s 1  June 3 A* B C D  June 24 A B C D  J u l y 15 A B C D  Aug. 12 A B C D  4  + + + + + + + • + • • +  + • - • - - - + • + •  - - - -  *•  1 2 3  • + • + - • - - - -  • + - + + • - • + .+- 4 .  **  5 5  • +  •  + +  7  + • • + . •  3 9  • +  Variety  Plant  Sept. 2 A B C D  A) F i e l d plants St. George * « N  LN-33 M  **  + • • + + +  + +  • +  + +  • •  + +  • +  \Q  "  11  •  +  • +  "  12  +  +  +  •  •  M  13  +  4  +  •  4  n  14  •  H  15  4  *  4  4  *  • •  *  • • • •  *  * . *  + • - •  •  +  •  •  4  4  +  4  -  + • •  "  4  - - - -  -  + •  4  •  4  *• *  •  4  _  +  • +  +  4  • + + • •  + +  4  4  +  •  +  -  4  -  4  4  •  +  +  +  +  •  -  4  4  4  + + 4  4  +  4  +  •  +  + • • - +  +  + + + +  +  *  -  4  •  -  4  4  4  • +  4  4  4  + +  +  +  4  4  •  •  4  4  4  4  4  4 4  4  + •  4  4  4  4  +  +  •  4 +  4  •  -  4  +  4  -  +  +  4  + +  +  +  -  + • .  +  4 •  4  .  4  4  4  4  •  4  B) Screenhouse p l a n t s Riesling  16  4  +  17  H  +  + +  + +  + •  lg  H  PinOt Chardonnay ** St. George " "  19 20 21 22 23  + + +•• • • • • • + • • + + - •  24  St. George H  +  4  4  •  +  •  •  •  •  4  -  -  -  -  -  4  +  +  -  *  4  -  • + • •  4  4  -  4  4  4  -  4  4  4  4  - +  4  4  -  4  4  4  4  *  4  -  *  -  •  •  •  •  28  + • - •  H  29  4  •  4  4  4  •  31  *•  32 TOTALS  4 * - +  25 25 23 25  (-)-negative.  +  4  •  + 4 •  *  •  •  ,  4  4  •  +  4  *  -  4  31 31 23 31  4  4  *  -  + 4  -  +  4  4  -  +  +  4  4  4 •  •  . *  -  -  + +  4  •  +  4  -  -  4  4 •  -  *  4  4  .  4  -  4 4 4  -  -  -  4  4  4  -  4 +  +  4  4  -  - + 4  4  *  4  4  *  *  *  4 4  19 26 11 24  4  4 *  4  -  4  4  4 -  4  + 4  4  4  •  + • + -  +  *  *  •  4  -  4  30  4  4  4  *  "  4  4  4  4  +  4  • 4  4  4  *  +  4  + 4  4  4  4  4 4  4 4+ * -  4  +  + 4  + + • + -  •  •  4  +  • • • • •  +  -  26  +  .  4 • • 4 4 + 4 4 • + • •  **  '(••(•positive  • -  25  Unknown # 1 2 7 Unknown #2  -  •  4  30 30 15 30  4  4  .  4  +  -  *  4  -  4  29 29 15 29  Blank spaces were not tested.  'Refers t o the f o l l o w i n g g r i n d i n g b u f f e r s : A - 0.15 M phosphate b u f f e r e d s a l i n e (PBS), pH 8.4 with 0.05% Tween-20, 0.2% ovalbumin and 2% p o l y v i n y l p y r r o l i d o n e ; B -1 b u f f e r A with 1% n i c o t i n e , pH 8.4; C - 0.01 H PBS, pH 7.4, with 0.05% Tween-20, 0.2% ovalbuain and 2% p o l y v i n y l p y r r o l i d o n e ; D - b u f f e r C with 1% n i c o t i n e , pH 8.2.  70  The  three  most  all  dates  except  and  e s p e c i a l l y the 0.15  increased  sensitive buffers  ELISA  undetectable Chardonnay  for July  Until  July  GFLV d e t e c t i o n  was  On  date,  the r e a d i n g s  George p l a n t s .  difference effect  This  healthy  all  S t . George  plants  while  became  using  buffer  f o r the three  was u s e d .  from  the S t .  less This  varietal  v a r i a t i o n had an l e v e l s which on J u l y  became  affected  15.  When  by v a r i e t y , three  p o s i t i v e when t h e  By c o m p a r i s o n , the field  plants.  Also,  were u s e d , t h e p o s i t i v e  sufficiently  better  samples  p o s i t i v e f o r the  do n o t change f o r t h e LN-33  dropped  from  (Table XVI).  separately  4 of 6 plants  o f s c r e e n h o u s e mother p l a n t  positive  those though  greater  means were used  field  grinding  level  taken  LN-33  two t e s t i n g d a t e s .  when t h e S t . George c o n t r o l s  value  than  i n t h e S t . George p l a n t s  control  buffers,  threshold  a Pinot  less r e l i a b l e  from  continued,  and t h e a s s o c i a t e d  the  results  became  o f GFLV. As t h e  o f samples  obtained  trend  on t h e l a s t  detection  standard  (no.20),  strain  on s e t t i n g p o s i t i v e t h r e s h o l d  better  nicotine,  o f the two v a r i e t i e s t e s t e d  f o r GFLV were much h i g h e r  pronounced,  buffers,  15, GFLV  became  on  not much d i f f e r e n c e between  absorbance values  controls  GFLV  a "severe"  15, t h e r e  healthy  tested  As o f J u l y  well  buffer.  mean ELISA  that  with  i n one s c r e e n h o u s e p l a n t  standard  the  M PBS b u f f e r  i n f e c t e d with  equally  15, when b o t h n i c o t i n e  sensitivity.  summer p r o g r e s s e d , the  performed  f o r the a b s o r b a n c e  no.20  buffers  .  t o become  barely  71  T a b l e XVI. Mean ELISA a b s o r b a n c e v a l u e s o f h e a l t h y c o n t r o l p l a n t s g r o u n d i n v a r i o u s b u f f e r s t h r o u g h o u t t h e summer GFLV ELISA test date  Buffer  June 3  2  LN-33  1  controls  AMV  S t . George  LN-33  controls  S t . George  A B C D  0.01 0.01 0 . 02 0.01  0 .01 0 . 00 0 . 00 0•  0 . 00 0.00 0.01 0.01  0 0 0 0  0  0  .00 .01 .00 .00  June  24  A B C D  0 . 02 0 .02 0.01 0 . 00  0 .01 0 .01 0 . 00 0 .01  0.01 0.00 0 . 00 0.01  0 .00 0. 00 0 .01 0 .00  July  15  A B C D  0 . 20 0.19 0 .03 0.14  0 .01 0 . 02 -0 . 02 0 .01  0.03 0 . 02 0.01 0.01  -0 .01 0 .00 0 .00 -0 .01  A B C D  0 . 04 0 . 04 0.01 0.05  0 .01 0 .01 0 .00 0 .01  0. 0. 0. 0.  A B C D  0 . 04 0 . 04 0.02 0 . 04  0 .02 0 . 02 0 . 00 0 . 02  0.01 0.01 0.01 0.01  August  15  September 2  = grapevine  fanleaf  v i r u s . AMV  02 02 00 03  = arabis  0 0 0 0  .02 .00 .00 .01  0 0 0 0  .00 .01 .00 .00  1  GFLV  mosaic  virus  2  R e f e r s t o the f o l l o w i n g g r i n d i n g b u f f e r s : A - 0.15 M p h o s p h a t e b u f f e r e d s a l i n e (PBS), pH 8.4 w i t h 0.05% Tween-20, 0.2% o v a l b u m i n and 2 % p o l y v i n y l p y r r o l i d o n e ; B - b u f f e r A w i t h 1% n i c o t i n e , pH 8.4; C - s t a n d a r d g r i n d i n g 0.01 M PBS, pH 7.4, w i t h 0.05% Tween-20, 0.2% o v a l b u m i n and 2 % p o l y v i n y l p y r r o l i d o n e ; D - b u f f e r C w i t h 1% n i c o t i n e , pH 8.2.  72 DISCUSSION  Virus  detection  Although widely been  used  in tissue-cultured plantlets  t h e ELISA  to d e t e c t  few r e p o r t s  recommended  as s o u r c e s  culture,  plants,  of t e s t i n g material  a labour  there being  f o r ELISA  1985,1986).  and r e s o u r c e - i n t e n s i v e  method  f o r improving  have  In  vitro  procedure,  virus  detection  g r a p e v i n e s by E L I S A . ELISA  although This  detection  possible,  study  failure  dates within  ELISA  plantlets reliably  This  there  grown a t 25°C  rather  the v a r i a b i l i t y  20°C and 25°C.  this  than  indicated  showed  i n short  The  at d i f f e r e n t  When p l a n t l e t s  and t h e p a r t s  tested  i n absorbance values contention  that  and  GFLV-infected  these v i r u s e s  shoots  the type o f t i s s u e  obtained  (Figure 1).  rapidly proliferating  i n elongated  that  of virus  i n a l l p l a n t l e t s t e s t e d on  i n v e s t i g a t i o n s w i t h AMV  detected  repeatable.  was an uneven v i r u s d i s t r i b u t i o n  the v a r i a b i l i t y  t e s t s .supported  Further  and  in plantlets retested  a t 25°C were d i s s e c t e d  individually,  roots  and.GFLV  p l a n t l e t s at both  growing  inplantlets,  tissue-cultured grapevines.  AMV  that  reliable  t o document  and r e p e a t e d l y  suggested  GFLV, and TomRV  was n o t always  within  to detect  each date,  o f AMV,  i s the f i r s t  concentration  by  in field  1983; M o n e t t e ,  may n o t be a r e l i a b l e in  viruses  i s a s e n s i t i v e method  of tissue-cultured p l a n t l e t s  (McMorran and A l l e n , plant  technique  and l e a v e s taken  were more t i s s u e s and (Figure 2).  for testing  73  from  p l a n t l e t s growing  detection could if  by  ELISA.  poor  growth  pattern  of  detection biggest  replicate should  not  at  may  Allen  (1983) d e t e r m i n e d  at  Virus  d i f f e r e n t temperatures.  detecting  one  using  of  ELISA,  f o r the  plantlets  five  that  leaves,  root  infected with  relative stems, the  replicated  experiment  distribution Even  o f AMV  though  elongated  the  In  would be  and  GFLV at  virus  over  to  may,  parts.  Normal  virus  and  unsuitable  for  in tissue-cultured  root four  the  another  levels in certain  l e v e l s were samples o f  viruses.  case of  required  not  the  Different  this  to c l a r i f y  different  concentration  t i s s u e s when the  to  degradation  part  t i s s u e was  and  the  apparent  replication  antigen  other  behaved d i f f e r e n t l y .  no  A l t h o u g h McMorran  potato viruses  the  Virus  plant  higher  AMV  lower  have been a b l e  advantageous  have o c c u r e d  with  T h e r e was  in c e r t a i n plant  processes  at  the  i n one  the  rapidly  25°C.  temperature.  parts  viruses  i n the  20°C or  improved  testing  GFLV and  leaves,  GFLV. The  should  have been enhanced  replication  uniform  with  and  Both v i r u s e s  biochemical  plants  shoots  roots.  either  for  a l t e r e d by  improved  and  however, have been  tissue  these v i r u s e s , e s p e c i a l l y  20°C  titres  shoots  e i t h e r moderate  plant  at  plant  virus  short  well  wrong  virus  used.  occurring  reduced  for t h i s .  was  Culture  improvement  proliferating  the  e f f e c t on  v i r u s d i s t r i b u t i o n was  in elongated  temperature  an  to d e t e c t  q u a l i t y antiserum  temperature.  reason  25°C had  Sampling  result in f a i l u r e  The  for  at  study,  a  the  temperatures. was  increased  p l a n t l e t s were grown at  in 20°C,  AMV  and  GFLV d e t e c t i o n  (Table  III).  This  was  was  not  assured  shown when t h e  i n a l l the p l a n t l e t s v i r u s e s were  detected  i n f i v e p l a n t l e t s w h i c h had  previously  positive  while  chance of  virus less  g r o w i n g a t 20°C.  i n f e c t i o n due at  20°C t h a n a t  The  inability  plantlets Monette  to the  was  initiated  period.  consistent  GFLV and  with  could  not  the  GFLV, he  all  p l a n t l e t s throughout that  consistent  was  able  detection  reculturing  may  Studies  AMV  the  o v e r a 14 day  plants  with  to the  g r o w t h on  H a r r i s and  end  rates This  of  may  a s i x week r e c u l t u r e  may  coupled with  initiated  shorter medium i n a  Stevenson  better  under  interval.  n u t r i e n t s were  at  depleted. in  the  detection.  from i n f e c t e d greenhouse-grown p l a n t s . one  virus  Viral replication  i n grapevines growing  the v i r u s i n t h e  the  were u s u a l l y t e s t e d  normal v i r u s d e g r a d a t i o n  also detected  to detect  (1982)  a l s o have i n c r e a s e d  plants  the  AMV  This  liquid  have a c c o u n t e d f o r r e d u c e d v i r u s  TomRV was  failure  s t u d y , the  have been l o w e r e d as  fact,  plants  In my  reculture  r a p i d r a t e o f m e t a b o l i s m by  a d e q u a t e n u t r i t i o n may  replication. the  This  term  virus consistently in  showed t h a t g r a p e p l a n t l e t s grew f a s t e r and these c o n d i t i o n s .  short  t h e s i s study with  reculture period.  the  by  GFLV i n a l l p l a n t l e t s  have been due  by  consistently in  Monette r e p o r t e d  this  to d e t e c t  i n t e r v a l s and  device.  however,  r e s u l t s obtained  detect  r e s u l t s of  and  tilting  the  i n GFLV c o n c e n t r a t i o n  Unlike  misdiagnosing  25°C.  from i n f e c t e d p l a n t s .  fluctuations  tested  t i s s u e t y p e s a m p l e d was,  to d e t e c t  ( 1 9 8 5 ) , who  The  not  in The  poorly-growing  vitro  75 c u l t u r e a l s o r e i n f o r c e s the importance o f s e l e c t i n g appropriate  tissue f o rtesting.  The w h o l e p l a n t l e t was  g r o w i n g p o o r l y and t h e t e s t e d l e a f was p a r t i a l l y n e c r o t i c . The  s u c c e s s f u l and r e p e a t e d  d e t e c t i o n o f TomRV i n a l l o t h e r  plantlets  compared t o AMV and GFLV may have b e e n due t o  naturally  higher  This  study  concentration  l e v e l s o f TomRV r e p l i c a t i o n i s the f i r s t  t o q u a n t i f y t h e e f f e c t s o f BAP  i n t h e m e d i a and g r o w t h room t e m p e r a t u r e on  AMV and GFLV c o n c e n t r a t i o n s BAP  i n grapevines  d i d not appear t o p l a y a major r o l e  o f AMV a t t h e c o n c e n t r a t i o n s effects  a t 20°C.  ranging  in  i n ELISA  used i n t h i s  on AMV c o n c e n t r a t i o n s  concentrations  grown  detection  study.  i n the p l a n t l e t s  vitro.  Any  c a u s e d by BAP  f r o m 2 t o 8 mg/l were masked by t h e  overwhelming e f f e c t s of temperature. The factor  g r o w t h room t e m p e r a t u r e was t h e most  affecting virus detection i n plantlets.  concentrations results, 15°C.  in plantlets,  were q u i t e h i g h  as shown by E L I S A  Virus concentrations  the  concentrations  was  with  t o 30°C where  dropped below t h e d e t e c t i o n This  reduction  n o t a c c o m p a n i e d by a s i m i l a r  When v i s u a l l y  detection  were r e d u c e d s i g n i f i c a n t l y as  t e m p e r a t u r e was r a i s e d i n 5°C i n c r e m e n t s  t h e ELISA t e s t .  AMV and GFLV  a t a c u l t u r e room t e m p e r a t u r e o f  the  of  critical  compared, p l a n t l e t  t h e t e m p e r a t u r e and t h e b e s t  i n virus  reduction  sensitivity  concentration i n plant  growth a c t u a l l y  vigour.  increased  g r o w t h was o b s e r v e d a t  30°C. It  was e x t r e m e l y  continuously  important  t o grow g r a p e v i n e  a t 20°C o r l e s s f o r r e l i a b l e  plantlets  AMV and GFLV  76 detection.  Exposure  virus  titre  to l e v e l s  least  2 months.  effect long  t o t e m p e r a t u r e s above 25°C r e d u c e d t h e below  Exposure  detection  thresholds  t o 30°C t e m p e r a t u r e had a d r a s t i c  on AMV and GFLV c o n c e n t r a t i o n s .  lasting.  constant without  Treatment  The e f f e c t  meristem  or s m a l l  e l i m i n a t e AMV f r o m p l a n t l e t s grown i n p o t s . plantlets  Based  may be  shoot t i p e x c i s i o n ,  and s u b s e q u e n t  on t h e r e s p o n s e o f G F L V - i n f e c t e d  in tissue-cultured  failure  to  rooted explants  same t r e a t m e n t c o n d i t i o n s would have a s i m i l a r  The  at a  sufficient,  t o t h e 30°C t e m p e r a t u r e , i t i s l i k e l y  GFLV t i t r e s  was v e r y  of AMV-infected p l a n t l e t s  t e m p e r a t u r e o f 30°C f o r 60 days subsequent  f o r at  that the effect  on  plantlets.  t o d e t e c t AMV i n some p l a n t l e t s  growing at  20°C and 25°C i n t h e s e c o n d e x p e r i m e n t was p r o b a b l y due t o their  h a v i n g been grown p r e v i o u s l y  temperatures fluctuated virus the  up t o 29°C d u r i n g  plant.  month.  Some o f t h e p l a n t l e t s  However, t h e v i r u s  plantlets detect  used  The  and e s c a p e d d e t e c t i o n  by t h e end o f t h e second month.  AMV  in a l l plantlets  after  a f t e r one  i n more  The a b i l i t y t o  2 months o f g r o w t h a t 15°C  in selecting  infected  s t a r t o f t h e e x p e r i m e n t b u t , more l i k e l y ,  growth  within  f o r the experiment  became d e t e c t a b l e  may have b e e n due t o c h a n c e  and  the day.  c o n c e n t r a t i o n was p r o b a b l y r e d u c e d and u n e v e n  p r o b a b l y had v e r y low l e v e l s  the  i n a room where  a t a t e m p e r a t u r e more f a v o r a b l e  t i s s u e at  was due t o  to virus  replication  reduced v i r u s degradation. The  rapid  concentration  effect  o f t h e 30°C t e m p e r a t u r e on AMV  i n infected  plantlets  was u n e x p e c t e d and  77 difficult ELISA  to e x p l a i n .  t e s t s as  buffers  the  It could  not  have been due  same c o a t i n g  and  conjugated a n t i s e r a  were u s e d f o r a l l t e s t s and  well.  In h i s d i s c u s s i o n  virus  i n a c t i v a t i o n in  small  spherical viruses  with  increased  already  present,  the  are  He  often  mentions  blocking  RNA  virus  p a r t i c l e assembly because the correct  temperature. the  ability The  Any of  the  reduction  study  (Table X I ) .  AMV  or  indicates  virus  that  was  not may  GFLV has  due  only  f o r 15-30  proper  higher be  affecting  w h e t h e r the  be  30°C.  the  in  this  rapid  virus  the  reduction  degradation  increasing Increases  viral  i n growth  have r e d u c e d the r e p l i c a t i o n have i n c r e a s e d  short,  In t h e  days at  for  explain  observed  to n a t u r a l  a thermal end-point of  infectivity  does not  a l s o be  is fairly  temperature i s increased.  reducing  account  E x p e r i m e n t s have shown t h a t  of n e p o v i r u s e s  a  cannot  a t the  M a t t h e w s may  p l a n t l e t s at  t h i s v i r u s but  degradation.  by  a n o t h e r f a c t o r may  i n the  to  virus  of  coat p r o t e i n  vivo  to r e p l i c a t e .  It is unclear  room t e m p e r a t u r e may r a t e of  prevention  in virus concentration  concentrations  degradation  the  r e p l i c a t i o n r a t e but  rapid  in  and  in  due  by  t h e s e mechanisms c o u l d  above e x p l a n a t i o n  reduced v i r a l  sap  of  be  synthesis  packing configuration one  on  that  i n a c t i v a t i o n of  viral  assume the  t h i s may  of v i r a l  polymerase a c t i v i t y ,  temperature  (1981) s t a t e s  that  and  worked  easily inactivated  mechanisms: the  the  controls  e f f e c t of  vivo, M a t t h e w s  heat.  number o f p o s s i b l e  on  the  to f a u l t y  the  stability  e s p e c i a l l y as  sap  viral  the  of herbaceous  60-65°C and  20°C ( H e w i t t  in  plants,  retains et a l . , 1970).  78  AMV, at  i n Petunia  hybrida,  55-61°C a n d  (Murant,  of  this  Whether  or not  then  they  which  monitor  virus  plantlets titre.  for  end-point The  may  with  when  temperature  f o r 90  recurrence. of  virus  plantlets virus  be  by  in  was  In t h i s  vitro  would  by  surprising  rapid  below  The  have  only  t o me  therapy  and  that  reduction  virus  both  to  supply  in otherwise actively  found  (1961)  and  symptoms on  was  (1968)  detection  a plant.  different  temperature,  effects  GFLV w e r e  days  One  at  increased  heat  weeks  growing  in  virus  degradation  even  though  the u s u a l and  the  the  conditions  thermal  temperatures.  virus  with  35°C  within  minutes  increased  replication  time  f o r such  Galzy  infected  Compan  that  10  Kassanis' theory.  viral  over  is well  temperature  that  with  i t seems  inactivation  found  and  suspend  levels  elimination  Galzy  Galzy  agree  increasing  r e p o r t e d by  believed  concentration in a plant,  also  those  the  (1957)  is speculation.  account  temperature  virus  f o r 1-2  degradation rates  There  higher  do  However,  would  associated  temperature  destroyed viruses  would  virus  temperatures. normal  room  s t u d y may  the v i r u s  conditions  after  Kassanis  actually  results  determine  infectivity  storage at  1970).  temperature  loses  reduced  and  increased found  necessary thesis ELISA  that  in this Galzy  and  over  plant above  Compan  growth 26°C.  a treatment  the  The  (1968). plantlets  improved However,  of  at  least  symptom  increased  symptom  permitted following  c o n c e n t r a t i o n more c l o s e l y .  resemble  tissue-cultured  to prevent  study,  study  sensitivity  e x p r e s s i o n on  the  reduction  temperature  in  effect  79 was d e t e c t e d  as i t was o c c u r r i n g .  Barlass eliminated  et a l .  GFLV  fluctuating  (1982) found  from  V. vinifera  day/night  t h a t growth plantlets  temperatures  a t 35°C  while  o f 27°/20°C d i d n o t .  They d i d n o t i n d i c a t e  the d u r a t i o n of treatment  try  a t 30°C.  virus  that  elimination  growth  at temperatures  growth at  nor d i d they  They d i d m e n t i o n ,  above  35°C r e s u l t e d  however,  i n death.  The  i n c r e a s e d p l a n t g r o w t h and s u r v i v a l  a t 30°C c o u p l e d  with  AMV  and GFLV e l i m i n a t i o n  study  that  it  i s unnecessary  near-lethal  obtained  to subject  i n my  in vitro  c o n d i t i o n s i n order  indicated  p l a n t l e t s to  to e l i m i n a t e these  two  viruses.  Virus  detection in f i e l d  The typical  results  of these  the s t a n d a r d  was e s s e n t i a l  at Sidney,  ELISA v a l u e s  on a l l t h e t e s t  when t h e s t a n d a r d f o r AMV-infected  standard  buffer,  obtained  when u s i n g  same low l e v e l s greatest  although  summer was o b t a i n e d  nicotine  still  The l o w e s t  dates  throughout  p l a n t s ground significantly  the other b u f f e r s ,  i n ELISA  ELISA the  d i d not drop plants.  was added  used.  i n the  below  absorbance values  when n i c o t i n e  from  g r i n d i n g b u f f e r was  as f o r t h e G F L V - i n f e c t e d  improvement  B.C.,  d e t e c t i o n o f GFLV and AMV  and s c r e e n h o u s e - g r o w n g r a p e v i n e s .  summer o c c u r r e d  t h a t , under t h e  ELISA g r i n d i n g b u f f e r w i t h  for reliable  absorbance values  The  studies indicate  summer c o n d i t i o n s e x p e r i e n c e d  enhancing  field  plants  those to the  The d u r i n g the  to both  buffers.  As  the summer  unnecessary  progressed,  increasing  when n i c o t i n e  was added.  amended w i t h absorbance With  nicotine  values  the b u f f e r w i t h  o f one d a t e ,  July  0.15 M g r i n d i n g b u f f e r gave ELISA significantly  lower  The s t a n d a r d  o f t e n gave s t a t i s t i c a l l y  than  the e x c e p t i o n  the b u f f e r m o l a r i t y was  than  those  buffer  higher  increased molarity.  15 f o r GFLV, u s i n g t h e  absorbance  values  measured u s i n g  the n i c o t i n e  buffers. The  most  increased values with  ability  from  ELISA  values  important  benefit  to separate  negative  values.  absorbance values  determined  o f b u f f e r enhancement  positive  ELISA  Positive  p l a n t s were  g r e a t e r than  by the h e a l t h y c o n t r o l  i s the  absorbance those  the t h r e s h o l d p l a n t s on e a c h  date . The  standard  g r i n d i n g b u f f e r was a l w a y s  detecting  positive  the  three b u f f e r s .  other  reliable the  Assays  one i n f e c t e d  reliability also the  dropped  with  this  observations  reduce  technique  These r e s u l t s virologists  Detection  (Table  additives  with  T h e r e was  c o o l e r weather  towards  were s i m i l a r t o testing I).  t o o p t i m i z e ELISA  i s t o be used  Buffer  to detect at  as the summer p r o g r e s s e d .  by o t h e r  t h e need  at the s t a r t o f  buffer failed  p l a n t on each d a t e .  warmer p a r t s o f t h e w o r l d not  b u f f e r was n o t as  v i r u s e s even  no i n c r e a s e i n d e t e c t i o n w i t h end o f summer.  v i r u s e s when compared t o  The s t a n d a r d  f o r d e t e c t i n g these  summer.  least  p l a n t s f o r both  inferior for  grapevines i n  Our c o o l e r summers do conditions i f this  confidence.  were e s p e c i a l l y  important  f o r GFLV  detection. using  the  even  GFLV was standard  though  failed This  they  titre  i n the  antiserum buffers,  GFLV  plants  against  background infection  the  buffers  individual exception  used  viruses of  the  varieties  as  conditions. spurious  allow  out  f o r proper The  need  analysis  15,  absorbance values  controls  went up  r e s u l t s of  the these  determine  virus  tests, especially  the  values. three  in detecting  controls  detection  of  the with  should  included  and  be  the  difficult  the  the  the  spurious  same  detect  which have  Uniform  same  been  tissue reactions  and  results.  July  obtained  significantly  to  be  viruses.  for  g r o w i n g under  reactions,  the  must  grapevine  to c a r e f u l l y s e l e c t h e a l t h y the  of  from  threshold  well  to m i n i m i s e  by  virus  even w i t h free  not  the  grapevines.  of  plants.  date.  negative  supported ELISA  should  i t would be  selected  buffers  screenhouse p l a n t s ,  tested  tests with  that,  sensitivity  representative  controls  p o s i t i v e and  must be  and  ELISA  Otherwise,  to a lower  f o r each date,  test  those being  i n ELISA  samples  15  enhanced  antiserum  equally  in f i e l d  to n o t e  critical,  near  when  screenhouse  s i n g l e ELISA  separately  July  healthy  one  and  lower  titre  are  adequate number o f  Preferably,  to a  Also,  performed  when c a r r y i n g  found  highest  the  only  It remains  absorbance values  enhanced  not  also  than AMV  i s important  detection,  r e s u l t s of  When compared  An  but  GFLV.  the  detected  i n some f i e l d  reactions. on  It  have been due  to use  when the  buffer. increased  to d e t e c t  r e s u l t may  more p o o r l y  15 for  compared  controls  was  test date.  On  the to  LN-33  those  July  healthy  f o r the  St.  82 George controls, especially when tested against the GFLV antiserum  (Table XVI).  Combining the values for both  control v a r i e t i e s for each antiserum  to determine the  threshold values for positive tests tended  to lower the  number of GFLV-positive St. George plants and, conversely, to increase the number of GFLV-positive LN-33 plants.  When  the virus detection was analysed separately by variety, the number of positive St. George plants was increased to 6, 6, 4, and 6 for buffers A, B, C, and D respectively.  The  number of positive LN-33 plants was unchanged. The increased absorbance values observed  for samples  taken from the LN-33 control on July 15 are d i f f i c u l t to explain.  Retesting the same tissue samples 2 days later  against newly conjugated GFLV antiserum gave the same results.  A review of c u l t u r a l practices and pesticide spray  applications prior to July 15 gave no different  treatments  for one variety over the other.  high  The abnormally  absorbance values for healthy plants may have been due to the type of plant material sampled.  Mink et a l .  (1985)  found a similar problem when testing apple plants for TomRV. Samples taken from shoot tips and not f u l l y expanded leaves of healthy apple plants gave abnormally high ELISA absorbance readings which could be interpreted as positive when compared to readings of healthy cucumber plants.  These  higher readings occurred in samples taken between July and August and not in A p r i l . the same results.  Older expanded leaves did not give  They concluded  that the rapidly growing  leaves were producing nonviral, noninfectious antigens which  83 were r e a c t i n g for  the  They  apparent  seasonal  The  also  of  Also,  July  affected  In  this  gave h i g h was  used.  for  both ELISA  high the  case,  absorbance The  plates  a  plant  readings  have r e a c t e d  against  the  purified  i n t o the  for  grinding, one  only  with  rabbit  some n o r m a l  plant  proteins  antibodies.  In  impurities  i n the  render  the  resulting polyclonal  rabbit  nonusable because of  some c a s e s , virus  plant  against  GFLV, w h i c h  plants  sap.  t h a n AMV,  against  a  the  high  In a t t e m p t s i s found  two  viruses.  LN-33 p l a n t s and  July  A  produced  15.  This  produced that  was  antiserum.  there  which  the  antiserum  much  animal of  to produce  produces  these  enough  to  reactions an  titres of  as  remain  p r o d u c e d by  background  l e s s pure p r e p a r a t i o n  as  often  is great  i n lower  to GFLV  applied  concentration  preparation  plants  sample was  is purified  animal,  LN-33  control  the  the  in  healthy  preparation  preparation  expanded  months.  antibodies  to p r o d u c e  i n j e c t i o n i n t o an  healthy  of  and  study  antiserum  the  the  the  the  GFLV.virus  a virus  as  time  grape  fully  the  when an  used  that  were o b s e r v e d  from  between June 24  may  possible  only  is that  protein  not  in later  taken  against  protein  Although  less  at  in this  account  readings.  healthy  absorbance values  After  explanation  injected  open but  samples  coated  t e s t s on  fully  not  false  in progress  taken  same t i s s u e was  antisera.  the  samples  results  the  possible normal  studies  young,  the  They c o u l d  e f f e c t on  that  grape  leaves. and  antiserum.  similar results for  plants.  consisted  to  the  a l s o mentioned  were g i v i n g pear  with  the to  antiserum i n herbaceous  GFLV may  have  been u s e d specific  for  injection  antiserum.  these  t e s t s was  plant  material  background  against all  healthy  negative The Noir  the  the  r e s u l t i n g in a  GFLV a n t i s e r u m  against  healthy  ^-globulin purification  reactions  against  healthy  cross-adsorption, herbaceous  antibodies,  reason on  was  may  not  in  herbaceous to r e d u c e  plant  w h i c h was  plants,  used  less  sap  carried  have  low  out  eliminated  r e s u l t i n g in abnormally  high  for  July  the  15  sampled,  AMV  detection  i s not  clear.  c a u s e d by  failure  i n the  I suspect  a miscount  that  i n the  Pinot  the  plant  wrong  row  in  field. The  from  most d i f f i c u l t  negative  threshold  plants  level  considered  based  that  was  important  one  w h i c h gave  there  to  They  arbitrary  at  In my  determined  fewest  stated  least  and  on  that  using  i t to be  answers  when u s i n g  plants  These  authors  for setting  ELISA  negatives  of  the  negative use  of  the  is  on  plants.  "correct" threshold,  false  often  reading  a l . , 1986).  easy  a population  positive  results is setting  absorbance  s e l e c t a most  study  by  ELISA  et  were no  and and  any  that  It is  or p o s i t i v e s , positive  threshold  was  misleading.  detecting  screenhouse grapevines,  believed  on  thresholds  the  analysing  samples.  in separating  above which an  positive-negative  after  part  p o s i t i v e (Sutula  concluded  and  fact,  animal,  readings.  vine  plant  before  The  unwanted  In  cross-adsorbed  ELISA  components.  i n t o the  AMV  the  and  GFLV  threshold  Chebychev's e q u i v a l e n t  statistically  sound.  in infected  field  f o r each date  was  because I  This  value  is  equal  85 to the  the  mean of  the  background  colour  means) p l u s  three  identifying  the  the to  the  in buffer  standard  of  standard  times  published whether  the  means b e f o r e the  threshold  results  values,  fewer  threshold XVII).  offset,  three false  value  This  standard  may  control  of  wells  It i s  a value  two  or  Results not  indicate  been s u b t r a c t e d  have an  was  methods o f negatives  equal  to  The  important  from  the  s e l e c t i o n of  effect  however, by (Table  the  the  make i t d i f f i c u l t , positives,  on  the  other false  two  The  Chebychev's constant  c o r r e c t e d mean  increased  of  43  this  had  on  method  the  ground  a greater  when t e s t i n g eliminated  false  was  controls AMV  these  may  false  thresholds.  thresholds  number o f  f o r GFLV.  false  the  positives  of  for setting  to d e t e r m i n e  in  selecting false  healthy  impact  the  (Table  detection  chance of  l a r g e number o f  methods u s e d  especially  when u s i n g  f o r samples  total  depending  p o s i t i v e s but  negatives,  true  A  threshold  p o s i t i v e f o r e i t h e r GFLV or  The  to a c c e p t  the  increased  XVIII).  summer.  determining  were d e t e c t e d  twice  especially  would have been c a l l e d  no  colour.  had  because  determined.  threshold values.  grinding buffer.  positives  The  buffer  of  obtained.  Comparing  during  used  j o u r n a l s o f t e n do  values  the  criteria  to be  of  (corrected method  a threshold value,  scientific  setting  and  background  buffer well  This  r e a c t i o n s was  deviation  healthy  i n the  the  deviations.  as  subtraction  control wells  enough h e a l t h y  more common to s e l e c t , three  controls, after  p o s i t i v e ELISA  availability allow  healthy  both  gave  false  Using  p o s i t i v e s but  still  86  Table XVII. A comparison of false negatives c r e a t e d by using three d i f f e r e n t methods of d e t e r m i n i n g ELISA threshold values for arabis mosaic virus (AMV) and g r a p e v i n e fanleaf virus (GFLV) detection during the summer  False  Method Tes t ing  for  date  threshold  A)  per  buffer  1  setting A  B  C  D  3s  0 0  0 0  2 1  0 0  2 1  mean)  0  0  4  0  4  corr. m e a n + 3s 2(corr. mean)  0 0  0 0  1 1  0 0  1 1  2(uncorr.  0  0  1  0  1  Totals  AMV  June  corr.  3  mean  2(corr.  June  July  Aug.  Sept.  24  15  12  2  +  mean)  2(uncorr.  mean) 3s  1  1  2  1  5  2(corr. mean) 2(uncorr. mean)  0 1  1 1  0 2  0 1  1 5  corr. mean + 3s 2(corr. mean) 2(uncorr. mean)  0 0 3  0 0 3  9 0 7  0 0 3  9 0 16  corr.  0 0  5 3 7  corr.  mean  mean  +  +  3s  2(corr. mean) 2(uncorr. mean)  *A  negat ives  >  0  0  5  0  0 0  3 7  0 0  s a l i n e ( P B S ) , PH 8 . 4 0.15 M phosphate b u f f e r e d with 0.05% Tween-20, 0.2% o v a l b u m i n and 2 % p o l y v i n y l p y r r o l i d o n e ; B - buffer A with 1% n i c o t i n e , p H 8 . 4 ; C standard grinding b u f f e r - 0.01 M PBS, pH 7 . 4 , with 0.05% Tween-20, 0.2% o v a l b u m i n and 2 % p o l y v i n y l p y r r o l i d o n e ; D - b u f f e r C with 1% n i c o t i n e , p H 8.2.  87 Table  XVII.  Testing date  continued  Method for setting threshold  False A  negatives B  C  per b u f f e r D  1  Totals  B) GFLV June  3  c o r r . m e a n + 3s 2 ( c o r r . mean) 2 ( u n c o r r . mean)  0 0 0  0 0 0  2 0 2  0 0 0  2 0 2  June  24  c o r r . mean + 3s 2(corr.mean) 2 ( u n c o r r . mean)  0 0 0  0 0 0  8 3 9  0 0 0  8 3 9  July  15  c o r r . mean + 3s 2 ( c o r r . mean) 2 ( u n c o r r . mean)  12 5 1  5 0 0  20 3 13  7 0 3  44 8 17  Aug .  12  c o r r . mean + 3s 2 ( c o r r . mean) 2 ( u n c o r r . mean)  1 1 1  1 0 1  16 4 20  1 1 1  19 6 23  Sept . 2  c o r r . mean + 3s 2 ( c o r r . mean) 2 ( u n c o r r . mean)  2 3 2  2 1 2  16 10 20  2 0 2  22 14 26  X  A  - 0.15 M p h o s p h a t e b u f f e r e d s a l i n e ( P B S ) , pH 8.4 w i t h 0.05% Tween-20, 0.2% o v a l b u m i n and 2 % p o l y v i n y l p y r r o l i d o n e ; B - b u f f e r A w i t h 1% n i c o t i n e , pH 8.4; C - s t a n d a r d g r i n d i n g b u f f e r - 0.01 M PBS, pH 7.4, w i t h 0.05% Tween-20, 0.2% o v a l b u m i n and 2 % p o l y v i n y l p y r r o l i d o n e ; D - b u f f e r C w i t h 1% n i c o t i n e , pH 8.2.  88  T a b l e X V I I I . A c o m p a r i s o n o f f a l s e p o s i t i v e s c r e a t e d by u s i n g t h r e e methods o f d e t e r m i n i n g ELISA t h r e s h o l d v a l u e s f o r a r a b i s m o s a i c v i r u s (AMV) and g r a p e v i n e f a n l e a f v i r u s (GFLV) d e t e c t i o n d u r i n g t h e summer  Testing date  Method for setting threshold  False  p o s i t i v e s per b u f f e r  A  B  c o r r . mean + 3s 2 ( c o r r . mean) 2 ( u n c o r r . mean)  0 1 0  1  c o r r . mean + 3s 2 ( c o r r . mean) 2 ( u n c o r r . mean)  0 1 0  c o r r . mean + 3s 2 ( c o r r . mean) 2 ( u n c o r r . mean)  0 2 0  c o r r . mean + 3s 2 ( c o r r . mean) 2 ( u n c o r r . mean)  0 1 0  c o r r . mean + 3s 2 ( c o r r . mean) 2 ( u n c o r r . mean),  0 0 0  C  D  1  Totals  A) AMV June 3  June  July  Aug.  Sept.  4  A  24  15  12  2  0  0  0  0  0 0 0  0  0 0 0  0 1 0  0 2 0  0 0  0 2 0  0 7 0  0 0 0  0 0 0  0 1 0  0  0 3 0  0 6 0  2  0 0 0 1 0  2  0 0 0 0 1 0  2 0  - 0.15 M p h o s p h a t e b u f f e r e d s a l i n e (PBS), pH 8.4 w i t h 0.05% Tween-20, 0.2% o v a l b u m i n and 2 % p o l y v i n y l p y r r o l i d o n e ; B - b u f f e r A w i t h 1% n i c o t i n e , pH 8.4; C - s t a n d a r d g r i n d i n g b u f f e r - 0.01 M PBS, pH 7.4, w i t h 0.05% Tween-20, 0.2% o v a l b u m i n and 2 % p o l y v i n y l p y r r o l i d o n e ; D - b u f f e r C w i t h 1% n i c o t i n e , pH 8.2.  89 Table  XVIII.  Tes t i n g date  continued  Method for s e t t i n g threshold  False  pos i t i v e s p e r  buffer  1  A  B  c  0  Totals  B) GFLV June 3  c o r r . mean + 3s 2 ( c o r r . mean) 2 ( u n c o r r . mean)  0 1 0  0 2 0  0 1 0  0 1 0  0 5 0  June 24  c o r r . mean + 3s 2 ( c o r r . mean) 2 ( u n c o r r . mean)  0 0 0  0 1 0  0 1 0  0 1 0  0 3 0  July  15  c o r r . mean + 3s 2 ( c o r r . mean) 2 ( u n c o r r . mean)  0 2 0  0 2 0  0 3 0  0 2 0  0 9 0  Aug.  12  c o r r . mean + 3s 2 < c o r r . mean) 2 ( u n c o r r . mean)  0 0 0  0 1 0  0 3 0  0 1 0  0 5 0  2  c o r r . mean + 3s 2 ( c o r r . mean) 2 ( u n c o r r . mean)  0 0 0  0 1 0  0 2 0  0 1 0  0 4 0  Sept.  X  A  - 0.15 M p h o s p h a t e b u f f e r e d s a l i n e (PBS), pH 8.4 w i t h 0.05% Tween-20, 0.2% o v a l b u m i n and 2 % p o l y v i n y l p y r r o l i d o n e ; B - b u f f e r A w i t h 1% n i c o t i n e , pH 8.4; C - s t a n d a r d g r i n d i n g b u f f e r - 0.01 M PBS, pH 7.4, w i t h 0.05% Tween-20, 0.2% o v a l b u m i n and 2 % p o l y v i n y l p y r r o l i d o n e ; D - b u f f e r C w i t h 1% n i c o t i n e , pH 8.2.  90  maintained  a fairly  especially  when d e t e c t i n g GFLV. B e c a u s e o f  the to  S t a t i o n , to r e t e s t confirm  accepted twice  their  at  the  This  samples  only  The  be  determine  are  included  station  or  and  plants  field  That  growing  screenhouses  and  the  are  of  the  at  in order  often  a t h r e s h o l d of  samples  would  Station  give  purposes.  l a b o r a t o r y where  consequences  calling ELISA  of m i s s i n g  a negative  test  separate  test.  a  sample  threshold  case.  It i s standard  retest ELISA  plants giving  It i s  values  simple  o f ELISA  may  be  either  in  important affect  tests  on  detected vitro  or  in  at  the  borderline  virus  have a l s o d e t e r m i n e d described  vitro  w e l l by i n the  c o n c l u s i o n of  recommendations  improvements  policy  results.  made t o  that  plant lets  ELISA  in  field  this  d e t e c t i o n by  TomRV d e t e c t i o n i n g r a p e v i n e s .  presented with  i s an  w h i c h may  identified and  results  nepoviruses  grapevines  falsely  tendency,  t h r e s h o l d p r o v i d i n g enough h e a l t h y c o n t r o l s  negative  the  tests  to a  the  f o r each  i n each  Comparing  Factors  than  to r e p e a t e d l y  positive  the  repeatedly  Quarantine  acceptable  parameters  the  negatives,  I b e l i e v e that using  done once and  determined  results  positive  f o r the  be  is greater  positive. should  not  false  mean o f h e a l t h y  results  may  are  positive  status while  uncorrected  choice  number o f  negative  face value,  more a c c u r a t e  to  high  and  study.  ELISA  were  improve AMV,  However,  the  GFLV,  data  n e i t h e r method,  in this  thesis  is  even  100%  91  accurate.  E i t h e r method may  confidence  providing  this  t h e s i s are The  the  adhered  greater  plantlets  modified  buffers  indicate  that,  and  grown  in  The  the  increased  testing  need  conjunction  subsequent  to d e t e r m i n e applied may  be  in  vitro  with  in  the  reduced  and  vitro  faster  testing  Stellmach,  of  and  testing  I t may  and  rooted  p l a n t s e s t a b l i s h e d from  levels  test in  in  canes,  1985a,b;  and  i t is  before vitro  for longer  more r e l i a b l e these.  by  p r e f e r a b l e when u s e d  However,  virus titres  time.  attractive  1987).  be  this  from  plants  less  virus elimination  below d e t e c t i o n be  sampling  initiation  Etienne,  propagation.  The  ELISA  i n c l u d i n g dormant  p l a n t s may  limitations  routinely.  the  successful virus detection  Walter  r a p i d bulk  of  on  a l . , 1980a;  in  than  becomes even  (Bovey e t  of  in  and  vitro  tissues,  Testing  initiate  in  grapevine  a l . , 1986;  described  reliability  for routine  ELISA  Huss e t  to  purposes,  results  considered  as  v i r u s e s by  when p u b l i s h e d  are  reasonable  parameters  more e f f i c i e n t  plantlets  in other  with  to.  for routine  p l a n t s would be  grapevine  optimum  i n d e t e c t i n g these  field  of  used  amount o f work needed  maintain  vitro.  be  necessary  i t can  be  plantlets periods  to d e t e c t  of  them i n  92 BIBLIOGRAPHY B a r l a s s , M. and K.G.M. Skene. 1978. In vitro p r o p a g a t i o n o f g r a p e v i n e (Vitis vinifera L.) from f r a g m e n t e d s h o o t a p i c e s . V i t i s 17:335-340. B a r l a s s , M. and K.G.M. Skene. 1980a. S t u d i e s on t h e f r a g m e n t e d shoot apex o f g r a p e v i n e . I . The r e g e n e r a t i v e c a p a c i t y of l e a f p r i m o r d i a l fragments vitro. J . Exp. B o t . 31:483-488.  in  B a r l a s s , M. and K.G.M. Skene. 1980b. S t u d i e s on t h e f r a g m e n t e d shoot apex o f g r a p e v i n e . I I . F a c t o r s a f f e c t i n g growth and d i f f e r e n t i a t i o n in vitro. J . Exp. B o t . 31:489-495. B a r l a s s , M., K.G.M. Skene, R.C. Woodham, and L.R. K r a k e . 1982. R e g e n e r a t i o n o f v i r u s - f r e e g r a p e v i n e s u s i n g in vitro a p i c a l c u l t u r e . Ann. A p p l . B i o l . 101:291-29 5. Baumann, G., R. C a s p e r , and P. D e t e c t i o n o f prune dwarf m e r i s t e m - p r o p a g a t e d sour c u l t u r e . P h y t o p a t h o l . Z.  Kornkamhaeng. 1984. v i r u s by ELISA i n cherry plants during 110:168-171.  in  vitro  Bovey,  R. 1972. T h e r a p e u t i c methods a p p l i e d t o t h e g r a p e v i n e , pp. 32-38. I n : M. Kenneth and J . K a t a n ( e d s ) . Proceedings o f the 18th H o r t i c u l t u r a l C o n g r e s s . V o l I I I . Symposium on p r o d u c t i o n o f h e a l t h y p l a n t s by t h e r a p e u t i c and o t h e r methods and t h e i r m a i n t e n a n c e and u s e . T e l A v i v , I s r e a l .  Bovey,  R., J . J . B r u g g e r , and P. G u g e r l i . 1980a. D e t e c t i o n o f f a n l e a f v i r u s i n g r a p e v i n e t i s s u e e x t r a c t s by enzyme-1 i n k e d immunosorbent a s s a y ( E L I S A ) and immune e l e c t r o n microscope (IEM). I n : A . J . M c G i n n i s , ( e d . ) : Proceedings o f the 7th Meeting o f the I n t e r n a t i o n a l C o u n c i l f o r t h e Study o f V i r u s e s and V i r u s - l i k e D i s e a s e s o f t h e G r a p e v i n e . N i a g a r a F a l l s , Canada, pp. 259-275. A g r i c u l t u r e Canada, V i n e l a n d R e s e a r c h Station.  Bovey,  R., W. G S r t e l , W.B. H e w i t t , G.P. M a r t e l l i , and A. V u i t t e n e z . 1980b. V i r u s and V i r u s - l i k e D i s e a s e s of G r a p e v i n e s . E d i t i o n s Payot, Lausanne.  Bovey,R., P. G u g e r l i , and J . J . B r u g g e r . 1985. Some improvements i n t h e d e t e c t i o n o f g r a p e v i n e f a n l e a f v i r u s by enzyme-1 i n k e d immunosorbent a s s a y ( E L I S A ) and immune e l e c t r o n m i c r o s c o p y ( I E M ) . A b s t r . P h y t o p a t h o l . M e d i t e r r . 24:208-209.  93  Chee,  R. and R.M. P o o l . 1982. The e f f e c t s o f g r o w t h s u b s t a n c e s and p h o t o p e r i o d on the d e v e l o p m e n t o f a p i c e s o f Vitis c u l t u r e d in vitro. Sci. Hortic. 16:17-27.  shoot  Chee,  R., R.M. P o o l , and D. B u c h e r . 1984. A method f o r l a r g e s c a l e in vitro p r o p a g a t i o n o f Vitis. New York's Food and L i f e S c i e n c e s B u l l e t i n . 109:1-9.  Clark,  M.F. and A.N. Adams. 1976. C h a r a c t e r i s t i c s of a m i c r o p l a t e method o f enzyme-1 i n k e d immunosorbent a s s a y f o r the d e t e c t i o n o f p l a n t v i r u s e s . J . Gen. Virol. 34:475-483.  Corte,  G. and A. de Mendoca. 1985. Importance de l a c u l t u r e de m e r i s t e m e s pour l a m u l t i p l i c a t i o n a c c e l e r e e de c l o n e s de v i g n e exempts de v i r u s . B u l l . O.I.V. 650-651:396-402.  E n g e l b r e c h t , D.J. 1980. Indexing grapevines f o r grapevine f a n l e a f v i r u s by enzyme-1 i n k e d immunosorbent a s s a y . In: A . J . M c G i n n i s ( e d . ) : P r o c e e d i n g s of the 7th M e e t i n g o f the I n t e r n a t i o n a l C o u n c i l f o r the S t u d y o f V i r u s e s and V i r u s - l i k e D i s e a s e s o f the G r a p e v i n e . N i a g a r a F a l l s , Canada, pp. 277-282. A g r i c u l t u r e Canada, V i n e l a n d R e s e a r c h S t a t i o n . F r a s e r , R.S.S. and R.J. Whenham. 1982. P l a n t growth r e g u l a t o r s and v i r u s i n f e c t i o n : a c r i t i c a l r e v i e w . P l a n t Growth R e g u l a t i o n . 1:37-59. Galzy,  R. 1961. C o n f i r m a t i p n de l a n a t u r e v i r a l e du c o u r t - n o u e de l a v i g n e par des e s s a i s de t h e r m o t h e r a p i e s u r des c u l t u r e s in vitro. C. R. S c i . ( P a r i s ) 253:706-708.  Galzy,  R. 1963. T h e r m o t h e r a p i e de q u e l q u e s v a r i e t e s v i g n e . P r o g . A g r i c . V i t i c . 160:255-261.  Galzy,  R. 1964. la vigne.  Galzy,  R. 1966. A c t i o n de l a t e m p e r a t u r e 35°C s u r Vitis rupestris a t t e i n t du c o u r t - n o u e . B u l l . F r a n c . P h y s i o l . V e g e t . 12:391-399.  Galzy,  T e c h n i q u e de t h e r m o t h e r a p i e des Ann. E p i p h y t i e s 15:245-256.  Acad.  de  viroses  de  Soc.  R. 1969. R e c h e r c h e s s u r l a c r o i s s a n c e de Vitis rupes tris S c h e e l e e t c o u r t noue c u l t i v e in vitro d i f f e r e n t e s t e m p e r a t u r e s . Ann. Phytopathol. 1:149-166.  a  94  Galzy,  R. 1972. La c u l t u r e in vitro des apex de Vitis rupestris. C. R. A c a d . S c i . ( P a r i s ) 274:210-213 .  Galzy,  R. e t Compan, H. 1968. T h e r m o t h e r a p i e de q u e l q u e s v a r i 6 t e s de v i g n e p r ^ s e n t a n t des symptdmes de v i r o s e . V i g n e s e t V i n s . 166:1-8.  G i l m e r , R.M., J.K. Uyemoto, and L . J . K e l t s . 1970. A g r a p e v i n e d i s e a s e i n d u c e d by t o b a c c o r i n g s p o t Phytopathology 60:619-627.  new virus.  Goheen, A.C. and C F . Luhn. 1973. Heat i n a c t i v a t i o n o f v i r u s e s i n g r a p e s . R i v . P a t h o l . Veg. 9:287-289. Goheen, A . C , C F . Luhn, and W.B. H e w i t t . 1965. I n a c t i v a t i o n o f g r a p e v i n e v i r u s e s in vivo. P r o c . I n t e r n . C o n f . on V i r u s and V e c t o r on P e r e n n i a l H o s t s , pp. 255-267. D a v i s , C a l i f o r n i a . G o n s a l v e s , D. 1979. D e t e c t i o n o f tomato r i n g s p o t v i r u s i n g r a p e v i n e s : a c o m p a r i s o n o f Chenopodium quinoa and enzyme-1 i n k e d immunosorbent a s s a y ( E L I S A ) . P l a n t D i s . Rep. 63:962-965. G o u s s a r d , P.G. 1982. M o r p h o l o g i c a l r e s p o n s e s o f shoot a p i c e s o f g r a p e v i n e c u l t u r e d in vitro: e f f e c t s o f c y t o k i n i n s i n r o u t i n e s u b c u l t u r i n g . V i t i s 21:293-298. Harris  R.E. and J.H. S t e v e n s o n . 1982. In vitro p r o p a g a t i o n o f Vitis. V i t i s 21:22-32.  H e w i t t , W.B.. G. M a r t e l l i , H.F. D i a s , and R.H. T a y l o r . 1970. G r a p e v i n e f a n l e a f v i r u s . C.M.I./A.A.B. D e s c r i p t i o n s of P l a n t V i r u s e s No. 28. Huss,  B., B. W a l t e r , L. E t i e n n e , and M.H.V. VanRegenmorte1. 1986. G r a p e v i n e f a n l e a f v i r u s d e t e c t i o n i n v a r i o u s grapevine organs u s i n g p o l y c l o n a l and m o n o c l o n a l a n t i b o d i e s . V i t i s 25:178-188.  J a n k u l o v a , M., M. E s t e r h a z y , N. B a k a r d z h i e v a , and P. G e o r g i e v a . 1982. ELISA f o r the q u a n t i t a t i v e d e t e r m i n a t i o n o f g r a p e v i n e f a n l e a f v i r u s . Z. P f l a n z e n k r . P f l a n z e n s c h u t z 89:18-29. J i m e n e z , F. and A.C. Goheen. 1980. The u s e o f enzyme-1 i n k e d immunosorbent a s s a y f o r d e t e c t i o n o f grape f a n l e a f v i r u s . In: A . J . McGinnis ( e d . ) : P r o c e e d i n g s o f the 7 t h M e e t i n g o f the I n t e r n a t i o n a l C o u n c i l f o r the Study of V i r u s e s and V i r u s - l i k e D i s e a s e s o f the G r a p e v i n e . N i a g a r a F a l l s , Canada, pp. 283-291. A g r i c u l t u r e Canada, V i n e l a n d R e s e a r c h Station.  95  K a s s a n i s , B. 1957. Some e f f e c t s o f v a r y i n g t e m p e r a t u r e the q u a l i t y and q u a n t i t y o f t o b a c c o m o s a i c v i r u s i n f e c t e d p l a n t s . V i r o l o g y 4:187-199.  on in  K e a r n s , C.G. and D.W. Mossop. 1984. D e t e c t i o n o f n e p o v i r u s e s o f Vitis vinifera i n New Z e a l a n d u s i n g enzyme-1 i n k e d immunosorbent a s s a y ( E L I S A ) . N. Z. J . A g r i c . Res. 27:431-435. K o l b e r , M., L. B e c z n e r , S. P a c s a , and J . L e h o c z k y . 1985. D e t e c t i o n o f g r a p e v i n e chrome m o s a i c v i r u s i n f i e l d - g r o w n v i n e s by E L I S A . P h y t o p a t h o l . M e d i t e r r . 24:135-140. Kuo,  C.G., B . J . Shen, M.J. Shen, S.K. G r e e n , and D.R. Lee. 1985. V i r u s - f r e e sweet p o t a t o s t o r a g e r o o t s d e r i v e d from m e r i s t e m - t i p s and l e a f - c u t t i n g s . S c i . H o r t i c . 26:231-240.  L e h o c z y , J . , M. K61ber, L. B e c z n e r , and S. P a c s a . 1984. D i s t r i b u t i o n o f the g r a p e v i n e chrome m o s a i c d i s e a s e Hungary and d e t e c t i o n o f i t s v i r u s (GCMV) i n the l e a v e s of o u t d o o r v i n e s by ELISA t e c h n i q u e . Kertgazdasag 16:41-52. Li,  J . and G.W. E a t o n . 1984. s h o o t a p i c e s in vitro.  in  Growth and r o o t i n g o f g r a p e H o r t S c i e n c e 19:64-66.  Lozoya-Saldana, H. and A. M a d r i g a l - V a r g a s . 1985. K i n e t i n , t h e r m o t h e r a p y , and t i s u e c u l t u r e t o e l i m i n a t e p o t a t o v i r u s (PVX) i n p o t a t o . Amer. P o t . J . 62:340-345. Matthews, R.E.F.. 1981. P l a n t V i r o l o g y . 2nd Academic P r e s s . New York  edition.  McMorran, J.P. and T.C. A l l e n . 1983. M a i n t e n a n c e , symptoms and d i s t r i b u t i o n o f p o t a t o v i r u s e s X.S.Y.A.M and l e a f r o l l i n p o t a t o t i s s u e c u l t u r e p l a n t l e t s . Amer. P o t . J . 60:839-847. Milo,  G.E. and B . I . S a h a i S r i v a s t a v a . 1969a. E f f e c t o f c y t o k i n i n s on t o b a c c o m o s a i c v i r u s p r o d u c t i o n i n l o c a l - l e s i o n and s y s t e m i c h o s t s . V i r o l o g y 38:26-31.  Milo.  G.E. and B . I . S a h a i S r i v a s t a v a . 1969b. E f f e c t o f c y t o k i n i n s on t o b a c c o m o s a i c v i r u s p r o d u c t i o n i n t o b a c c o p i t h t i s s u e c u l t u r e s . V i r o l o g y 39:621-623.  Mink, G.I., W.E. H o w e l l , and P.R. F r i d l u n d . 1985. Apple l e a f t i p a n t i g e n s t h a t cause s p u r i o u s r e a c t i o n s with tomato r i n g s p o t v i r u s a n t i s e r a i n enzyme-1 i n k e d immunosorbent a s s a y . P h y t o p a t h o l o g y 75:325-329.  96 M o n e t t e , P.L. 1983. V i r u s e r a d i c a t i o n t h r o u g h in vitro t e c h n i q u e s . Combined P r o c . I n t . P l a n t Soc. 33:90-100.  Prop.  M o n e t t e , P.L.. 1985. Use o f g r a p e v i n e s h o o t t i p c u l t u r e s f o r d e t e c t i o n o f f a n l e a f v i r u s by enzyme-1 i n k e d immunosorbent a s s a y . Can. J . P l a n t S c i . 65:977-980. M o n e t t e , P.L. 1986. E l i m i n a t i o n in vitro two g r a p e v i n e n e p o v i r u s e s by an a l t e r n a t i n g t e m p e r a t u r e r e g i m e . J . P h y t o p a t h o l . 116:88-91. M o r i n i , S., P. M a r z i a l e t t i , and C. B a r b i e r i . 1985. In vitro p r o p a g a t i o n o f g r a p e v i n e . R i v . O r t o f l o r o f r u t t . I t a l . 69:385-396. Moses,  Mur,  L . E . and R.V. O a k f o r d . 1963. T a b l e s o f random permutations. Stanford, C a l i f o r n i a . Stanford University Press.  G. 1979. T h e r m o t h e r a p i e s de v a r i e t e s de Vitis vinifera p a r l a methode de c u l t u r e in vitro. Prog. A g r i c . V i t i c . 96:148-151.  M u r a n t , A.F. 1970. A r a b i s mosaic v i r u s . C.M.I./A.A.B. D e s c r i p t i o n s o f P l a n t V i r u s e s No. 16. M u r a s h i g e , T. and F. Skoog. 1962. A r e v i s e d medium f o r r a p i d growth of b i o a s s a y s with tobacco t i s s u e c u l t u r e s . P h y s i o l . P l a n t . 15:473-497. P i e r p o n t , W.S., R . J . I r e l a n d , and J.M. C a r p e n t e r . 1977. M o d i f i c a t i o n o f p r o t e i n s d u r i n g the o x i d a t i o n o f l e a f phenols: r e a c t i o n of potato v i r u s X with c h l o r o g e n o q u i n o n e . P h y t o c h e m i s t r y 16:29-34. R a m s d e l l , D . C , R.W. Andrews, J.M. G i l l e t t , and C.E. M o r r i s . 1979. A c o m p a r i s o n between enzyme-1 i n k e d immunosorbent a s s a y (ELISA) and Chenopodium quinoa f o r d e t e c t i o n o f peach r o s e t t e m o s a i c v i r u s i n ' C o n c o r d ' g r a p e v i n e s . P l a n t D i s . Rep. 63: 74-78. Shanmuganathan, N. and G. F l e t c h e r . 1982. Enzyme -1 i n k e d immunosorbent a s s a y t o d e t e c t f a n l e a f v i r u s i n g r a p e v i n e s grown i n c o n t a i n e r s . P l a n t D i s . 66:704-707. S i m p k i n s , I . , D.G.A. Walkey, and H.A. N e e l y . 1981. Chemical suppression of v i r u s i n c u l t u r e d plant t i s s u e s . Ann. A p p l . B i o l . 99:161-169. Snir,  I . and A. S t e i n . 1985. In vitro d e t e c t i o n and e l i m i n a t i o n o f prunus n e c r o t i c r i n g s p o t v i r u s i n sweet c h e r r y (Prunus avium). R i v . O r t i f l o r o f r u t t . I t . 69:191-194.  97  S t a c e - S m i t h , R. 1985. T o b a c c o r i n g s p o t v i r u s . A.A.B. D e s c r i p t i o n s o f P l a n t V i r u s e s No. 309. S t e l l m a c h , G. 1985a. P o s s i b i l i t i e s and l i m i t a t i o n s o f the v i r u s d i a g n o s i s f o r nepo v i r u s e s by means o f ELISA w i t h the sawdust from b u n d l e d g r a p e s h o o t s . Gesunde. P f l a n z e n . 37:454-460. S t e l l m a c h , G. 1985b. ELISA t e s t i n g o f g r a p e v i n e r o o t i n g s r e a r e d from n e p o v i r u s - i n f e c t e d mother p l a n t s f o r c e d r a p i d g r o w t h . P h y t o p a t h o l . M e d i t e r r . 24:123-124  to  S t e l l m a c h , G. and B e r e s . 1985. I n v e s t i g a t i o n s on mixed i n f e c t i o n s o f N e p o v i r u s e s i n Vitis spp. and Chenopodium quinoa W i l l d . by means o f ELISA. P h y t o p a t h o l . M e d i t e r r . 24:125-128. S t e v e n s o n , J.H. and P.L. M o n e t t e . 1983. D e l a y o f o n s e t of l e a f r o l l symptom e x p r e s s i o n . Can. J . P l a n t S c i . 63 : 557-560. S u t u l a , C.L., J.M. G i l l e t t , S.M. M o r r i s s e y , and D.C. R a m s d e l l . 1986. I n t e r p r e t a t i n g ELISA d a t a and e s t a b l i s h i n g the p o s i t i v e - n e g a t i v e t h r e s h o l d . P l a n t D i s . 70:722-726. Tanne, E. 1980. The use o f ELISA f o r the d e t e c t i o n o f some nepoviruses in grapevines. In: A . J . M c G i n n i s ( e d . ) : P r o c e e d i n g s o f the 7th M e e t i n g o f the I n t e r n a t i o n a l C o u n c i l f o r the Study o f V i r u s e s and V i r u s - l i k e D i s e a s e s o f the G r a p e v i n e . N i a g a r a P a l l s , Canada, pp. 293-296. A g r i c u l t u r e Canada, V i n e l a n d R e s e a r c h Station. Uyemoto, J.K., A.C. Goheen, C F . Luhn, and L . J . P e t e r s e n . 1976. Use o f Chenopodium quinoa i n indexing for grapevine fanleaf v i r u s . Plant Dis. Rep. 60:536-538. Valat,  C. and G. Mur. 1976. T h e r m o t h e r a p i e P r o g . A g r i c . V i t i c . 6:200-202.  du  Cardinal  Rouge.  Vetten,  H.J. 1981. I n d e x i n g o f n e p o v i r u s e s on Chenopodium quinoa a f t e r e l i m i n a t i o n o f v i r u s i n h i b i t o r s i n g r a p e l e a f e x t r a c t s . Z. P f l a n z e n k r . Pflanzenschutz 57:99-110.  W a l t e r , B. and L. E t i e n n e . 1987. D e t e c t i o n f a n l e a f v i r u s e s away from the p e r i o d J. Phytopathol. 120:355-364.  of of  grapevine vegetation.  98 W a l t e r , B., A. V u i t e n n e z , J . K u s z a l a , G. S t o c k y , J . B u r c k a r d , and M.H.V. Van Regenmorte1. 1984. D e t e c t i o n s 6 r o l o g i q u e des v i r u s du c o u r t - n o u 6 de v i g n e par l e t e s t ELISA. Agronomie 4:527-534. W r i g h t , N.S. and R. S t a c e - S m i t h . 1966. A c o m p a r i s o n o f s e n s i t i v i t y of three s e r o l o g i c a l t e s t s f o r p l a n t v i r u s e s and o t h e r a n t i g e n s . P h y t o p a t h o l o g y 56 : 944-948.  la  the  Z a t y k o , J.M. and I. M o l n a r . 1985. P r e l i m i n a r y r e s u l t s on the in vitro mass p r o p a g a t i o n o f g r a p e s from s h o o t - t i p m e r i s t e m . F r u i t . S c i . Rep. 12:83-85.  99 APPENDIX Appendix  1.  Vitis  2  ie.  sol'n  A B C D E F G  3  i-inositol thiamine adenine  HCI sulfate  NaH P0 H 0 2  4  2  culture  media  Growth  stage  Initiation  Component MS  tissue  1  Proliferation  3/4 s trength  full s trength  15.0 ml/L 15.0 ml/L 3.75 ml/L 3.75 ml/L 3.75 ml/L 3.75 ml/L 3.75 ml/L  20.0 ml/L 20.0 ml/L 5 . 0 ml/L 5. 0 ml/L 5 . 0 ml/L 5 . 0 ml/L 5.0 ml/L  75.0 0.3  mg/L  100.0  mg/L  mg/L  0 . 4 mg/L  60.0  mg/L  80 . 0 mg/L  128.0  mg/L  BAP  4  2.0  mg/L  IBA  S  0.0 23  mg/L  170.0  mg/L  Root i n g 1/4 s trength 5.0 ml/L 5.0 ml/L 1.25 ml/L 1.25 ml/L 1 .25 ml/L 1 .25 ml/L 1.25 ml/L 25.0 0.1  150.0  0.03  g/L  PH  5. 7  agar  7.0 g/L  mg/L  mg/L 0.1  22.5  mg/L  2.0 mg/L  I AA sucrose  mg/L  30 . 0 g/L  17.5  5. 0  mg/L g/L  5. 0  To p r e p a r e the c u l t u r e medium, add the d e s i r e d amount s o f each s o l u t i o n and i n g r e d i e n t s t o about 800 ml o f d i s t i l l e d w a t e r . B r i n g t o 1000 ml and a d j u s t pH. I f needed, h e a t and add a g a r . 1  MS c o n s i s t s o f a m o d i f i e d M u r a s h i g e and Skoog s a l t mixture w i t h the f o l l o w i n g i n g r e d i e n t s : 2  plant  100  Appendix  1.  continued  Solution  Cons t i t u e n t s  Cone.  (g/L)  A  NH N0  B  KN0  C  H B0 KH P0 KI Na MoO.« . 2H 0 CoCl .6H 0  1.24 34.00 0.166 0.05 0.005  CaCl .2H 0 (or C a C l )  88.0 66.43  4  3  82.5  3  95.0  3  3  2  4  2  2  2  D  2  2  2  2  E  F*  MgS0 MnS0 (or ZnS0 CuS0  4  .7H 0 .4H 0 MnS0 .H 0) .7H 0 .5H 0 4  4 4  74.0 3.36 2.55 1.72 0.005  2  2  2  2  2  Na .EDTA FeS0 .7H .0  7.45 5.57  2  4  G  4  3  Nicotinic acid P y r o x i d i n e HCl Glycine  0.1 0.1 0. 1  A s t o c k s o l u t i o n o f BAP ,(.5 mg/ml) may be p r e p a r e d by d i s s o l v i n g 50 mg o f BAP i n a v e r y s m a l l volume o f 1 M HCl t o form a s l u r r y . T h i s can then be added t o 100 ml o f d i s t i l l e d w a t e r and kept r e f r i g e r a t e d . 4  To p r e p a r e a s t o c k s o l u t i o n o f IBA (.lmg/ml), d i s s o l v e 10 mg i n a v e r y s m a l l volume o f 1 M NaOH t o form s l u r r y . Then s l o w l y add 100 ml o f d i s t i l l e d w a t e r w h i l e stirring. 5  * To p r e p a r e s o l u t i o n F: d i s s o l v e each c o n s t i t u e n t i n 200 ml d i s t i l l e d w a t e r , heat the Na .EDTA s o l u t i o n , t h e n w i t h c o n t i n u o u s s t i r r i n g add the F e S 0 . 7 H 0 s o l u t i o n . When c o o l e d d i l u t e t o 1000 ml. 2  4  2  a  

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